OPLEIDINGSCOMMISSIE BIOCHEMIE EN BIOTECHNOLOGIE
Voorzitter: Prof. Dr. Peter Vandenabeele Ondervoorzitter: Prof. Dr. Bart Devreese
27 februari 2009 Beste studenten, Enkele overwegingen bij het aanvatten van de masterproef In deze brochure hebben wij de onderwerpen verzameld voor de masterproeven. Het is een lange lijst geworden met een gevarieerd aanbod. Dit laat jullie toe om keuzes te maken volgens jullie eigen interesse en ambitie. Ik zou bij deze gelegenheid de begeleiders (postdocs, doctoraatsstudenten), technici en promotoren willen bedanken die de inspanningen zullen leveren om de studenten een intensieve en zeer degelijke opleiding te bezorgen. Het is inderdaad zo dat de hoeveelheid werk die gepaard gaat met masterprojecten in Master 1 en masterproeven in Master 2 niet kan onderschat worden. Het is een zware en jaarlijks terugkerende inspanning van deze personen, die bijdraagt tot jullie vorming als jonge wetenschappers. De kwaliteit van deze vorming is de basis van het wetenschappelijk onderzoek van de komende 20 jaar. Wij zijn ervan overtuigd dat jullie dit weten te appreciëren en dat jullie de mogelijkheden die tijdens de masterproef worden geboden, ten volle zullen benutten. De keuze van de masterproef ligt in de regel in het verlengde van jullie majorkeuze (BSB, BIS, BIB, MIB, PLB). Gemotiveerde afwijkingen van deze regel kunnen worden aangevraagd bij de OC Biochemie en Biotechnologie. De onderwerpen van de masterproeven zijn gerangschikt volgens de departementen die de masterproeven begeleiden. Het merendeel wordt aangeboden door de 4 departementen die grotendeels bij jullie opleiding zijn betrokken (WE09, WE10, WE14, WE15), maar ook enkele andere departementen bieden masterproeven aan. Elke masterproef wordt ook nog gerangschikt volgens de major waarop de masterproef aansluit. Vaak sluit de masterproef aan op verschillende majors: PLB (Plantenbiotechnologie), BIS (Bio informatica en systeembiologie), MIB (Microbiële biotechnologie), BSB (Biochemie en structurele biologie), BIB (Biomedische biotechnologie). Keuzes maken is niet eenvoudig. Ik zou hierover enkele overwegingen willen meegeven: • Laat u leiden door uw interesse, maar besef ook dat uw kennis betreffende deze onderwerpen heel beperkt is. Wetenschappers in fundamenteel onderzoek worden vaak geconfronteerd met onbekende, op het eerste zicht niet te situeren gegevens,
die mits gerichte experimenten en nieuwe inzichten een wereld kunnen doen opengaan. Dus, een voor u onbekend onderzoeksdomein kan heel boeiend worden. • Laat u niet misleiden door de onmiddellijke toepasbaarheid en het grote kader van de onderzoeksonderwerpen. De meeste onderwerpen in onze departementen betreffen fundamenteel onderzoek. Het grote kader (“kanker bestrijden”, “voedselproductie verbeteren”, “milieuprobleem oplossen”) is enkel een langetermijn doelstelling van het onderzoek, en kan uiteraard nooit in het bestek van een masterproef worden behaald. De meeste onderzoeksonderwerpen, hoe fundamenteel of toegepast ze ook mogen klinken, houden het gebruik in van dezelfde methoden en technieken. • Besef dat in alle departementen die jullie opleidingen ondersteunen, uitstekend wetenschappelijk onderzoek floreert. Dit zorgt voor een kwaliteitslabel voor jullie opleiding en betekent dat je in feite niet de verkeerde keuze kunt maken. • Geen enkele keuze is definitief. Na je masterproef bestaat nog steeds de mogelijkheid om op basis van interesse, nieuwe inzichten en ambitie, een onafhankelijke keuze te maken voor een doctoraatsproef. De keuze van de masterproef is belangrijk, maar het is zeker geen definitieve keuze voor een doctoraatsonderwerp. De keuze om een doctoraatsonderzoek uit te voeren is te belangrijk om het automatisch in het verlengde van de masterproef te leggen. Overweeg en negocieer over verschillende mogelijkheden (al of niet doctoraat, zelfde onderzoeksgroep, andere onderzoeksgroep, ander onderwerp, andere universiteit, buitenland). • Gebruik de periode van de masterproef vooral om je een aantal wetenschappelijke attitudes eigen te maken. Dit kan gebeuren in elke degelijke onderzoeksomgeving. Deze wetenschappelijke attitudes houden o.a. het volgende in: precieze vraagstelling bij het opstellen van experimenten; correctheid bij het uitvoeren en noteren van de experimenten, vasthouden aan experimentele protocols (reproduceerbaarheid is de essentie van wetenschappelijk onderzoek); zich informeren door vragen te stellen of zaken op te zoeken (elk woord, product of protocol zou je moeten weten te duiden); reviews en onderzoeksartikels te lezen over je onderwerp; actieve interesse en betrokkenheid te betonen voor je werk; communiceren over wetenschappelijke vragen, inzichten en resultaten met uw leidinggevende(n), uw collega’s en medestudenten. Het voordeel van wetenschappelijke discussies is dat ze letterlijk overal en op elk moment kunnen worden gevoerd. Deel je kennis, inzichten en vragen met anderen. Je zult er veel plezier aan beleven! Wetenschappelijk onderzoek is een manier van leven.
Praktische afspraken Het masterproef onderdeel in het studieprogramma voor Master 2 Biochemie en Biotechnologie omvat een aanzienlijk deel "zelfstandig" praktisch werk, gevolgd door het schrijven en verdedigen van een scriptie. Het scriptie gedeelte van de opleiding telt mee voor 30 van de 60 studiepunten van de 2 de master, en beslaat in praktijk minstens het volledige 2 de semester. Binnen de opleiding Biochemie en Biotechnologie is het de uitdrukkelijke wens van de Opleidingscommissie dat de masterproeven van de hoogst mogelijke kwaliteit behoren te zijn. Alle onderwerpen sluiten aan bij lopend onderzoek binnen de departementen, en er wordt over gewaakt dat er steeds een voldoende pakket aan basistechnologie eigen kan worden gemaakt. Naast de onderwerpen aangeboden door de vakgroepen vertegenwoordigd binnen de Opleiding Biotechnologie en Biochemie [Vakgroep Plantenbiotechnologie en Genetica (WE09), Vakgroep Biochemie en Microbiologie (WE10), Vakgroep Biomedische Moleculaire Biologie (WE14)] en vakgroep Fysiologie (WE15)], worden er ook onderwerpen aangeboden die worden uitgevoerd in andere vakgroepen (vanaf sectie VI van de lijst met masterproeven). De scriptie geeft je de mogelijkheid om je te verdiepen in een onderwerp naar je keuze. Gebruik de komende weken om de onderwerpen reeds door te nemen. De scriptie onderwerpen worden hierna nogmaals verder toegelicht tijdens een gezamelijke voorstelling door de betrokken vakgroepen op Vrijdag 24 april 2009 om 14 u in het UGent/VIB gebouw, Technologiepark 927 Zwijnaarde Wij raden je aan deze presentatie bij te wonen, het zal je ongetwijfeld helpen bij het maken van je keuze. Bij elk onderwerp zal je ook coördinaten vinden van de verantwoordelijke promotor/begeleider, waarbij je terecht kan met al je vragen. Nog enkele spelregels Definitieve toewijzing van een masterproef zal gebeuren na de 1 ste zittijd of na de 2 de zittijd, naargelang. De keuzes van alle studenten worden verwacht tegen maandag 6 juli om 18u (via online formulier; zie verder). De toekenning na de 1 ste zittijd zal gebeuren ten laatste op vrijdag 10 juli 2009; de toekenning na de 2 de zittijd zal gebeuren ten laatste op vrijdag 18 september 2009. De volgorde van de toekenning berust op de sommatie van de behaalde examenquoteringen in de Bachelor en Master 1: de best geplaatste krijgt het eerst haar/zijn voorkeuronderwerp toegewezen, enz. Hiernaast is de toekenning van de onderwerpen ook onderworpen aan een aantal voorwaarden en wordt elke situatie specifiek bekeken. Deze procedure is noodzakelijk omdat een Masterproef een zware inspanning en investering vergt van de betrokken vakgroepen en enkel kan geleverd worden met zicht op het behalen van het finale Master diploma en omdat de noodzakelijke theoretische achtergrond nodig is voor het afleggen van de Masterproef.
Tenslotte, de Masterproef omvat 30 stp, in principe volledig op te nemen in het 2 de semester van Master 2. Er wordt hierin wel flexibiliteit voorzien nl. dat maximaal 9 stp aan theoretische vakken kunnen worden gevolgd in het 2 de semester van Master 2, maar dit dient gecompenseerd door een verhoogde inspanning voor de Masterproef in semester 1 van Master 2. • Afwijkingen van de toelatingsvoorwaarden voor de Masterproef zijn enkel mogelijk mits omstandige motivering gericht aan de voorzitter van de OC Biochemie en Biotechnologie. Zij zullen worden behandeld op de OC Biochemie en Biotechnologie. • Voltijdse studenten (i.e. een jaarlijks studieprogramma van 54 66 stp) die grotendeels modeltraject volgen Toelatingsvoorwaarden voor de masterproef: - In het bezit zijn van een bachelordiploma - Voor minstens 51 STP credits voor opleidingsonderdelen uit MA1 hebben verworven - Voor minstens 21 STP credits voor opleidingsonderdelen uit MA2 hebben verworven of gelijktijdig aan het verwerven zijn, of een combinatie van beide
(1) Definitieve toewijzing van het onderwerp onmiddellijk na de eerste zittijd voor studenten die beschikken over een bachelordiploma Biochemie en Biotechnologie* en voor minstens 51 STP credits hebben verworven voor opleidingsonderdelen uit MA1 Biochemie en Biotechnologie*. (2) Voorlopige toewijzing van het onderwerp onmiddellijk na de eerste zittijd voor studenten die beschikken over een bachelordiploma Biochemie en Biotechnologie* en voor minstens 30 STP credits hebben verworven voor opleidingsonderdelen uit MA1*. De voorwaarde om een definitieve toewijzing na de tweede zit te bekomen is in de tweede zittijd bijkomende credits te verwerven voor minstens 21 STP voor opleidingsonderdelen uit MA1 Biochemie en Biotechnologie*. (3) Studenten kunnen een aanvraag indienen voor een definitieve toewijzing van een thesisonderwerp indien zij in de tweede zittijd een Bachelordiploma Biochemie en Biotechnologie* verworven hebben en credits verworven hebben voor ten minste 51 STP aan opleidingsonderdelen uit MA1 Biochemie en Biotechnologie**. (4) Bij aanvang van het 2 de semester Master 2 hebben de studenten voor minstens 21 STP credits opleidingsonderdelen uit MA2 verworven of zijn deze aan het verwerven zijn, of een combinatie van beide. • Deeltijdse studenten Studenten kunnen een aanvraag indienen voor een definitieve toewijzing van een thesisonderwerp indien zij beschikken over een Bachelordiploma Biochemie of Biotechnologie* en voor minstens 51 STP credits hebben verworven voor opleidingsonderdelen uit MA1 Biochemie en Biotechnologie** en voor minstens 21 STP credits hebben verworven voor opleidingsonderdelen uit MA2*. *: of equivalent via schakel of voorbereidingsprogramma **: of equivalent via een Erasmusuitwisseling
Genoeg regels, nu de onderwerpen ... en uw keuze In deze bundel vind je een korte inhoud van de masterproeven en een samenvattende lijst van alle onderwerpen, gerangschikt per vakgroep. Gelieve uw keuze te maken uit het totale aanbod (1 tem 5) en uw keuze tegen 6 juli 2009 ten laatste kenbaar te maken via het online formulier op de website van de opleiding Biochemie en Biotechnologie. Vriendelijke groeten, Prof. Dr. Peter Vandenabeele Voorzitter Opleidingscommissie Biochemie en Biotechnologie
MASTERPROEVEN
2009-2010
Biochemie en Biotechnologie
Master 2
Onderwerpen waarvan het nummer gevolgd wordt door * houden een verplicht volgen van de cursus proefdierkunde in.
1 Molecular analysis of PIN exocytosis
Department: Plant Systems Biology
Promoter: Prof. Dr. Ji ří Friml Co-Promoter: Dr. Steffen Vanneste
Address + Phone number (Co) Promoter(s): VIB dept Plant Systems Biology Auxin group Technologiepark 927 9052 Zwijnaarde Tel: 09/33 13914
Focus : PLB
Short description of the subject: Auxin is for a long time known as a major hormonal regulator of plant development. Uniquely among plant signalling molecules, auxin is transported in a strictly regulated, polar fashion from cell to cell through plant tissues. Previously, our group has identified auxin efflux components encoded by the PIN gene-family through molecular genetic studies in the model plant Arabidopsis thaliana . We could show that they localise asymmetrically within the cell and their subcellular localisation is sufficient to dictate the direction of auxin transport. Through changes in local auxin accumulation, PINs are modulating plant development. Moreover, we found that these transmembranous proteins cycle constitutively between the plasma membrane and endogenous compartments. This dynamism allows plants to shuffle PINs rapidly from one side of the cell to the other, in response to developmental and environmental stimuli. Therefore, insights in the mechanisms that control PIN localisation are of fundamental importance to understand how plant growth and development is regulated.
Aim and rationale: In this project we will adress the mechanisms of PIN insertion into the plasma membrane (exocytosis). Old observations demonstrated that auxin itself is capable of inducing Ca 2+ fluxes in various developmental processes. Moreover, it could be demonstrated that Ca 2+ is required for some of these processes, and even that Ca 2+ is required for auxin transport. However, it is not known how Ca 2+ could affect auxin transport. Furthermore, Ca 2+ has been implicated as a positive regulator of exocytosis in stomatal opening, neurotransmission, ... Therefore, in this project we will adress the link between Ca 2+ and PIN exocytosis at the physiological level and at the molecular level. Alternatively, we will study the role of synaptotagmins in this process: predicted plant homologs of animal Ca 2+ sensors for exocytosis.
Techniques and methods: Confocal microscopy, immunolocalisation, in situ hybridisation, light microscopy, phenotypical analysis, PCR-based genotyping, crosses, gateway cloning, artificial microRNAs, targeted mutagenesis, expression analysis …
2 Oxidative Stress Signal Transduction in Plants
Department: WE09
(Co) Promoter(s): Prof. Frank Van Breusegem
Address + Phone number (Co) Promoter(s): PLANT SYSTEMS BIOLOGY Technologiepark 927 9052- Gent Tel. 09 33 13 920 [email protected]
Focus : PLB, BIS
Short description of the subject: Suboptimal growth conditions caused by drought, temperature, salt stress and pathogen- related stress are leading to worldwide yield losses in cultivated crops. This, together with the ongoing climatic changes, has encouraged the development of appropriate breeding strategies and has made crop 'stress tolerance' a major objective in plant biotechnology research. Under plant stress conditions, 'Reactive Oxygen Species' (ROS) are used in cell signalling as a 'warning' message that activates the plant defense response. Nowadays, the knowledge of the regulatory events during ROS signal transduction is only limited. Through a combined top-down and bottom-up genomics approach, we are aiming to identify key regulatory genes that are able to regulate changes in the cellular response during stress. The potential of these genes for engineering abiotic stress tolerance in plants will be assessed through state of the art molecular and physiological technologies.
Aim: Identify key regulatory genes that are able to sense and regulate changes in the cellular state during stress and assess the feasibility to use these genes in the making of stress- resistant plants.
Techniques and methods: Genome-wide transcriptome analysis (microarrays, ...) Transgenic plants. Promotor:reporter constructs In silico analysis
3 Functional evaluation of Arabidopsis metacaspases targets
Department: WE09
(Co) Promoter(s): Prof. Frank Van Breusegem
Address + Phone number (Co) Promoter(s): PLANT SYSTEMS BIOLOGY Technologiepark 927 9052- Gent Tel. 09 33 13 920 [email protected]
Focus : PLB, BSB
Short description of the subject: The main executioner proteins in metazoan programmed cell death are the caspases. We have cloned and characterized 9 Arabidopsis structural homologs of caspases: the plant metacaspases. Plant metacaspases are believed to be involved in a variety of growth and developmental processes and in the response against environmental stresses. However, little is known on their exact physiological functions and, in particular, their protein targets remain unknown. Together with the Dept. of Medical Protein Research we have identified on a proteom-wide scale, several protein substrates for different metacaspases. To validate the discovered protease targets and assess the stability of generated protein fragments, proteomes of both knock-out and over- expressing plants of the different metacaspases will be profiled. Protease substrates and stable protein fragments will be further investigated by altering their expression in knock- out lines, RNAi or over-expression. This project should lead to a profound insight in the physiological processes governed by plant metacaspases and might provide new knowledge that can be translated into crop improvement.
Aim: To unravel the involvement of metacaspases and their substrates in plant cell death.
Techniques and methods: Proteomics (gel electrophoresis, Western blot analysis, ...) Transgenic plants (RNAi, inducible overexpression,...) Protein-protein interactions (TAP technology, Y2H,...) Promotor:reporter constructs Immunolocalisations In silico analysis.
4 Molecular function of plant Elongator
Department: WE09
(Co) Promoter(s): Prof.Dr. Mieke Van Lijsebettens Begeleider: Steven De Groeve
Address + Phone number (Co) Promoter(s): Ghent University/VIB, Department Plant Systems Biology, Technologiepark 927, 9052, Ghent Email: [email protected] Tel. 09-3313970
Focus : PLB, BSB, BIS
Short description of the subject: The relation between chromatin structure and gene expression regulation is subject of a rapidly growing field and forms the main research Focus in our lab. Histone Acetyl Transferases (HATs) alter DNA accessibility by chromatin modification and thereby activate gene expression. Elongator is such a HAT complex that consists of six subunits and co-purifies with the elongating RNA Polymerase II in yeast and humans. In plants, mutations in the homologous Elongator genes severely affect cell proliferation and organ growth which results in very narrow leaves and reduced primary root growh. Transcriptome analysis shows that the Elongator complex is involved in stress responses, hormone signaling and light perception. Its colocalization with euchromatin (actively transcribed DNA) further confirms its role in regulating gene expression. How the Elongator complex itself is regulated and what the direct target genes are remains unknown.
Aim: We’re aiming to build a molecular network around the Elongator complex to better understand its role in plant growth and how it is regulated. This will be done by identifying proteins which interact with the Elongator complex, identifying the direct target genes of the complex and looking for genetic interactions with other genes involved in regulating plant growth by transcriptional regulation.
Techniques and methods: Yeast-2-hybrid and immunoprecipitation techniques for protein - protein interaction studies; Chromatin Immunoprecipitation for protein – DNA interaction studies; Protein electrophoresis and Western blotting for protein analysis; Quantitative Polymerase Chain Reactions (QPCR) for gene expression analysis; Double mutant analysis for genetic interaction studies.
5 Molecular network of HISTONE MONOUBIQUITINATION1 and chromatin in transcriptional regulation
Department: WE09
(Co) Promoter(s): Dr Mieke Van Lijsebettens, Dr Kristiina Himanen
Address + Phone number (Co) Promoter(s): Ghent University/VIB, Department Plant Systems Biology, Technologiepark 927, 9052, Ghent Email: [email protected] Tel. 09-3313970
Focus : PLB, BSB, BIS
Short description of the subject: Histone modifications affect the accessibility of chromatin thereby facilitating or repressing transcription activities. We have identified two conserved histone modifying protein complexes; HISTONE MONOUBIQUITINATION1 (HUB1) and ELONGATOR via characterization of a collection of leaf growth mutants (Nelissen et al., 2005, PNAS; Fleury et al., 2007, Plant Cell). HUB1 is an unconventional ubiquitin E3 ligase that is not involved in protein degradation but mediates histone H2B monoubiquitination. This histone H2B regulation initiates a chain reaction of other histone modifications ultimately resulting in activation of RNA Polymerase II mediated transcription. hub1-1 mutant is a narrow leaf mutant with pale colouring and decreased cell numbers in both leaf epidermis and palisade cell layers. Transcriptome analysis revealed that cell division and photosynthetic genes were misregulated and may thus explain the phenotypes. However, the direct target genes of HUB1 need still to be identified. To this end a comparative microarray transcriptome analysis of HUB1 overexpression and mutant lines was performed to allow identification of oppositely regulated genes between the HUB1 misexpression lines.
Aim: The aim of this project is to characterize the molecular networks of HUB1 and mediating its effects on plant growth. To this end we will identify and characterize regulatory pathways, functional protein complexes and direct target genes of HUB1. Genetic interaction studies will be used to confirm HUB1 involvement in regulatory pathways. To identify HUB1 protein complexes we have used the Tandem Affinity Purification technique. The functional meaning of the identified protein interactions will be tested by in vitro and in vivo assays. The candidate HUB1 target genes identified from microarray analysis will be confirmed by Chromatin Immunoprecipitation (ChIP) assays.
Techniques and methods: Tandem Affinity Purification and immunoprecipitation techniques for protein - protein interaction studies; Chromatin Immunoprecipitation for protein – DNA interaction studies; Protein electrophoresis and Western blotting for protein analysis; Quantitative Polymerase Chain Reactions (QPCR) for transcriptome analysis; Double mutant analysis for genetic interaction studies.
6 Functional analysis of maize growth regulatory genes under limiting environmental conditions
Department: Plant Systems Biology
Promoter: Prof. Dr. Dirk Inzé Co-Promoter: Prof. Dr. Gerrit Beemster
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Gent, 09/3313800, [email protected] ; 09/3313971, [email protected]
Focus : PLB
Short description of the subject:
Maize is an important food and bioenergy crop for which yield losses due to limiting environmental factors such as drought and cold represent a major economical problem. Therefore, our research group aims to identify and characterize novel growth-genes, of which altered expression results in improved growth under limiting conditions. Micro-array experiments revealed genes that putatively regulate growth under drought and cold conditions. We will validate the function of these genes and their potential in stress tolerance by detailed growth analyses of Arabidopsis plants overexpressing these maize genes and TDNA insertion mutants for the homologous genes. In addition, the function of these genes in the growing maize leaves will be examined by determining the expression levels by QRT-PCR on different cell types in the maize leaf meristem, sampled by Laser Capturing Dissection.
Aim: Validation of the role of novel maize growth genes in stress tolerance in Arabidopsis.
Techniques and methods: DNA- and RNA- based techniques (PCR, QRT-PCR), genetics, growth and stress analyses, Laser Capturing Dissection.
7 Detection and evolution of expression modules in eukaryotic species
Department: Plant Systems Biology Department, Vakgroep Plant Biotechnology and Genetics, Universiteit Gent (UGent-VIB)
(Co) Promoter(s): Dr. Klaas Vandepoele and Prof. Dr. Yves Van de Peer
Address + Phone number (Co) Promoter(s): Technologiepark 927, B-9052 Gent, Tel: 09 3313822 / 09 3313807
Focus : BIS & PLB
Short description of the subject: The diversity and complexity of higher plants and other multi-cellular eukaryotes is caused by the underlying molecular interactions driving different biological processes. Although it is easy to understand that changes in the place or time of gene expression can create new molecular interactions, little information about the evolution of transcriptional regulation is known. This knowledge however, is essential, because each gene is flanked by regulatory sequences which, together with the expression and activity of other proteins, determine the amount, place, and timing of expression. Short cis- regulatory elements form the functional components of a promoter, because they determine the specificity of protein binding by transcription factors. Therefore, characterizing these motifs is required in order to understand the regulatory interactions between trans-acting proteins and the promoters of thousands of genes within a eukaryotic genome. This information is essential when studying biological processes from a holistic point of view by incorporating and combining complementary functional data sets (‘systems biology’).
Aim: The objective of this project is to study the conservation of cis-regulatory elements in expression modules from different eukaryotic species. Based on known regulatory elements and expression clusters from the model species Arabidopsis we want to study how orthologs in other species are transcriptionally regulated. For example, are expression modules involved in cell division, protein biosynthesis and photosynthesis conserved in other species like moss, green or brown algae? Can we identify the mechanisms behind the evolution of transcriptional control?
Techniques and methods: Sequence analysis (orthology mapping, detection of cis-elements) and clustering of expression data. Affinity with computers/bioinformatics and an interest in writing small computer programs is a plus.
8 Identification and characterization of bioactive chemicals that alter PIN polar localization
Department: Plant Systems Biology
Promoter: Prof. Dr. Ji ří Friml (Co) Promoter(s): Dr. Stéphanie Robert
Address + Phone number (Co) Promoter(s): VIB dept Plant Systems Biology Auxin Group Technologiepark 927 9052 Zwijnaarde Tel: 09/33 13916
Focus : PLB
Short description of the subject:
Establishment of cell polarity is one of the most fundamental biological topics. Polarized cells distribute intracellular components asymmetrically along a particular axis. This polar distribution can facilitate specialized cellular functions, such as nutrient uptake in epithelial cells. It has also been proven that in animal systems, cell polarity can provide positional information in pattern formation of multicellular organisms. Interestingly, in plants, fully specified cells often retain the potential to re-define their polarity. Cell polarity manifests itself at the multicellular level through directional (polar) transport of a plant signaling molecule – the phytohormone auxin, which mediates a large variety of plant growth responses. The current model proposes that plant cells integrate internal and external signals at the level of the polarity of auxin transport components (PIN proteins) and via the redirection of auxin fluxes which translate them into adaptive developmental changes.
Aim:
This project proposes to use chemical genomic to characterize the establishment of cell polarity and the subcellular localization of proteins. We will use small molecules to reveal new molecular actors involved in the establishment of PIN polar localization. A detailed analysis of these compounds, regarding their ability to disrupt PIN polar localization and their general effect on plant development combined with forward genetic will allow us to select novel players involved in PIN polar targeting.
Techniques and methods:
Confocal microscopy, chemical genetics, imunolocalization, phenotypical analyses, screen, PCR-based genotyping, crosses, structure/function cluster analyses.
9 Identification of differentially expressed genes underlying growth response variation in pepper
Department: Dept. of Plant Systems Biology / Plant Biotechnology and Genetics
Promoter: Dr. Marnik Vuylsteke
Address + Phone number Promoter: Technologiepark 927 9052 Gent – 09/3313860
Focus : BIS
Short description of the subject: Expression QTL analysis in pepper
In an approach to identify genes underlying growth response variation in pepper, microarrays will be used to perform a genome-wide study of differential gene expression between contrasting QTL genotypes of a mapping population. Significant differentially expressed genes will be mapped relative to the QTL for variation in growth model parameters by an eQTL mapping procedure.
Aim: Identification of differentially expressed genes underlying growth response variation in pepper.
Techniques and methods: QTL analysis & microarray expression data analysis using Genstat software.
10 Mapping the Seminavis robusta MT locus.
Department: Dept. of Plant Systems Biology / Plant Biotechnology and Genetics
Promoter: Dr. Marnik Vuylsteke
Address + Phone number Promoter: Technologiepark 927 9052 Gent – 09/3313860
Focus: PLB
Short description of the subject: Mapping the Mating Type (MT) gene in the unicellular algae Seminavis robusta .
Most sex determining systems are genuinely genetic, determined by a single locus, called the mating type (MT) locus. In this project, we aim to map the sexual dimorphism present in Seminavis robusta , an unicellular algae, by a Bulked Segregant Analysis (BSA) approach. The BSA approach in combination with the AFLP marker technology allows screening for DNA-markers linked to a particular genomic region in a fast and efficient way. Markers linked to the MT locus will be mapped relative to other markers representing the sex chromosome and the autosomal genome.
Aim: Mapping the Seminavis robusta MT locus.
Techniques and methods: AFLP analysis; linkage analysis; QTL analysis.
11 Biosynthesis of novel plant-derived molecules with pharmaceutical activity
Department: Plant Biotechnology and Genetics (WE09)
(Co) Promoter(s): Alain Goossens, Dirk Inzé
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Zwijnaarde, 093313851
Focus : BSB, PLB
Short description of the subject: Combinatorial biosynthesis involves interchanging 'metabolism' genes between different organisms to create unnatural gene combinations. Novel metabolites can be made due to the effect of new enzyme-metabolic pathway combinations. In previous research tens of genes have been identified that all encode enzymes catalyzing the biosynthesis of biologically active plant triterpene saponins. In this master thesis, several constructs combining two or more of these genes will be designed, and integrated into a single host cell. This will create a combinatorial biosynthesis library that potentially produces novel saponin molecules. Transformed cells will be screened for the presence of novel molecules and biological activities, including anti-cancer, ant-inflammatory and adjuvant activities. Both plant ( Medicago truncatula ) and yeast ( Saccharomyces cerevisiae ) cultures can be used as production systems.
More info: http://www.psb.ugent.be/secondary-metabolites/index.php
Aim: Synthesis of novel biologically active saponins in yeast and/or plant cultures.
Techniques and methods: • Transformation of yeast cells and/or Medicago root cultures • Screening of transgenic cells for novel metabolites by fast TLC- or fluorescence-based metabolite detection • Screening of transgenic cells for novel metabolites by bio-activity assays
12 Engineering of jasmonate signaling to improve plant growth and defense
Department: Plant Biotechnology and Genetics (WE09)
(Co) Promoter(s): Alain Goossens, Dirk Inzé
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Zwijnaarde, 093313851
Focus : PLB
Short description of the subject: Jasmonates (JAs) are plant hormones that regulate plant development and defense. JA signaling is activated upon wounding or pathogen attack. As a consequence, plant growth is inhibited and defense responses are launched. Using protein-protein interaction mapping, we have identified several novel proteins that regulate the JA signalling cascade. The functions of these proteins and their protein domains is being intensely investigated. With this information, novel gene constructs will be tailored and expressed in Arabidopsis thaliana and Nicotiana tabacum with the aim of improving plant growth and boosting the production of secondary metabolites involved in plant defense, respectively. Successful constructs will be transferred to commercially important medicinal plant species such as Catharanthus rosues to improve the production of the anti-cancer compounds vinblastine and vinscristine.
More info: http://www.psb.ugent.be/secondary-metabolites/index.php
Aim: Characterization of transgenic plant and cell lines
Techniques and methods: - Selection and propagation of transgenics - Transcript profiling - Phenotypical analysis - Molecular cloning
13 Identification of novel endocycle regulators though the use of chemical genomics
Department: Plant Systems Biology (PSB)
Promoter: Prof. Dr. Lieven De Veylder Co-promotor: Dr. Tim Lammens
Address + Phone number promotor: Technologiepark 927, 9052 Gent Tel. 09/331.39.61
Focus: Plant Biotechnology (PLB)
Short description of the subject: Recently, both in plants and animals, the field of endoreduplication received huge attention because of its association with cell size determination, DNA stress resistance, and carcinogenesis. Endoreduplication is a wide- spread variant of the mitotic cell cycle during which cells duplicate their genome in the absence of mitosis, resulting into cells with high DNA ploidy content. To identify novel regulators of endoreduplication onset, we performed a high-throughput chemical genomics screen. Chemical genomics is based on the ability of small molecules (chemical compounds) to bind proteins, modulate their activity and thus disturb signaling cascades within cells. Within a set of 10.000 chemical compounds we have identified several molecules that promote endoreduplication in A. thaliana . Within this project, the effects of the selected chemical compounds on A. thaliana plants and cell cultures will be analyzed in detail. Through the combination of flow cytometry with cell biology, the tissues specifically responding to the chemicals will be identified. Chemical targets will be identified through yeast 3-hybrid screens, microarray analysis, and ESM mutagenesis. Moreover, a detailed transcriptomic analysis of chemical-treated plants will help in the identification of novel endocycle target genes and their regulators.
Aim: The ultimate goal of this project is to identify the molecular cascades by which selected chemical compounds trigger the endoreduplication process.
Techniques and methods: in vitro plant growth, plant cell culture, microarray analysis, Real-Time qRT-PCR, flow-cytometry, yeast 3-hybrid
14 Identification and characterization of novel DNA stress-inducing genes
Department: Plant Systems Biology (PSB)
Promoter: Prof. Dr. Lieven De Veylder
Address + Phone number Promoter: Technologiepark 927, 9052 Gent, Tel:09/3313961
Focus : Plant Biotechnology (PLB)
Short description of the subject: Genome integrity of cells is threatened by DNA damage that is the consequence of environmental stresses and endogenous causes. To cope with these stress conditions, cells have developed a set of surveillance mechanisms to monitor the status and structure of DNA during cell cycle progression. The plant WEE1 gene, encoding a negative regulator of the cell cycle, is rapidly activated in response to treatments that induce DNA stress. Plants without a functional WEE1 gene are hypersensitive to DNA damaging drugs because they fail to arrest their cell cycle before the damaged DNA is repaired. As a consequence, in the presence of either exogenous applied or endogenous DNA stress, WEE1-deficient plants proceed with a mutated genome in to mitosis, resulting in to a growth arrest.
DNA stress is not only caused by external stimuli (such as the environment), but also by endogenous factors (such as the DNA replication process itself). To identify in an unbiased manner the different endogenous processes that cause DNA stress, we transformed a WEE1 -deficient plant with a T-DNA construct. This construct was inserted randomly into the genome. When the T-DNA is inserted in to a gene, it is expected to inactivate this gene. When the inactivated gene represents an endogenous source of DNA stress, the transformed WEE1-deficient plant will display a growth inhibition phenotype that segregates with the T-DNA insertion. At this moment, already 10 of these genes were identified. In the proposed research proposal, we will try to understand why the identified genes cause DNA stress.
Aim: Characterizing novel DNA stress-inducing mutations and understanding the role of the corresponding wild-type genes during development.
Techniques and methods: Plant growth assays, RT-PCR, fluorescence microscopy, flow- cytometry, comet assays, genotyping,…
15 Identification and analysis of new genes involved in division plane determination in plant cells
Department: Plantenbiotechnologie en Genetica
(Co) Promoter(s): Prof. Geert De Jaeger and Dr. Daniel Van Damme Begeleiding: Astrid Gadeyne
Address + Phone number (Co) Promoter(s): PSB, Technologiepark 927, 9052 Gent
Focus : PLB, BSB
Cell division in plants is unique among eukaryotes because a new cell wall is built in between the two daughter nuclei. To accomplish this, the plant cell uses two unique cytoskeletal structures: the preprophaseband (PPB) and the phragmoplast. The PPB is a transient ring of microtubules (MT) that forms at the start of prophase and disappears upon nuclear envelope breakdown. Later on in mitosis, the cell plate is constructed in the center of the cell by targeted vesicle delivery along the parallel microtubule arrays of the phragmoplast. The growing cell plate is guided by the phragmoplast from the centre of the cell, outwards to that part of the cortex previously occupied by the PPB. This guidance to the cortical division zone after PPB breakdown implies that the PPB marks the cell cortex to determine the position and orientation of the division plane. There is not much known about the molecular mechanisms that support this process. Recently, a set of genes was hypothesed to be involved in the determination of the division plane based on the subcellular localization of the proteins or inferred from mutant analysis. These genes are localized at the division plane and their mutants possess oblique cell walls and/or display a dwarfed phenotype. The molecular- biochemical function in division plane establishment for this set of genes has not been investigated, as well as their correlation to each other. This set of genes is listed here: ATN, POK1, POK2, AIR9, TON1a, TON1b, TON2 (FASS), TPLATE, KCA1, KCA2 A protein interaction network (interactome) can give us more insight in how the division plane is determined. Therefore we started from this small set of genes to isolate protein complexes using the tandem affinity purification (TAP) based technology. Once a division plane interactome is built, we can easily visualise all interactions, not only between the genes we used as bait but also interactions with potential new genes involved in the process of division plane determination. The most interesting genes from the interactome will be selected for further analysis. Their subcellular localization will be determined and further functional analysis based on mutant phenotypes will be done for the chosen genes.
Techniques and methods: Protein localization by Fluorescence Microscopy, Analysis of Arabidopsis Mutants, Protein interaction assays
16 Tracking the endocytic routes of brassinosteroid receptor complex in Arabidopsis
Department: Plant Systems Biology
Promoter: Dr. Jenny Russinova Address + Phone number Promoter: VIB2-Department of Plant Systems Biology Technologiepark 927 9052 Gent Contact: Jenny Russinova: [email protected] Tel: 09-33-139-31
Focus : PLB
Short description of the subject: Brassinosteroids (BRs) are polyhydroxylated steroids that are ubiquitous in vascular pants. In addition to their strong growth promoting effect, BRs also control important developmental processes, such as photomorphogenesis, germination, fertility, and stress resistance. Due to their essential regulatory role and widespread occurrence, BRs have been recognized as an independent family of plant hormones. By now the the pathways of BR synthesis are well known and also many components of the BR signaling pathway have been discovered. BRs are perceived at the cell surface by direct binding to the plasma membrane-localized BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor. Activation of BRI1 initiates a signaling cascade that leades to nuclear responses. Recent advances in animal cell signaling research suggest that signal-transducing molecules are preorganized and sequestered in distinct compartments within the cell. Thus, the subcellular localization and trafficking of the signaling complexes determine the specificity and the efficiency of signaling. Despite the vast progress in BR research, very little is known about the subcellular compartmentalization and trafficking of BR signaling complexes and their relevance for BRs physiological responses. By applying fluorescence imaging techniques in living plant cells it was shown that brassinosteroid receptor, BRI1 is actively internalized and recycled via endocytosis in Arabidopsis roots. The aim of the project is to investigate the effect of small molecules on subcellular localization and transport routes of a BR signaling complex. The localization of GFP tagged BR receptor, BRI1 will be examined in either Arabidopsis roots or cell cultures of Tobacco BY-2 cells using confocal fluorescence microscopy. Compounds that prevent BRI1 endocytosis will result in exclusive plasma membrane localization. Alternatively compounds that inhibit exocytosis will lead to intracellular accumulation of the receptor. Colocalization of GFP tagged BRI1 with specific organelle markers will be also analysed.
Techniques and methods Gateway cloning, transformation of Arabidopsis plants and BY-2 cell cultures, confocal fluorescence microscopy.:
17
Mt CLE peptide receptors in Medicago truncatula
Department: Plant Systems Biology
(Co) Promoter(s): Prof. Dr. S. Goormachtig; Prof. Dr. M. Holsters
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Zwijnaarde Prof. Dr. S. Goormachtig: 09/331.39.10; Prof. Dr. M. Holsters: 09/331.39.00
Focus : PLB, MIB
Short description of the subject: Legume nodules host rhizobia who fix atmospheric nitrogen for the plant. In Medicago truncatula , nodules develop from re-initiation of cortical cells and become indeterminate due to an apical meristem that continuously provides new cells to the nodule. Because of the high C-input, legume plants control the number of nodules via several processes among which autoregulation involving the SUNN gene, encoding a leucine rich repeat receptor like kinase (LRR-RLK) that belongs to the phylogenetic class XI. CLE peptides are a group of short distance signalling peptides to which CLV3 belongs. Recently we have shown that 2 CLE peptides ( Mt CLE12 and Mt CLE13) have a main role in nodule development and control of nodule number. Interestingly, the receptors of two A. thaliana CLE genes are, just like SUNN, class XI LRR-RLKs. Moreover, also in A. thaliana , evidence exists that three other members of this group might as well bind certain CLE peptides. Hence, it is tempting to hypothesize that also the two M. truncatula CLE genes, involved in nodulation, are perceived by this class of receptors. We have genetic evidence that SUNN is not the direct receptor but that it might be an interacting partner of the real receptor. Based on in silico analysis, we have identified a potential Mt CLE12 or Mt CLE13 receptor candidate. It belongs to the class XI LRR-RLKs and is specifically expressed during nodulation.
Aim: The aim of this study is to further unravel the role of this newly discovered LRR-RLK in nodulation. An extended functional analysis will be performed. This will include expression analysis as well as a study of the effect of RNAi on nodulation. Moreover, the interaction of this LRR-RLK with Mt CLE12, Mt CLE13 and SUNN will be analyzed.
Techniques and methods: Promoter and ORF cloning by gateway techniques, transgenic root generation via Agrobacterium rhizogenes transformation, RNAi analysis, Prom:GUS analysis, qRT- PCR, in situ hybridisation.
18
Nodule meristem development in Medicago truncatula
Department: Plant Systems Biology
(Co) Promoter(s): Prof. Dr. S. Goormachtig; Prof. Dr. M. Holsters
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Zwijnaarde Prof. Dr. S. Goormachtig: 09/331.39.10; Prof. Dr. M. Holsters: 09/331.39.00
Focus : PLB, MIB
Short description of the subject: Legumes engage into a symbiotic interaction with rhizobia, resulting in the development of new root organs, the nodules in which the bacteria fix nitrogen for the plant. In Medicago truncatula , nodules arise from re-initiation of cortical cell division and are of the indeterminate type because they carry an apical meristem that provides continuously new cells to the nodule. The development, organisation and maintenance of the nodule apical meristem (NAM) is still not well understood. This is in strong contrast to the elaborate knowledge that is available about the shoot (SAM) and root apical meristem (RAM). Both in the SAM and RAM, a group of stem cells, determined by respectively the organizing centre and quiescent centre, control the activity of the meristems. Many markers are available for different SAM and RAM cell types such as Wuschel, CLV3, WOX5 among many others. Although a nodule is a unique structure, it is clear from the literature that pre-existing plant developing programs, involved in shoot and root development, have been recruited for its development. This hypothesis is strongly supported by the pleiotropic phenotype of many nodulation mutants.
Aim: To manipulate nodule architecture for enhanced nitrogen fixation capacity, we want to get insight into the development, organisation and maintenance of the nodule meristem. Based on literature searches and in silico analyses, genes will be identified in M. truncatula that are specifically expressed in the NAM and are homologous to SAM and RAM markers. Those genes will be used in prom:GUS analysis and in situ hybridisations to get an insight into the development and organisation of the NAM. This study will be the basis to discover a set of NAM marker genes that will be functionally analysed in the future.
Techniques and methods: In silico gene discovery tools (Blast searches, protein domain identification, phylogenetic analysis), In silico expression analysis (Expression Atlas of M. truncatula ), Promoter and ORF cloning by gateway, transgenic root generation via Agrobacterium rhizogenes transformation, Prom:GUS analysis, qRT-PCR, in situ hybridisation.
19 Data mining and integration to uncover the molecular mechanisms underlying growth
Department: WE09
Promoter: Dirk Inzé Co-promoters : Yves Van de Peer, Stefanie De Bodt
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Gent. Tel. 093313692
Focus : BIS
Short description of the subject: Within the Department of Plant Systems Biology, we are studying plant growth in optimal and stress conditions in a systems biology approach. To unravel the molecular pathways governing increased organ size, microarray experiments that profile growth-related mutants (plants with larger leaves, faster growth etc.) are performed. Microarrays are a valuable source of large-scale and detailed information on gene expression. Through microarray experiments, mRNA levels in particular tissues, at particular time points and in a particular condition are measured. As such, we aim to identify the genes and pathways that are affected in the growth-related mutants. Finally, we want to pinpoint key regulators at the interface of different pathways controlling plant growth.
Aim: Using computational approaches, the generated microarray data as well as publicly available data will be mined to detect common as well as specific responses. Different group testing methods such as gene set enrichment and global approaches will be applied to growth-related transcript profiling data. These approaches are used to interpret the data in a sensitive way, benefiting from existing biological knowledge. Finally, Cytoscape will be used to comprehensively represent the large amounts of data.
Techniques and methods: R and BioConductor (microarray data processing), biostatistics, Cytoscape (network visualization and analysis), PERL programming.
20 SYSTEMS BIOLOGY OF DROUGHT TOLERANCE IN ARABIDOPSIS.
Department: WE09
Promoter: Dirk Inzé Co-Promoter: Aleksandra Skirycz
Address + Phone number (Co) Promoter(s): Plant Systems Biology, Technologiepark 927, 9052 Gent (Phone number co-Promoter: 09/3313955)
Focus : PLB
Short description of the subject: Drought stress causes large crop losses and limits the area where crops can be grown. With the rapidly growing world population sustainable and equitable global food, feed and bio- energy security relies on the development of high yielding crop plants that can resist adverse environmental conditions including drought. To this end, understanding the mechanisms underlying plant adaptation to stress is not only of primary scientific but also of socio-economical importance.
In order to cope with drought, mature plant organs such as leaves developed a number of adaptative responses such as stomatal closure and accumulation of compatible solutes. These are well characterised (Verslues et al., 2006) and have led to the identification of a number of “tolerance genes” that improve plant survival under extreme drought conditions (Yamaguchi-Shinozaki and Shinozaki, 2006). In recent years, it became clear that environmental stress also directly affects meristem activity, limiting growth and biomass production (Granier et al., 2000 ; West et al., 2004; Achard et al., 2006; Rymen et al.,2007; Mlynarova et al., 2007). These studies clearly show that growth reduction is an important adaptation response to stress allowing diversion of energy and assimilates to drive the various drought tolerance mechanisms.
One of the major objectives of our group is to understand the molecular networks underling the reduction of leaf growth upon drought. Recently we obtained detailed growth data for leaves subjected to moderate drought stress followed by expression profiling of leaves at three different developmental stage (proliferating, expanding, mature) in short and long-term drought experiments (Skirycz and Inzé, unpublished results). This work identified number of interesting processes and candidate genes that need to be further characterised e.g. role of ethylene and GAs signalling. Applicant will contribute to this work and be integrated in a young and dynamic team that is using the latest methods and technologies to study plant growth.
Aim: Functional characterization of genes and processes selected from expression profiling data to learn more about the molecular networks underling growth under drought stress.
Techniques and methods: plant growth in both in vitro cultures and soil, different drought stress assays, analysis of plant growth, DNA and RNA extraction, PCR, Q-RT-PCR, expression profiling, data analysis and mining.
21 Designing plant cell walls for a better conversion to biofuels
Department: WE09
Promoter(s): Prof. W. Boerjan; Dr. R.Vanholme
Department of Plant Systems Biology Technologiepark 927 9052 Gent 09 3313881; [email protected]
Focus : PLB
Short description of the subject:
Lignin is an aromatic polymer that is mainly deposited in secondary-thickened plant cell walls. It is one of the main factors that prevent the efficient conversion of lignocellulosic plant biomass to liquid bio-fuels such as bio-ethanol, because it covers the cellulose microfibrils that need to be enzymatically depolymerized into glucose prior to fermentation to bio-ethanol. We have already demonstrated that reducing the amount of lignin in the plant cell wall facilitates this depolymerisation process, but also the composition of lignin is of capital importance.
Aim: Using a systems-biology approach, we have identified a set of novel genes that are co- expressed with known lignin biosynthesis genes. These genes are therefore good candidates to play an as yet unknown role in the biosynthesis of lignin. The aim of the project is to analyze Arabidopsis mutants that are defective in these genes, by measuring their lignin content and composition, and by assaying the efficiency with which the biomass can be converted to bio-ethanol. Such genes can then be introduced in bio-energy crops such as poplar to reduce our dependence on fossil fuels.
Techniques and methods: - Growing transgenic Arabidopsis - Molecular analysis by PCR, segregation analysis - Determination of lignin amount and composition - Metabolite profiling - Saccharification assays
22 Role of multifunctional GSK-3 in plant cell division
Department: Plant systems Biology
(Co) Promoter(s): Dr. Jenny Russinova; Dr. Miroslava Zhiponova
Address + Phone number (Co) Promoter(s): VIB2-Department of Plant Systems Biology Technologiepark 927 9052 Gent Contact: Miroslava Zhiponova: [email protected] Tel: 09-33-139-07 Jenny Russinova: [email protected] Tel: 09-33-139-31
Focus : PLB
Short description of the subject: The growth and development of a multicellular organism require the coordinated action of each cell. Different internal and environmental stimuli are perceived by the cell through a net of signaling transduction pathways that result in adequate cell response. The glycogen synthase kinase-3 (GSK-3) was originally identified in mammals as a regulator of glycogen metabolism. Despite its name, GSK-3 is a multifunctional protein kinase that acts as a regulator of numerous signaling pathways including cell fate determination, microtubule function, cell-cycle regulation and apoptosis. Miss-regulation of GSK-3 is linked to several prevalent pathological conditions, such as diabetes and/or insulin resistance, and Alzheimer's disease. GSK-3 is evolutionary conserved among eukaryotes. Plants have a family of GSK-3 and these kinases were found to play role in: perception of brassinosteroid hormones that are signal messengers involved in essential growth and developmental processes; other growth and developmental processes; stress tolerance. A lot of unknown GSK-3 actions are still to be revealed in plants.
Aim: Based on experiments performed in our lab (treatment with chemical compound, transcriptional profiling) and the fact that in animals GSK-3 affects cell division, we want to explore whether some of the plant GSK-3 might be involved in the process of plant cell division. The formation of daughter cells is a main force in growth and the overall control of the cell division cycle is broadly similar between plants and other eukaryotic organisms. Key enzymes regulate the progress of cell division – cyclin-dependent kinases (CDKs) requiring for their function a regulatory protein known as cyclin. Our strategy involves: 1) defining the localization of the plant GSK-3 by using their own promoter regulating the expression of the GSK-3 protein fused to reporter genes; 2) approving putative interactions between GSK-3 and cell division regulators (cyclins, CDKs) based on a fluorescent approach.
Techniques and methods: Gene cloning (by Gateway system and VectorNTI software); Work with the model plant Arabidopsis (transformation, selective growth, chemical treatments); In vivo protein interaction in tobacco leaves (by Bimolecular Fluorescence Complementation), Confocal and light microscopy
23 Functional characterization of genes involved in lateral root initiation in maize and Arabidopsis thaliana
Department: WE09
Promoter: Tom Beeckman, Co-Promoter: Boris Parizot, guidance: Leen Jansen, [email protected] Address + Phone number (Co) Promoter(s): Department of Plant Systems Biology, Technologiepark 927, B-9052 Gent, 09/331 39 30, [email protected]
Focus : PLB
Short description of the subject: Plants depend on nutrients and water that are taken up from the soil by the roots for their growth and development. As plants are sessile organisms they adopted different strategies to explore the surrounding soil for nutrient rich regions and take them up efficiently. The formation of lateral branches significantly extends the contact with the soil and makes it possible to explore a larger area of the soil for nutrients. Several years of research already revealed some key signals involved in the formation of lateral roots. However, most research was done in the model organism Arabidopsis thaliana, and a lot has still to be elucidated. Understanding the mechanisms by which these lateral roots initiate would be of great interest, as plants with a more extensive and efficient root system might be more resistant to drought or arid soils. Therefore however the knowledge of Arabidopsis will have to be transferred to economically important crops. Using a technique that allows synchronous induction of lateral roots and combinerd transcriptome analysis allowed us to identify a set of genes that might play a role in the initiation of lateral roots in Arabidopsis and maize. Further characterization will however be necessary to confirm and elucidate their role in lateral root initiation. The genes will be overexpressed in Arabidopsis and resulting phenotypes will be analyzed by kinematic analysis of root growth, and different microscopic techniques will shed a light on their effect on cellular level. To follow the place and timing of lines expressing GUS or GFP under control of the different promoters will be developed and analyzed. Finally the performance of the transgenic plants will be analyzed by testing the water- and nitrogen use efficiency.
Aim: Aim of the project is to validate the role of candidate genes in lateral root initiation and to elucidate their mode of action.
Techniques and methods: Cloning with GATEWAY technology; quantitative PCR; imaging techniques as light microscopy, fluorescence microscopy and confocal microscopy; sectioning; GUS-staining; kinematic analysis of root growth; tests for water and nitrogen use efficiency; analysis of root architecture using specialized software as ImageJ and Winrhizo.
24 Unraveling the Function of Trehalose Biosynthesis Genes in Root Development and Stress Tolerance
Department: WE09
(Co) Promoter(s): Tom Beeckman, guidance: Lorena Lopez Galvis, [email protected] Address + Phone number (Co) Promoter(s): Department of Plant Systems Biology, Technologiepark 927, B-9052 Gent, 09/331 39 30, [email protected]
Focus : PLB
Short description of the subject: Trehalose is a disaccharide widely distributed in nature. It is known to play an important role in carbohydrate storage and stress protection. This sugar is common in bacteria, fungi and yeast, however in plants it seems to be restricted to resurrection plants which highly accumulate this sugar under desiccation conditions. The most distributed trehalose biosynthesis pathway consist in two enzymatic reactions, the first reaction involves trehalose phosphate synthases (TPS) which convert UDP-glucose and Glucose-6-phosphate to uridine diphosphate (UDP) and α,α–trehalose-6-phosphate (T-6-P), and in the second step the T-6- P is de-phosphorylated by trehalose phosphate phosphatases (TPP) to produce trehalose and inorganic phosphate. This pathway is well understood for other kingdoms, as trehalose is an important sugar for them, but in plants and specifically in Arabidopsis thaliana, wherein only trace amounts can be found, the research on this molecule was not that attractive until the complete Arabidopsis genome was released and surprisingly 21 genes homologous to already known yeast trehalose proteins were found. These genes have been classified in three families, Class I which consist of four genes (AtTPS1-AtTPS4) homologous to the yeast TPS ( ScTPS1 ), the Class II with eight genes (AtTPS5-AtTPS11) which are homologous to the yeast TPP ( ScTPS2 ) and the Class III involving 10 proteins (AtTPPA-AtTPPJ) which only conserved the phosphate boxes from ScTPS2 . The complex TPS/TPP needs to be studied in plants as it has a potential function in stress resistance leading to the possibility of using some of these genes to improve significant crops grown in restricted environments.
The project will be focused on the Class III proteins. We already know that these genes are active phosphatases able to complement the tps2 yeast mutant, but the main question remains why does Arabidopsis need 10 TPP genes if trehalose is hardly detectable in it? What is their function if they are not working in the synthesis of trehalose? Are they redundant proteins or is it possible that each one has a different role in particular cell types or organs of the plant? To answer this questions the work plan started by cloning each of the 10 promoters of the genes and fused them to reporter genes as GUS and GFP in order to determine where these genes are expressed and to check the redundancy or specificity of them. Interestingly some of these genes showed preference for some cell types, however detailed analysis in particular lines must be done by means of GUS staining, sectioning and microscopy analysis or using confocal microscopy looking at GFP signaling. The expression pattern will give us some insight in the possible role of these genes, and by using over expression and T-DNA insertion mutant lines, phenotypic characterization and specific assays will be set up to better understand their presence in plants.
Aim: The aim of this project is to unravel the function in root development and stress resistance of some TPP genes highly expressed in particular root cell types using reporter, knock-down and overexpression lines.
Techniques and methods: Genotypic and phenotypic characterization of knock-out and overexpression lines, kinematic growth analysis of roots, qPCR, microscopy, confocal microscopy, sectioning, GUS staining.
25 Characterization of camel antibodies produced in transgenic Arabidopsis seeds
Department: WE09
(Co) Promoter(s): Prof. A Depicker, Dr. S. De Buck, Dr. A. De Paepe
Address + Phone number (Co) Promoter(s): FSVM building, Department of Plant Biotechnology and Genetics, VIB Department Plant Systems Biology, Technologiepark 927, B-9052 Gent. Tel. 09.33.13.940. [email protected] , [email protected] , [email protected]
Focus : PLB, BSB
Short description of the subject:
Transgenic plants for the production of high-value recombinant proteins are a promising alternative to conventional recombinant protein production systems, such as bacteria, yeast, animal and insect cell cultures. In particular seed-based platforms are attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing. By using a seed-specific expression cassette based on the regulatory signals of seed storage proteins of Phaseolus, very high yields of recombinant proteins could be obtained in Arabidopsis seeds (1,2). Moreover, it could be shown that the produced recombinant proteins were functionally active. I n planta produced antibody variants had the same antigen-binding and in vitro neutralizing activities as the corresponding full-length antibodies produced in insect cells.
Aim: The aim of this project will be to screen a library of camelantibodies for the best binders against particular Arabidopsis proteins. Those binders will be fused to Fc and the bivalent antibodies will be produced in Arabidopsis seeds. Furthermore, the in seed produced camel antibodies will be purified and characterized for their functionality, correct processing and glycosylation.
Techniques and methods: RNA-, DNA- and Protein extraction, SDS-PAGE gelelectrophoresis, Western blot analysis, ELISA, protein purification, in vitro plant work
26 Evaluation of in planta produced camel antibodies as proteomic tools
Department: WE09
(Co) Promoter(s): Prof. A Depicker, Dr. S. De Buck, Dr. A. De Paepe
Address + Phone number (Co) Promoter(s): FSVM building, Department of Plant Biotechnology and Genetics, VIB Department Plant Systems Biology, Technologiepark 927, B-9052 Gent. Tel. 09.33.13.940. [email protected] , [email protected] , [email protected]
Focus : PLB, BSB
Short description of the subject:
Transgenic plants for the production of high-value recombinant proteins are a promising alternative to conventional recombinant protein production systems, such as bacteria, yeast, animal and insect cell cultures. In particular seed-based platforms are attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing. By using a seed-specific expression cassette based on the regulatory signals of seed storage proteins of Phaseolus, very high yields of recombinant proteins could be obtained in Arabidopsis seeds (1,2). Moreover, it could be shown that the produced recombinant proteins were functionally active. I n planta produced antibody variants had the same antigen-binding and in vitro neutralizing activities as the corresponding full-length antibodies produced in insect cells.
Aim: The aim of this project will be to evaluate in seed produced and purified camel antibodies as tools for the purification of particular target proteins. Furthermore, the in seed produced camel antibodies will be tested in Western analysis, immunoprecipitation, ELISA and immunolocalisation as functional tools in Arabidopsis proteome characterization.
Techniques and methods: RNA-, DNA- and Protein extraction, SDS-PAGE gelelectrophoresis, Western blot analysis, ELISA, protein purification, in vitro plant work
27 The molecular basis of plant yield
Department: Department Plant Systems Biology
(Co) Promoter(s): Dirk Inzé and Nathalie Gonzalez
Address + Phone number (Co) Promoter(s): VIB Department of Plant Systems Biology, Ghent University Technologiepark 927, 9052 Gent, BELGIUM +32 (0)9 331 39 56
Focus : PLB
Short description of the subject: The demand for more plant-derived products is increasing spectacularly to feed a rapidly growing world population, produce more plant-derived feed and supply our ever-growing energy needs. To cope with this demand while using less arable land, a profound increase in crop yield will have to be achieved. Whereas a considerable amount of physiological research has been done on yield performance of crops, little is known about the molecular networks determining growth rates. Many genes have been described in Arabidopsis that, when mutated or ectopically expressed, lead to faster growth, often due to the formation of larger structures. These "intrinsic yield genes" (IYGs) are involved in various processes whose interrelationships are mostly unknown. However, published experiments to measure the effects of IYGs on growth under optimal conditions were carried out under often very different growth conditions and with different ecotypes, making comparisons virtually impossible. To this end, we recently initiated a large-scale project (yield booster) to compare the effects of "yield genes" under standardized conditions in the same genetic background and to analyze the cellular and molecular bases underpinning the increased leaf growth under optimal conditions. The cellular basis of the enhanced growth is being studied by kinematic analysis and various 'omics' technologies are used to decipher the molecular networks orchestrating the observed growth effects. A literature search identified 39 IYGs lines producing enlarged leaves
Aim: Analysis (at a molecular and phenotypic level) of different Arabidopsis lines that show an increase in leaf size.
Techniques and methods:
Molecular techniques - RNA / DNA isolation from plant material - PCR (qRT-PCR, …)
Phonotypical analysis: - macroscopic level: leaf size, leaf number (use of ImageJ software) - microscopic level: cell size/number measurement (microscopy), ploidy analysis (flow cytometry)
In vitro culture of plants
28 Study of the introner-elements found in the Micromonas genomes
Department: Plant Systems Biology (Co) Promoter(s): Pierre Rouzé & Yves Van de Peer Address + Phone number (Co) Promoter(s): BioInformatics & Evolutionary Biology Unit VIB Department of Plant Systems Biology Ghent University, Technologie park 927, B-9052 GENT Tel: 0933 13694 / 0933 13807 E.mail: pirou,[email protected]
Focus : BIS
Short description of the subject: Genome projects always aim at a global, overall ‘as good as’ possible annotation. But frequently during that process, we encounter some peculiarities that deserve more attention. Unfortunately, often there is no time to have a better look at those uncommon features. Therefore, we propose a project that consists of a human curation of a continuous genomic stretch (a chromosome, a sequence scaffold) from a genome which has previously been annotated using standard automatic machine processes, i.e. finding where are the genes located, predict their exon-intron structures, and infer the function of the protein they encode as well as including evidence tags. This project would especially apply to genomes which are heterogeneous and where standard annotation is far from being optimal in some regions of the genome. More specifically, we sequenced recently 2 different microscopic green algae, from the Micromonas (Prasinophytes), and discovered that one of the 2 species had a high number of quite highly conserved repeats that have as unique feature to perfectly correspond to introns. It is even so that genes from one species might be intron-free while the same gene, in the other species, can be divided into many exons interspersed with those repeats we called introners. Up to now we identified 4 different of such repeats. A hypothesis might be that those sequences would be repeat elements, like transposable elements commonly found in plants, using the splicing machinery from the host to propagate all over the genome.
Aim: The aim of the project is to analyze genome wide this type of element with a highly increased, human-curated, added-value that can serve for future work on the biology of the new elements. This detailed analysis would allow us to better understand the fine structure of the genomes and shed a light on the putative role of these elements have on the evolution of the organism. Furthermore, highly curated genes and their features will be used to validate and improve the efficiency of automatic genome annotation.
Techniques and methods: As the student will have to deal with many genes (> few 100), scripting and setting up routines will be required, as well as working on a computer cluster (languages as PERL, usage of shell).
• Bioinformatics basic methods to search for similarity in databases (Blast) and to find conserved motifs and structures (HMM) • Multiple alignments of proteins (ClustalW, Muscle, mCoffee) • Usage of tools to assist the manual curation of gene features in genome sequences (e.g. generic: Artemis, in-house: Bogas) • Understand and apply gene prediction software (e.g. Eugene) • Prediction of proteins features such as 2D structures, trans-membrane domains and sub- cellular localization (e.g. TargetP, Psort, TMHMM, ..) • Create a repertoire of repeats and Transposable Elements
29 Looking deeper in genome annotation of the Chromosome-2 in three different Ostreococci (green alga) genomes
Department: Plant Systems Biology
(Co) Promoter(s): Pierre Rouzé & Yves Van de Peer
Address + Phone number (Co) Promoter(s): BioInformatics & Evolutionary Biology Unit VIB Department of Plant Systems Biology Ghent University, Technologie park 927, B-9052 GENT Tel: 0933 13694 / 0933 13807 E.mail: pirou,[email protected]
Focus : BIS
Short description of the subject: Genome projects always aim at a global, overall ‘as good as’ possible annotation. But frequently during that process, we encounter some peculiarities that deserve more attention. Unfortunately, often there is no time to have a better look at those uncommon features. Therefore, we propose a project that consists of a human curation of a continuous genomic stretch (a chromosome, a sequence scaffold) from a genome which has previously been annotated using standard automatic machine processes, i.e. finding where are the genes located, predict their exon-intron structures, and infer the function of the protein they encode as well as including evidence tags. This project would especially apply to genomes which are heterogeneous and where standard annotation is far from being optimal in some regions of the genome. More specifically, we found that in 3 different microscopic green algae, from the Ostreococci (Prasinophytes), the resp. chromosome-2, show dramatically reduced synteny across the different species in hand, while those chromosomes do contain essential genes. It is even so that next to a shuffling of genes, also the structures of the genes themselves are different compared to the genes on other chromosomes. Indeed, genes in those regions are more complex with more introns, while elsewhere in the genome genes tent to be limited to single exons. We therefore suspect this chromosome to be at least species specific or maybe even sex determinant.
Aim: The aim of the project is to create a continuous genomic region with a highly increased, human-curated, added-value that can serve as a “golden standard” for future work on the species itself or other closely related Algae. This detailed analysis would allow us to better understand the fine structure of the genomes, catalogue the functions encoded by the genes in those regions and shed a light on the putative role the region might be playing in the organism. Furthermore, highly curated genes and their features will be used to validate and improve the efficiency of automatic genome annotation.
Techniques and methods: As the student will have to deal with many genes (> few 100), scripting and setting up routines will be required, as well as working on a computer cluster (languages as PERL, usage of shell). • Bioinformatics basic methods to search for similarity in databases (Blast) and to find conserved motifs and structures (HMM) • Multiple alignments of proteins (ClustalW, Muscle, mCoffee) • Usage of tools to assist the manual curation of gene features in genome sequences (e.g. generic: Artemis, in-house: Bogas) • Understand and apply gene prediction software (e.g. Eugene) • Prediction of proteins features such as 2D structures, trans-membrane domains and sub- cellular localization (e.g. TargetP, Psort, TMHMM, ..) • Create a repertoire of repeats and Transposable Elements
30 Functional annotation of Fungal genomes
Department: Plant Systems Biology
(Co) Promoter(s): Stephane Rombauts & Yves Van de Peer
Address + Phone number (Co) Promoter(s):
BioInformatics & Evolutionary Biology Unit VIB Department of Plant Systems Biology Ghent University, Technologie park 927, B-9052 GENT Tel: 0933 13694 / 0933 13807 E.mail: pirou,[email protected]
Focus : Bioinformatics and Systems Biology (BIS)
Short description of the subject: Genome annotation is twofold, including structural prediction on the one hand and functional annotation on the other. Commonly, functional annotations rely on best-BLAST-hits, where the functional description is transferred from one gene to the other (homologous one). The drawback of this approach is that in cases of multiple domain proteins this can lead to problems. To overcome this problem we have developed a new approach relying on profiles of domain hits that are clustered into gene families. We then still can transfer the annotation of the closest homolog or give a more family level annotation. We already have built the models for plants and vertebrates, but we would like to add fungi to broaden the coverage of specific genes and include specificities for certain phyla. Furthermore, we would like to expand our tool to include more features, such as trans-membrane domains, localization information via signal-peptides etc.
Aim: The aim of the project is to generate, in an automatic way, reliable gene families and based on those result to build Hidden Markov Models (HMMs) from conserved domains of the proteins that will characterize the gene family. Now we run one tool to match HMM models over a whole proteome, but we would like to include more tools to increase the specificity even more.
Techniques and methods: As the student will have to deal with many genes (> few 100), scripting and setting up routines will be required, as well as working on a computer cluster (languages as PERL, usage of shell).
• Bioinformatics basic methods to search for similarity in databases (Blast) and to find conserved motifs and structures (HMM) • Multiple alignments of proteins (ClustalW, Muscle, mCoffee) • Usage of tools to assist the manual curation of gene features in genome sequences (e.g. generic: Artemis, in-house: Bogas) • Prediction of proteins features such as 2D structures, trans-membrane domains and sub-cellular localization (e.g. TargetP, Psort, TMHMM, ..)
31 Looking for the origin of chromosomes 18/19 of the green alga Ostreococcus
Department: Plant Systems Biology
(Co) Promoter(s): Pierre Rouzé & Yves Van de Peer
Address + Phone number (Co) Promoter(s): BioInformatics & Evolutionary Biology Unit VIB Department of Plant Systems Biology Ghent University, Technologie park 927, B-9052 GENT Tel: 0933 13694 / 0933 13807 E.mail: pirou,[email protected]
Focus : BIS
Short description of the subject: Genome projects always aim at a global, overall ‘as good as’ possible annotation. But frequently during that process, we encounter some peculiarities that deserve more attention. Unfortunately, often there is no time to have a better look at those uncommon features. Therefore, we propose a project that consists of a human curation of a continuous genomic stretch (a chromosome, a sequence scaffold) from a genome which has previously been annotated using standard automatic machine processes, i.e. finding where are the genes located, predict their exon-intron structures, and infer the function of the protein they encode as well as including evidence tags. This project would especially apply to genomes which are heterogeneous and where standard annotation is far from being optimal in some regions of the genome. More specifically, we sequenced recently 3 different microscopic green algae, from the Ostreococcus (Prasinophytes), and stumbled in each of the 3 species on a small chromosome that had barely anything in common. Furthermore we were unable to reliably predict any gene models. The difficulty here mainly comes from the scattered homology with know genes, making manual curation almost impossible in many cases. We hypothesize a bacterial origin for these chromosomes but we need to be able to confirm this. Also the Global Ocean Sampling (GOS) Expedition data will be used to find more cases or sequences with a better homology and will be used to build a phylogeny that will include hopefully known bacteria.
Aim: The aim of the project is to analyze the specific chromosomes, human-curate, and generate added-value that can serve for future work on the biology of the special chromosomes. This detailed analysis would allow us to better understand the fine structure of the genomes and shed a light on the putative role of these chromosomes in the organism. Furthermore, highly curated genes and their features will be used to validate and improve the efficiency of automatic genome annotation.
Techniques and methods: As the student will have to deal with many genes (> few 100), scripting and setting up routines will be required, as well as working on a computer cluster (languages as PERL, usage of shell).
• Bioinformatics basic methods to search for similarity in databases (Blast) and to find conserved motifs and structures (HMM) • Multiple alignments of proteins (ClustalW, Muscle, mCoffee) • Usage of tools to assist the manual curation of gene features in genome sequences (e.g. generic: Artemis, in-house: Bogas) • Understand and apply gene prediction software (e.g. Eugene) • Prediction of proteins features such as 2D structures, trans-membrane domains and sub- cellular localization (e.g. TargetP, Psort, TMHMM, ..) • Create a repertoire of repeats and Transposable Elements
32 Characterization of the GOLVEN peptide binding sites
Department: WE09
Promoter: Dr. Ir. Pierre Hilson
Address + Phone number (Co) Promoter(s): VIB Department of Plant Systems Biology Ghent University Technologiepark 927, 9052 Gent, B (0)9 331 38 30 [email protected]
Focus : PLB, BIS
Short description of the subject:
The GOLVEN peptides (GLVp) encoded in plant genomes control signaling between neighboring cells via the phytohormone auxin. The structure and mode of action of GLVp suggest that they are perceived by receptor kinases associated to the plasma membrane. This project aims at determining the characteristics of the GLVp binding sites in planta and to Arabidopsis cultured cells. The position of such sites will be analyzed at the tissular and cellular levels and the affinity between GLVp derivatives and membrane components will be determined biochemically.
Aim:
The student will participate to the following experiments • Test the bioactivity of GLVp derivatives in plant growth assays • Locate GLVp binding sites with fluorescently labelled GLVp • Determine affinity and specificity of GLVp binding to plant cell extracts
Techniques and methods:
• In vitro culture of Arabidopsis plantlets and cell suspensions • Quantitative root growth analysis • Fluorescence microscopy • Preparation of protein and membrane extracts and affinity measurements
33 Peptide signaling in plant roots
Department: WE09
Promoter: Dr. Ir. Pierre Hilson
Address + Phone number (Co) Promoter(s): VIB Department of Plant Systems Biology Ghent University Technologiepark 927, 9052 Gent, B (0)9 331 38 30 [email protected]
Focus : PLB, BIS
Short description of the subject:
Recent studies showed that secreted peptides play an important role in the communication between neighboring plant cells. However many signaling peptides and their mode of action remain to be discovered. The project combines complementary tools to investigate the function of secreted signaling peptides in the model plant Arabidopsis thaliana , including bioactive synthetic peptides used in bioassays, Arabidopsis mutants and transcriptional reporter lines. The work will be performed in close collaboration with other lab members and is at the core of our research program.
Aim:
The student will participate to the following experiments • Study modulation of peptide gene expression in response to environmental stimuli and in the course of plant development • Study the bioactivity of the signaling peptides • Identify and characterize Arabidopsis plant mutated in genes coding for signaling peptides • Investigate regulatory networks controlling their expression on the basis of transcriptome data
Techniques and methods:
• Recombinational cloning and plant transformation • In vitro culture of Arabidopsis plantlets; root, shoot and leaf growth measurement • Characterization of transcription profiles via reporter activity in plant tissues, based on β- glucuronidase (GUS) assay and on in vivo localization of the green fluorescent protein (GFP) • Mining of transcriptome profile datasets; gene expression correlation studies
34 Hormonal interactions shaping root system architecture
Department: WE09 (Hormonal cross-talk group)
(Co) Promoter(s): Eva Benkova
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Gent Tel 09 331 38 71
Focus : PLB
Short description of the subject:
The growth and development of plants is governed by signaling substances such as hormones. In plants, to much larger extent than in other organisms, interactions between hormonal pathways represent a crucial factor that governs their action, especially in regulation of plant organ formation. However, molecular basis for hormonal cross-talk remains largely unknown. In our studies, we are interested in the molecular mechanism(s) underlying cross-talk between hormonal signaling pathways with a special Focus on regulation of postembryonic organogenesis. The physiologically well characterized interaction is between two groups of phytohormones - auxins and cytokinins. We study auxin-cytokinin interaction in lateral root primordia (LRP) development in Arabidopsis . Physiological and genetic data indicate that both auxin and cytokinins are involved in this process and their effects are antagonistic. Roles of both hormones in lateral root development are being examined in detail and molecular components of their and interaction are identified by a novel genetic screen and microarray approach.
Aim:
Techniques and methods: Functional characterization of genes involved auxin- cytokinin interaction, expression analysis using reportes constructs, immunolocalisation, quantitative RT-PCR, confocal microscopy,analysis of roots system development in Arabidopsis mutants usin marker genes
35 The involvement of persistence in multidrug tolerance and in biofilm formation of Shewanella oneidensis
Department: WE10: Biochemistry and Microbiology
(Co) Promoter(s): Prof. B. Devreese; Dr. L. De Smet
Address + Phone number (Co) Promoter(s): Ledeganckstraat 35 Gent B. Devreese: 092645473 L. De Smet: 092648731
Focus : BSB, MIB
Short description of the subject: Bacterial multidrug tolerance (MDT) is largely responsible for the inability of antibiotics to eradicate infections. More of half of the infections in the developed world are caused by biofilms which exhibit multidrug tolerance. At least part of his phenomenon is caused by a small population of dormant bacteria called persister cells. Persisters are phenotypic variants of wild-type cells and are not sensitive for bactericidal antibiotics. Recently, the protein HipA is identified as a critical factor regulating persistence . Overproduction of HipA leads to MDT in E. coli . During normal growth, Hip A is neutralized via complex formation with HipB. Dissociation of HipB triggers transition to a persistence state via a hitherto unknown mechanism. Crystalographic studies revealed that HipA adopts a protein kinase structure. The elongator factor EF-TU has been demonstrated to be a HipA substrate, indicating a role in translational control. HipA is highly conserved among Gram-negative bacteria which indicates its central role in the development of persistence.
Aim: Upon screening a transposon library of the non-pathogenic bacterium Shewanella oneidensis we discovered that inactivation of the hipAB locus altered biofilm forming behavior. In frame deletions of both genes and of the complete operon are constructed. Further physiological and proteomic characterization will unravel the mechanism of persistence, the involvement of this operon in MDT and in biofilm formation. Both HipA and HipB are currently being expressed which makes it possible to characterize both proteins in detail. Mass spectrometry will be used to investigate the structure of the complex. A Tandem affinity approach will be used to screen for HipA substrates.
Techniques and methods: Molecular Biology : PCR, cloning, site directed mutagenesis, RT-PCR Biochemistry : protein purification, mass spectrometry, activity measurements Molecular Microbiology : growth analysis, physiological characterization of the different mutant strains
References: Schumacher et al . Molecular mechanisms of HipA-Mediated Multidrug Tolerance and its neutralization by HipB. Science (2009) 323 , 397 Jayaraman et al. Bacterial persistence : some new insights into an old phenomenon. J. Biosci. (2008) 33 , 795 Smith-Romesberg. Combating bacteria and drug resitance by inhibiting mechanisms of persistence and adaptation. Nat. Chem. Biol. (2007) 3, 549-556
36 A novel mass spectrometric tool for determining protein:protein and protein:ligand interactions
Department: WE10: Biochemistry and Microbiology
(Co) Promoter(s): Prof. B. Devreese
Address + Phone number (Co) Promoter(s): Ledeganckstraat 35 Gent B. Devreese: 092645473
Focus : BSB
Short description of the subject: Ion mobility mass spectrometry emerged recently as a novel tool for studying biological interactions (Pringle et al. 2007). The methods separates molecular ions based on their shape after transition from the liquid phase to gas phase upon ionization, preferably electrospray ionization. This allows to determine changes in gas phase mobility due to structural conformation changes after ligand binding to a particular protein (Ruotolo et al. 2007). It is widely accepted that the structural properties in the gas phase largely reflect the solution state of the protein.Such a mass spectrometer will become available in the laboratory of Prof. Devreese in spring 2009. The technique is really novel, and within this project, a lot of room is open for developing strategies for mass spectral determination of protein:protein and protein:ligands interactions.
Aim: It is our aim to explore the capabilities of ion mobility mass spectrometry in the study of intact protein complexes and protein:ligand interactions. A large set of model proteins are available in the L-Probe lab that can be used to evaluate the capacities of the techniques to determine the stoechiometry and topology of protein complexes and quantitative analysis of ligand binding by competitive assays and titration experiments.
Techniques and methods: Protein purification, mass spectrometry, determination of protein:ligand interactions
References: Pringle, S.D., Giles, K., Wildgoose, J.L., Williams, J.P., Slade, S.E., Thalassinos, K., Bateman, R.H., Bowers, M.T. and Scrivens, J.H. Int. J. Mass Spectrom. 261, 1-12. (2007) Ruotolo, B.T., Hyung, S.-J., Robinson, P.M., Giles, K., Bateman, R.H. and Robinson, C.V. Angewandte Chemie 46,8001-8004. (2007) .
37 Phosphoproteomic analysis of bacterial resistance to antibiotics and biofilm formation.
Department: Biochemistry and Microbiology L-ProBE
(Co) Promoter(s): Prof. Dr. Bart Devreese
Address + Phone number (Co) Promoter(s): K.L. Ledeganckstraat 35, 9000 Gent, 09/264 52 34
Focus : BSB, MIB
Short description of the subject:
The purpose of this research topic is to obtain more details on the mechanisms of bacterial resistance to antibiotics. Recent proteomic research revealed differential expression of protein kinases upon exposure of gram negative bacteria to antibiotics. Identification of phosphorylated proteins could lead to possible mechanisms or signal transduction pathways which lead to resistance, e.g. the production of antibiotic degrading enzymes like beta- lactamases or the formation of biofilm which impairs the access of antibiotics to the bacteria. Our aim is to determine the phosphoproteome of nosocomial bacteria like Stenotrophomonas maltophilia, an opportunistic pathogen colonizing patients in intensive care settings, especially those with underlying debilitating conditions such as immunosuppression, malignancies, and implantation of foreign devices (catheters, respiratory therapy equipment, etc.). Treatment of S. maltophilia infections is compromised by the intrinsic resistance of this organism to many broad-spectrum antibiotics, including the carbapenems. The bacterium can form biofilm and induces two different carbapenem hydrolyzing β-lactamases (L1 and L2) upon antibiotic stress although the mechanism by which this induction is triggered is largely unknown.
Aim: We will map the Stenotrophomonas maltophilia phosphoproteome using nanoLC and mass spectrometric approaches to specifically screen for phosphorylated peptides. Effect of antibiotics on the phosphoproteome will be assessed by differential labeling strategies and label free proteomic strategies using state-of-the-art mass spectrometry. Boinformatic tools will be used to map signal transduction pathways in this bacterium.
Techniques and methods: Cultivation of bacteria Phosphoproteomics: 2D-gel electrophoresis / 2D-LC / protein digestion Mass spectrometry
38 Design of genetically encoded glutathione disulfide biosensors
Department: Biochemistry and Microbiology (WE10)
Promotor: Bjorn Vergauwen ([email protected]) Promoter: Bart Devreese ([email protected]) Supervisor: Bjorn Vergauwen ([email protected])
Address + Phone number (Co) Promoter(s): K.L. Ledeganckstraat 35, 9000 Gent +32 (0)9 264 51 27
Focus : BSB
Short description of the subject:
Basically, genetically encoded biosensors are molecular machines linking the highly evolved ligand-binding properties of a sensing detector protein to changes in fluorescence of specific fluorescent proteins. Then, by using fluorescence microscopy, these molecular tools provide the possibility to study a specific cellular signal within the complex environment of the living cell. The cellular signal of our interest relates to redox control, and more in particular, to the causal role glutathione plays in determining the spatiotemporal (considering cell compartmentalization) redox potentials, and exploiting these for signaling.
Aim:
The present projects’ aim is to develop highly specific genetically encoded sensors for the oxidized form of the tripeptide antioxidant glutathione, referred to as glutathione disulfide (GSSG). FRET-based as well as circularly permutated Green Fluorescent Protein (cpGFP)-based approaches will be explored to generate protein chimera’s that change fluorescence upon GSSG binding.
Techniques and methods:
• Standard bio-informatics techniques (sequence alignments, structural modeling, ...) • Standard molecular biology techniques (cloning, in vitro and in vivo mutagenesis, …) • Standard protein chemistry techniques (protein expression and purification, …) • Standard biophysical techniques (UV/VIS and fluorescence spectroscopy, X-ray crystallography,...)
39 Development and automation of a new approach for selective enrichment of N- terminal peptides from complex proteomic samples.
Department: Department of Biochemistry and Microbiology (WE-10) Promoter: Dr. B. Samyn Co-promotor: Prof. B. Devreese
Contact : L-ProBE KL. Ledeganckstraat 35 09/2645125 - 09/2645273 [email protected] - [email protected]
Focus : BSB
Short description of the subject: Recent studies have indicated that there are a large number of alternatively terminated protein isoforms, those are proteins that are coded by the same gene and have identical sequences but shortened N-or C-termini (Gupta et al, 2007). These are created by alternative initiation of transcription within genes or by transcription independent of annotated gene boundaries. Cell specific mRNA splicing and enzymatic proteolytic processing can further alter the termini, and therefore the biological function, of proteins. E.g. recent analysis of proteome data against the human genome indicated the presence of 87 unpredicted N-termini and 193 unpredicted C-termini in human embryonic carcinoma cells (Dormeyer et al, 2007).
Aim: As a complement to our novel approach to study C-termini (Samyn et al, 2005; 2006) we want to develop and automate a targeted approach for selective enrichment and identification of protein N-termini. Recently, a number of approaches have been developed that allow the specific isolation of N-terminal peptides (Gevaert et al, 2003; McDonald et al, 2005). In this project we want to use an improved chemical derivatization strategy for the specific isolation of N-terminal peptides (Samyn et al, 2004; 2007). After guanidination of the -amino side chain of the internal lysine residues, the amino terminus can be specifically tagged with a negatively charged sulfonation reagent (SPITC) which will allow a specific selection of the N-terminal peptide after tryptic digestion of the modified proteins. Therefore, we will apply alternative enrichment procedures, such as SCX fractionation (Aivaliotis et al, 2007; Dormeyer et al, 2007). After optimization, and by automating the derivatization and enrichment procedures (Tecan robot), this will allow a high-throughput study of N- terminal proteolytic processing. As proof of principle, we will perform a proteomic analysis of S. oneidensis as a prokaryotic model organism.
Techniques and methods: - TECAN Freedom EVO robotic sample preparation platform - Chemical derivatization techniques - MALDI-TOF/TOF MS + database analysis - Peptide separation techniques - 2D-PAGE
References : Aivaliotis M. Et al. J. Prot. Research 6, 2195-2204 ( 2007 ). Dormeyer W. et al. J. Prot. Research 6, 4634-4645 ( 2007 ). Gupta N. et al. Genome Res . 17 , 1362-1377 ( 2007 ). Gevaert et al. Nat. Biotechnol . 21 , 566-569 ( 2003 ). McDonald L. et al. Nat. Methods 2, 955-957 ( 2005) . Samyn B. et al. J. Am. Soc. Mass Spectrom . 15 , 1838-1852 ( 2004) . Samyn B. et al. Nat. Methods 2, 193-200 ( 2005 ). Samyn B. et al. Nat. Protocols 1, 317-321 ( 2006 ). Samyn B. et al J. Prot. Research 6, 70-80 ( 2007 ).
40 A Mycobacterium bovis BCG transposon insertion library as a resource for the study of the role of mycobacterial glycolipids in pathogenesis.
Department: Department of Biochemistry and Microbiology
(Co) Promoter(s): Prof. Dr. Nico Callewaert; Copromotor: Dr. Nele Festjens
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Zwijnaarde Prof. Dr. Nico Callewaert tel +32 9 331 36 30 Dr. Nele Festjens tel +32 9 331 36 35
Focus: BIB, BSB, MIB
Short description of the subject: Pathogenic mycobacteria are highly adapted intracellular pathogens surviving for long periods of time within their hosts. Mannosylated lipoarabinomannan (ManLAM), a constituent of the mycobacterial cell wall, has been described as a major virulence determinant. The mycobacterial phosphatase SapM is also described as an inhibitor of phagosome-lysosome fusion and thus also supports survival of mycobacterium in the phagocyte. However, studies with mutants of mycobacteria deficient in these molecules are still largely lacking, and thus, the real contribution of these molecules to mycobacterial pathogenesis remains unclear. We therefore have generated a large M. bovis BCG transposon insertion mutant library and screened for mutants in genes which are known to be involved in the biosynthesis of Man(L)AM and for the SapM mutant. We have analyzed the importance of SapM and ManLAM in uptake and persistence of mycobacteria following uptake by studying mycobacterium-host interactions of transposon insertion mutants of Mb1661c, Mb2203 (both involved in ManLAM biosynthesis) and SapM versus M. bovis BCG wild type. We demonstrate, both in in vitro phagocytosis assays and in in vivo infection models that neither the mycobacterial phosphatase SapM nor mannose-capped LAM are of critical importance for uptake or mycobacterial persistence in the phagosome. We also did not find any major difference on cytokine production or liver and lung granuloma formation upon intravenous or intratracheal infection of mice, omitting a major role of SapM or ManLAM in host immune responses following infection.
Aim: We thus aim at further exploring major virulence factors of mycobacterium.
Techniques and methods: • Microbiology (growth of M. bovis BCG cultures, CFU determination) • PCR screening • Tissue culture • Phagocytosis assays (FACS analysis) • Cytokine profiling
41 Dissection of the interaction between cytokines and the extracellular domains of their cognate receptors
Lab; Department: L-ProBE ; Biochemistry and Microbiology (WE10) Promotor: Savvas Savvides ( [email protected] ) Supervisors: Jonathan Elegheert ([email protected]), Bert Remmerie ([email protected]), Kenneth Verstraete ([email protected]) Address + Phone number: K.L. Ledeganckstraat 35, 9000 Gent +32 (0)9 264 51 24 ; 0472 928 519
Focus : BSB, BIB
Number of students : 2-3
Cytokines mediate intracellular signaling by oligomerizing their respective receptors at the cell surface to trigger signaling cascades which contain the information necessary for correct and controlled cellular proliferation and differentiation. To facilitate such a process most cytokine receptors expose Ig-like extracellular domains, followed by single transmembrane domains, ending with cytosolic kinase domains. In this project we propose an interdisciplinary research program that combines structural biology , binding studies , and molecular biology to elucidate the molecular architecture and binding epitope underlying the interaction of cytokines, such as Flt3 ligand, Colony Stimulating factor-1, IL-34, Leptin and their cognate receptors. This is a very opportune time to develop such projects. A search in PubMed returns tens of thousands of citations in the last 10 years reporting on these cytokines and their receptors. Yet only a handful of these publications attempt to shed light onto the interactions between these molecules indicating that there is clearly a lot to be done to provide a balanced view of these molecules. Given the enormous biomedical relevance of the target cytokine-receptors complexes our ultimate goal will be to design molecules with antagonistic/agonistic activity to facilitate classical and novel therapeutic avenues against numerous hematopoietic disorders, inflammatory disorders, and cancer. We wish to recruit 2-3 motivated MA-thesis students to work on this multi-facetted research project for which the following methods are employed:
Methods and Techniques • Molecular biology techniques • Expression of proteins in eukaryotic cells (e.g. HEK293, pichia pastoris etc) • Expression of human proteins in bacteria and refolding from inclusion bodies. • Protein purification methods (Affinity chromatography, Gel filtration etc.) • SDS-PAGE, Western blot, Native PAGE • Binding studies (Isothermal Titration Calorimetry and surface plasmon resonance) technology) • Protein Crystallization • X-ray crystallography • Small-angle X-ray Scattering
42 Structural biology of the malaria parasite Plasmodium falciparum : The intriguing interaction of Adenylate Kinase with N-Myristoyl Transferase
Lab; Department: L-ProBE ; Biochemistry and Microbiology (WE10) Promotor: Savvas Savvides ( [email protected] ) Supervisors: Kedar Moharana ( [email protected] ) Address + Phone number: K.L. Ledeganckstraat 35, 9000 Gent,+32 (0)9 264 51 24 ; 0472 928 519
Focus : BSB, BIB
Number of students : 1
Tropical malaria yearly infects 300-500 million people world-wide with very high mortality rates especially among children under the age of five. It is therefore not surprising that with such staggering numbers malaria easily ranks among the top three deadliest infectious diseases. Malaria is caused by Plasmodium falciparum , a parasite that infects red blood cells and that is transmitted by the female Anopheles -mosquito. Here, we propose an interdisciplinary research program combining structural biology, binding studies, and molecular biology to study the interaction of two key proteins from Plasmodium falciparum , the adenylate kinase (AK) and the N-myristoyl transferase (PfNMT). Adenylate kinases play a central role in the adenine nucleotide homeostasis and the energy metabolism of P. falciparum . On the other hand PfNMT is a cytosolic enzyme that adds a myristate group to the N-terminus of diverse proteins in P. falciparum to facilitate their interactions with other proteins and their transport to the cell membrane. Recently, we demonstrated that AK can be myristoylated by PfNMT. Remarkably the two proteins can be trapped in a stable complex which now opens a unique opportunity to study an otherwise very transient macromolecular interaction. We wish to dissect in detail the interaction of the two proteins via an interdisciplinary approach combining binding and structural studies. Binding studies using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR, BiaCORE) will be carried out to determine thermodynamic parameters and affinity/inhibition kinetic constants. In parallel we will attempt to crystallize and determine the structure of the complex to characterize in the landscape of interactions. Structural studies in solution will be carried out by small-angle x-ray scattering. We wish to recruit a motivated thesis student to contribute to the project based on the following methods and techniques:
Methods and Techniques • Molecular biology techniques • Protein purification methods (Affinity chromatography, Gel filtration etc) • SDS-PAGE, Western blot • Analytical Gel Filtration • Binding studies ( isothermal titration calorimetry and Surface Plasmon Resonance) • Protein Crystallization • X-ray crystallography and small-angle x-ray scattering
43 Investigating the role of atypical glutaredoxins in S-(de)glutathionylation in cellular oxidative stress
Department: Biochemistry and Microbiology (WE10)
Promotor: Savvas Savvides ( [email protected] ) Mede-Promotor: Bjorn Vergauwen ( [email protected] ) Supervisor: Geraldine Buysschaert ( [email protected] ) Address + Phone number: K.L. Ledeganckstraat 35, 9000 Gent, +32 (0)9 264 51 24 ; 0472 928 519
Focus : BSB, BIB, MIB
Number of students : 1
Sulfhydryl groups (-SH) play a vital role in cell homeostasis and in defense against reactive oxygen species, oxidants, free radicals and electrophiles. Modulation of the thiol- disulfide status (low oxidative stress) is recognized as a way of redox signaling. An ever growing body of evidence has linked the irreversible oxidation of –SH groups to sulfinic (-SOOH) and sulfonic (-SO 2OH) acids under conditions of oxidative stress, to aging, cardiovascular and neurodegenerative diseases (Alzheimer’s and Parkinson’s), diabetes and cancer.
Protein glutathionylation , or mixed disulfide formation between a protein moiety and the cysteine moiety of glutathione (GSH), is a typical case of sylfhydryl chemistry that plays an important role in cellular signal transduction under normal cellular conditions. Furthermore, it is thought to protect critical sulfhydryl groups during overt oxidative stress. Since protein (de)glutathionylation is not fully understood, this research project Focus es on unraveling the mechanism of S-glutathionylation by studying the role of atypical glutaredoxins, a ubiquitous family of redox-active proteins, in this process. We wish to recruit a motivated thesis student to contribute in this research project for which the following techniques and methods are employed: • Molecular biology techniques • Protein expression and purification (affinity, ion-exchange, size-exclusion chromatography) • Protein analysis (SDS-PAGE, Western blotting, …) • Enzymology (Michaelis-Menten kinetics, mechanistic studies, …) • Biophysical techniques (Surface Plasmon Resonance & Isothermal Titration Calorimetry) • Bacterial Physiology (Growth assays, disc diffusion assays, etc)
44 Dissection of the molecular basis of bacterial type II secretion in pathogenic bacteria
Lab; Department: L-ProBE ; Biochemistry and Microbiology (WE10) Promotor: Savvas Savvides ( [email protected] ) Supervisors : Ruben Van der Meeren ( [email protected] ) Address + Phone number: K.L. Ledeganckstraat 35, 9000 Gent, +32 (0)9 264 51 24 ; 0472 928 519 Focus : BSB, BIB, MIB Number of students : 1
Many Gram-negative bacteria are human pathogens posing an ever-growing health threat worldwide. Essential to the infection process are a diversity of exquisitely regulated and interdependent strategies that often employ large macromolecular assemblies. One such process, bacterial secretion , utilizes specialized macromolecular machineries spanning the bacterial double membrane system to facilitate the presentation of the bacterial adhesion apparatus to the cell surface, and to transport proteins, nucleic acids, and protein/DNA complexes to the target cells and the extracellular milieu. We are studying the type II secretion system ( T2SS ) of P.aeruginosa and B. cenocepacia, which are two of the most opportunistic pathogens in humans. The T2SS in P. aeruginosa and B. cenocepacia are referred to as the Xcp and Gsp machineries, respectively , and consist of at least 10-12 proteins that form an intriguing macromolecular assembly that spans the entire bacterial cell envelope. We are working towards a dissection of the structural, kinetic and thermodynamic determinants governing T2SS protein-protein complexes using structural studies (x-ray crystallography, electron-microscopy, and small-angle x-ray scattering), mass-spectrometry, binding studies (surface plasmon resonance measurements, microcalorimetry), and site-directed mutagenesis. Molecular biology and protein chemistry will be the source of recombinant proteins throughout the project. Furthermore, methods such as analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering will be used to assess oligomeric assemblies. Such a project initiative is very timely given the emphasis at the European level and world-wide on the characterization of protein-protein complexes and membrane proteins. We wish to recruit a MA-thesis student to work on this multi-facetted research project for which the following methods are employed: • Molecular biology techniques • Expression and isolation of membrane proteins and protein-complexes • Protein purification methods (Affinity chromatography, Gel filtration etc.) • SDS-PAGE, Western blot, Native PAGE • Binding studies (Isothermal Titration Calorimetry and surface plasmon resonance) technology) • Protein Crystallization • X-ray crystallography • Small-angle X-ray Scattering
45 Microbiological quality of fish and fishery products
Department: WE10 + ILVO (Institute for Agricultural and Fisheries Research / Instituut voor Landbouw- en Visserijonderzoek) - Unit Technology and Food (the dissertation will be performed at ILVO in Melle)
(Co) Promoter(s): Prof. Dr. Anne Willems, Dr. Marc Heyndrickx, Dr. ir. Geertrui Vlaemynck
Address + Phone number (Co) Promoter(s): [email protected] Lab. Microbiologie, UGent, Ledeganckstraat 35, 9000 Gent 09/2645103 [email protected] , ILVO, Brusselsesteenweg 370, 9090 Melle 09/272.30.17
Focus : MIB & BIB
Short description of the subject: Fish is a healthy food product which is necessary in human diet for the uptake of omega-3-fatty acids, several vitamins and trace elements. The public knowledge about this health advantage involves an increase in the demand for especially fresh and light preserved fish. However, the very rapid spoilage of fish, which is mainly due to bacterial activities, cannot easily be placed in our busy daily life. Therefore it is important to identify the specific spoilage organisms in order to ultimately advise on measures for improving the quality and shelf life (=storage potential). At ILVO - Unit Technology and Food, a project is running in which these specific spoilage organisms (SSOs) of some commercial fish and fishery products are being identified and characterized for the fresh product as well as within the production chain.
Aim: The specific spoilage organisms of some commercial fish and fishery – products from catch until consumer’s purchase will be studied by molecular and/or (bio-)chemical analyses.
Techniques and methods: During handling/production and storage, the composition of the microbiota can change quite dramatically depending on the initial microbiota, the storage conditions (e.g. aerobic iced storage, MAP (modified atmosphere)) etc. The microbiota of fish will be studied with traditional culture dependent microbiological methods as well as with culture independent techniques such as Denaturing Gradient Gel Electrophoresis (DGGE) and molecular techniques such as rep-PCR typing techniques, sequencing based on the 16S rDNA gene. Since it is known that a large part of the bacteria present on the spoiled fish play no role whatever in spoilage, the spoilage potential of the strains must be studied. In order to search the specific spoilage organisms some (bio)-chemical parameters such as trimethylamine (TMA) and total volatile nitrogen bases (TVBN) and the characterization of the detected micro-organisms (possibilities to produce specific metabolites), will be applied. Each fish /fish product will have its own specific spoilage bacteria and the number of these will, as opposed to the total number, be related to the shelf life. The work will be a collaboration between the Laboratory of Microbiology (WE10, UGent) and ILVO in Melle, which is easy accessible by train, bus or bike coming from Gent.
46 Molecular identification of spoilage microflora on common shrimp ( Crangon crangon )
Department: WE10 + ILVO (Institute for Agricultural and Fisheries Research / Instituut voor Landbouw- en Visserijonderzoek) - Unit Technology and Food
(Co) Promoter(s): Prof. Dr. Anne Willems, Dr. Marc Heyndrickx, Dr. ir. Geertrui Vlaemynck
Address + Phone number (Co) Promoter(s): [email protected] Lab. Microbiologie, UGent, Ledeganckstraat 35, 9000 Gent 09/2645103 [email protected] , ILVO, Brusselsesteenweg 370 , 9090 Melle 09/272.30.44
Focus : MIB
Short description of the subject: The common shrimp ( Crangon crangon ) is a typical product of the Belgian fishery that has recently received a local product label, “Purus”. It is exclusively caught in the North Sea and prepared by Flemish fishermen predominantly in a traditional way. Like most typical fishery products it is particularly sensitive to spoilage, for example through the conversion of free amino acids in spoilage metabolites by microbial activities. At the Institute for Agricultural and Fisheries Research (ILVO) – Unit Technology and Food, an ongoing project explores the specific spoilage micro-organisms (SSOs) of cooked and unpeeled shrimps (identification and characterization) to ultimately advise on measures for improving quality and shelf life (=storage potential). Preliminary identifications of the microbiota on cooked and unpeeled shrimps using molecular techniques have been performed and through 16S rRNA gene sequencing a number of genera were recovered, including Pseudoalteromonas and Psychrobacter , that have an enormous impact on total bacterial count of shrimps without preservatives (i.e. Purus shrimp) stored aerobically on ice.
Aim: Pseudoalteromonas and Psychrobacter are marine genera that are often isolated from seawater and sediment samples. Less is known about their association with (spoilage of) fish and fishery products and an extensive literature search will be required. Strains need to be identified to species level because the spoilage microflora of shrimp is poorly characterized thus far. Precise species identification is required to be able to link particular species to increased spoilage risk.
Techniques and methods: The study will include identifying strains recovered from the processing chain of shrimp, using molecular techniques. Because 16S rDNA analysis is too limited in its taxonomic resolution, alternative techniques will be needed, such as sequence analysis of housekeeping genes. The identified strains will be characterized and some (bio-)chemical parameters such as production of trimethylamine (TMA) and total volatile nitrogen bases (TVBN) will be applied in order to determine the spoilage potential of these micro-organisms. The work will be a collaboration between the Laboratory of Microbiology (WE10, UGent) and ILVO in Melle, which is easy accessible by train, bus or bike coming from Gent..
47 Community AFLP as a new tool for the characterization of human intestinal tract microbiota in health and disease
Department: Laboratory of Microbiology
(Co) Promoter(s): Promotor: Dr. Geert HUYS Co-promotor: Prof. Dr. Peter VANDAMME
Address + Phone number (Co) Promoter(s): K.L. Ledeganckstraat 35 9000 Gent Phone: 09 2645113 (P. Vandamme); 09 2645131 (G. Huys)
Focus : MIB, BIB
Short description of the subject: Through a sophisticated network of mutualistic interactions, the human intestinal tract (HIT) microbiota has a profound impact on human health. Although crucial to understand their etiology and thus to predict the outcome of clinical interventions, the identification of bacterial groups responsible for local or partial dysbiosis in many HIT disorders is still in its infancy. Denaturing Gradient Gel Electrophoresis (DGGE) is a metagenome-based fingerprinting technique that is commonly used to assess the relative diversity and population dynamics of the predominant HIT microbiota. Still, the taxonomic resolution and detection level of DGGE and related techniques are fairly limited and require further technical improvement. In this context, the implementation of the Amplified Fragment Length Polymorphism (AFLP) concept in a new fingerprinting strategy for HIT communities may prove a way forward. Whereas AFLP is most established as a high-resolution whole-genome typing tool of single organisms, its use to analyze polymicrobial systems (i.e. community AFLP [cAFLP]) remains virtually unexplored. In the proposed dissertation, a well-documented collection of fecal metagenome extracts from Crohn’s disease patients and their healthy relatives will be used as a model to evaluate the potential of cAFLP for HIT community fingerprinting. From in-silico analyses of available sequence data derived from individual genes (e.g. the 16S rRNA gene), whole-genomes and metagenomes, optimal combinations of restriction enzymes and primers will be identified. During optimization of cAFLP protocols, several endonuclease-primer combinations will be tested. The resulting fingerprints will be subjected to discriminative band class analysis with specialized software. Using DGGE as a reference technique, the ultimate goal of the thesis is to evaluate the use of cAFLP as an improved tool for detecting bacterial indicators that differentiate diseased HIT microbiota from healthy ones.
Aim: The student will expand his/her background on intestinal microbial ecology and diversity, and will contribute to the development and evaluation of a new fingerprinting strategy to characterize HIT ecosystems in health and disease.
Techniques and methods: - bioinformatic tools for restriction analyses and primer design - AFLP (restriction analyses, ligation, PCR, capillary electrophoresis) - DGGE (PCR, gel electrophoresis) - AFLP/DGGE fingerprint analysis
48 Development of a diagnostic tool for identification and detection of plantpathogenic Clavibacter species
Department: Biochemie en Microbiologie (WE10) (Co) Promoter(s): Prof. Dr. Paul De Vos (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; 09.264.5110; email: [email protected]; http://lmg.ugent.be)
Focus: MIB
Short description of the subject: The subject is part of a European project that envisages a more general development of a new diagnostic tool using bar-coding in support of plant health. The Focus of the EU project is on quarantine organisms in general, of course for the laboratory for microbiology, the Focus is on plantpathogenic bacteria that can form a danger for the European crops. One of these bacteria are the plant pathogenic Clavibacters. These bacteria are responsible for the important economic loss of crops as potato and tomato. The genus Clavibacter contains on species – C. michiganensis , with five subspecies. The C. michiganensis subspp. sepedonicus , michiganensis , and insidiosus that are high-risk statutory bacteria for the E.U. and EPPO. The subspecies status relies at present heavily on plant-pathological criteria and host plant infection experiments. But recent whole genome sequences (Gold database) offer a new angle for tracing gene sequences for both taxonomy and barcoding of quarantine strains. Comparative genome analyses are used for selecting better phylogenetic markers for a large-scale diversity study and to delineate the set of household genes useful for multi-locus sequence analysis (MLSA) and barcoding. In a comparative analysis single copy genes can be selected, and if sufficient specific conserved regions in those single copy genes can be appointed, new primers can be developed.
Aim: The study aims at selecting and developing and testing various primers to amplify well selected house keeping genes that are candidate genes for bar-coding and the development of the diagnostic tool for Clavibacter . Various reference strains as well as numerous new and formerly well documented isolates of the various Clavibacter subspecies and relatives will be grown, and genomic DNA will be prepared for PCR of the selected house keeping genes. Based on concatenated and single phylogenetic trees, the most relevant sequences for diagnosis will be selected and proposed as international standard for identification and detection.
Techniques and methods: Reference strains of established Clavibacters species ans subspecies will be cultivated on appropriate media, genomic DNA will be prepared by a fast be reliable method. DNA will be conserved on FTA cards and at -80°C. Primers to amplify the selected house keeping genes will be developed/tested (depending of the progress in the subject at that time), the amplificates will be sequenced and analyzed for the selection of the most relevant ones for diagnosis. All data will be processed using different software packages (e.g. BioNumerics, Kodon…).
49 Assessment of novel primer(s) for the amplification of nor and nir genes
Department: Biochemie en Microbiologie (WE10) (Co) Promoter(s): Prof. Dr. Paul De Vos (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; 09.264.5110; email: [email protected]).
Focus: MIB
Supervisor: Drs. Ines Verbaendert (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; 09.264.501; email: [email protected] ).
Short description of the subject: - - Denitrification is the dissimilatory reduction of nitrate (NO 3 ) or nitrite (NO 2 ) over nitric oxide (NO) and nitrous oxide (N 2O) to dinitrogen gas (N 2). Limited reliable information is available on the distribution of the denitrification trait amongst Gram-positive bacteria. They have rather been neglected in research on denitrification. Through the presence of alternative enzymes in Gram-positive bacteria or the specificity of available primers towards Gram-negative denitrifying strains, this possible large group of denitrifiers is often undetected in environmental monitoring studies. Novel primers based on Gram- positive gene sequences might create new possibilities for the detection of denitrifying genes in Gram-positive denitrifiers. Denitrification by Gram positive organisms, such as Bacillus species, has a negative effect on certain cheese making processes where development of clostridia has to be suppressed by nitrate.
Aim: Since previous detection tools for Gram-positive denitrifiers were based on only a small number of known denitrifying genes, novel primers for Gram-positive denitrifiers were developed. This master thesis aims at screening pure culture denitrifying Bacilli for nor and nir genes with those novel PCR tools and perform sequence and phylogenetic analysis of the retrieved genes.
Techniques and methods: Based on DNA blotting, the project will focus on molecular screening for new denitrification genes in selected pure culture denitrifiers and will comprise culture independent methods including PCR based amplification of the nir and the nor genes of pure culture denitrifying Bacilli , sequence analysis of the retrieved genes and phylogenetic analysis with specific software. Furthermore, the relation between these functional genes and the organismal phylogeny will be investigated in order to retrieve evidence for occurrence or absence of horizontal gene transfer.
50 Cultivation and identification of fast growing methane oxidizers
Department: Biochemie en Microbiologie (WE10) (Co) Promoter(s): Prof. Dr. Paul De Vos and Co-promoter Prof. Dr. Peter Vandamme (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; 09.264.5110; email: [email protected]; [email protected] ).
Focus: MIB
Short description of the subject: This research project is supported by a BOF GOA grant that started at the beginning of 2009. Methane is a naturally occurring atmospheric gas and is the second most important contributor to the increasing greenhouse effect. Microbiological methane oxidation is globally the second most important methane removing process (up to 3.0X 10 7 ton per year). In collaboration with LABMET (Faculty of Bio-ingeneering) methane oxidizing high speed fermentors will be set up and are used as source to isolating and cultivating methane oxidising pure cultures as well as consortia. In this part of the GOA project, the methane oxidizing microbiota are isolated and characterized. Further goals of this research program concerns the function of the various components of the methane oxidising process, and the analysis of the stability of the eventual consortia that are being found.
Aim: This master study aims at isolating, cultivating and characterizing methane oxidizers from high speed methane oxidizing fermentors. It is expected that not only pure cultures but also consortia may be isolated. Furthermore, culture independent approaches will be used to characterize and unravel the bacterial consortia.
Techniques and methods: for the isolation procedure we will make use of a so-called ‘Colony and clone managing system’ for a high throughput approach. At the same time - keeping in mind that methanotrophes might be difficult to grow as pure cultures - approaches using diffusion growth chambers may be applied. Furthermore culture independent approaches (DGGE, or T-RFLP or MALDI TOF MS) will be applied, analyzed and compared with the results obtained from the cultivation approach. Data will be analyzed with appropriate software packages.
51 MRSA (methicillin resistant Staphylococcus aureus ) on Belgian pig farms
Department: WE10 + ILVO - Unit Technology and Food (the dissertation will be performed at ILVO in Melle)
(Co) Promoter(s): - Prof. dr. Peter Vandamme - dr. Marc Heyndrickx / dr. Geertrui Rasschaert
Address + Phone number (co) promoter(s) : Lab. Microbiologie, UGent, [email protected] , 09/264.52.34 ILVO: Brusselsesteenweg 370, 9090 Melle, 09/272.30.26; [email protected]
Focus : MIB
Short description of the subject: Methicillin Resistant Staphylococcus aureus (MRSA) is a pathogen causing healthcare and less frequently community-acquired infections. Since a few years, this pathogen is also isolated from livestock animals, until now particularly from pigs, and farmers. These animals seem to be a new separate reservoir, as the isolates belong to a new clonal complex (CC)398 by multilocus sequence typing (MLST). Studies have shown that in Belgium, a country with large scale pig production, approximately 70% of the farms were positive for MRSA. Moreover, it seems that transmission from animals to humans is possible, as there was on half of the farms at least one person colonized with this MRSA type. A problem would occur when this type would further spread in the population and enter hospitals. This master thesis is situated in a project, during which epidemiological routes on pig farms, including pig farms with other livestock, will be studied in order to remediate MRSA contaminated pig farms.
Aim of the subject: The aim of the subject is to study contamination patterns on pig farms and pig farms with other livestock animals such as poultry or cattle. Therefore, samples will be taken and analyzed by pigs of all age groups, cattle or poultry, pet animals, the farm environment, the air and people in contact with the animals.
Techniques and methods: Samples taken on the pig farms will be further processed via a culture method. Typical colonies will be confirmed by a PCR specific for MRSA and typed with molecular typing methods. In parallel, samples will also be analyzed by quantitive real-time PCRs for MRSA and more specific for MRSA CC398.
52 Development of matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry for the study of spoilage microorganisms in Belgian artisan beers
Department: Biochemie en Microbiologie (WE10) (Co) Promoter(s): Prof. Dr. Peter Vandamme (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; 09.264.5113; email: [email protected]; http://lmg.ugent.be/); Prof. Dr. Anita Van Landschoot (Departement Toegepaste Ingenieurswetenschappen, Chemie/Biochemie, Hogeschool Gent, Voskenslaan 270, 9000 Gent, 09.242.42.42; email: [email protected])
Focus: MIB, BSB
Short description of the subject: Spoilage microorganisms represent the main economical problem in the beer industry. This spoilage flora mainly consists of hop resistant lactic acid bacteria but also some other Firmicutes and yeasts. Potential spoilage flora can be removed from the beer by filtration or pasteurisation but these processes can not be applied without detrimental effect on taste and quality of a range of artisan Belgian beers which depend on an essential microbial flora. Microbiological quality control throughout the production process is essential to prevent future spoilage but relies on rapid and accurate identification of potential spoilage microorganisms. Traditional detection methods are culture based and rely on ATP measurements. In the beer industry these procedures are generally too expensive and time consuming to be implemented in real time. Also immunological and molecular procedures have been introduced as alternatives but they are considered too expensive and lack specificity and sensitivity. In medical microbiology, MALDI TOF mass spectrometry (MS) is becoming increasingly popular as a very rapid and specific procedure for the identification of bacteria and yeasts. Its simplicity, rapidity and low cost render it an ideal diagnostic instrument for the food industry as well.
Aim: The study aims to first characterize MALDI TOF MS fingerprints of a range of established beer spoilage microorganisms and to study how the obtained fingerprints allow differentiation (pattern, peak or biomarker analysis). Subsequently, a collection of unidentified or partially identified spoilage microorganisms from Belgian artisan beers will be examined. It can be expected that part of the latter strain collection will remain unidentified and thus will require additional molecular analyses to reveal their identity and update the MALDI TOF MS fingerprint database.
Techniques and methods: Reference strains of established beer spoilage flora and novel unidentified isolates will be cultivated on appropriate media and analyzed by MALDI TOF MS. Isolates that require further molecular identification will be examined using DNA fingerprinting and sequence analysis 16S rRNA and housekeeping genes. All data will be processed using different software packages based on patterns and biomarker analyses.
53 Study of the predominant colon microbiota of cystic fibrosis patients
Department: Biochemie en Microbiologie (WE10) (Co) Promoter(s): Prof. Dr. Peter Vandamme (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; Phone: (32)9.264.5113; Fax: (32)9.264.5092; Email:[email protected]; http://lmg.ugent.be/)
Focus: MIB, BIB
Short description of the subject: Cystic fibrosis (CF) patients have a genetic disease caused by a mutation in the CFTR-gene which encodes for a transmembrane protein involved in transepithelial ion transport. The main health problems of CF patients concern the digestive and respiratory tracts which are characterized by very viscous mucus layers predisposing these patients for malnutrition, chronic inflammation and lung infection. Because of recurrent or chronic lung infections CF patients are treated several times per year with multiple oral, inhaled or intravenous antibiotics. These antibiotic treatments have a strong impact on the physical condition of the patients and cause a general feeling of malaise characterised by chronic abdominal pain and diarrhoea. They must also have a detrimental effect on the intestinal flora of these patients which is generally considered essential for human health and well-being. In addition, normal digestion of food is hampered by a lack of normal enzyme activity in the gut.
There are currently no data on the composition and stability of the intestinal flora of CF patients. It can be assumed that digestive problems and antimicrobial therapy have a dramatic effect and that this contributes to their general physical malaise, a reduced microbial flora with limited metabolic capacity and increased levels of antimicrobial resistant bacteria.
Aim: In the frame of a long term study on the use of novel probiotic bacteria to improve the health of CF patients, the present study aims to characterize the predominant members of the intestinal flora of a group of cystic fibrosis patients and to compare these with the flora of siblings without the cystic fibrosis phenotype. The effect of frequent and combined antibiotic therapy on the development of antibiotic resistances in the commensal colon flora of a group of cystic fibrosis patients will also be studied.
Techniques and methods: Most human intestinal bacteria are strictly anaerobic and difficult to grow. Molecular ecological studies demonstrated that culture-based methods reveal only a fraction of the microbial biodiversity in the human colon. Therefore the project focuses on culture independent methods including PCR based amplification of community DNA, Denaturing Gradient Gel Electrophoresis (DGGE) to reveal the diversity and stability of the predominant microflora, real-time PCR assays to generate quantitative information on specific groups of bacteria, and sequence analysis of specific genes to identify bacteria. Analysis of antimicrobial resistance will focus on indicator species that are relatively easily cultured and that acquire resistance determinants relatively easily through their genetic flexibility.
54 Linking the microbial diversity of traditional fermented foods to their artisan-type characteristics
Department: Biochemie en Microbiologie (WE10) (Co) Promoter(s): Prof. Dr. Peter Vandamme (Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35; Phone: (32)9.264.5113; Fax: (32)9.264.5092; Email:[email protected]; http://lmg.ugent.be/) Prof. Dr. ir. Luc De Vuyst and Prof. Dr. ir. Frédéric Leroy (Onderzoeksgroep Industriële Microbiologie en Voedingsbiotechnologie, Vakgroep Bio-ingenieurswetenschappen, Faculteit Wetenschappen, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Phone: (32)2.629.3245; Fax: (32)2.629.27.20; Email: [email protected]; http://imdo.vub.ac.be/)
Focus: MIB
Short description of the subject: The production of fermented foods and beverages is an important branch of the food industry. Several of these fermented food products are of a traditional, artisan nature and are perceived by consumers as natural and healthy foods who appreciate them for their gastronomic qualities. Fermented foods are produced through the use of microorganisms, such as yeasts and fungi, lactic acid bacteria (LAB), and acetic acid bacteria (AAB), either through back-slopping or by the direct addition of selected starter cultures. Implementation of carefully selected strains helps to achieve expression of desired properties, maintaining a natural, safe, and health-promoting food. Although it is possible to express desirable and suppress undesirable properties of certain strains via genetic modification, most industrial innovations in the food sector avoid the use of genetically modified starter cultures. This underlines the importance of screening, thereby making the crucial link between species diversity of the raw materials and organoleptic quality of the end-products. Understanding the biodiversity and ecosystem functioning of traditional fermented foods is both an industrial and academic challenge that will allow advanced food production processes and/or will help to find optimal microorganisms that produce the desired attributes, for instance with respect to texture and flavour, in specific food applications. Aim: The present study aims to describe the functional behaviour, such as flavour production (production of antmicrobials, exopolysaccharides, flavour compounds, etc.) of natural isolates from traditional fermented foods, such as fermented sausages, sourdoughs, fermented vegetables, and cocoa beans, in function of the food environment and process technology to implement them in large-scale production processes without loosing artisan properties. Techniques and methods: Through a combination of culture-dependent (plate counting and rep-PCR of picked-up colonies) and culture-independent (PCR-DGGE and real-time PCR) approaches, the microbiota of fermented foods can be followed in detail over time to analyze overall competitiveness and population dynamics of the different bacterial groups involved in the fermentation process. Its metabolic activity can be monitored through a wide range of techniques, mainly based on quantitative chromatography (HPLC, HPAEC, GC) and mass spectrometry (GC- MS, LC-MS), for instance to measure acidification, competitiveness factors, aroma production, and stress responses. Computer-controlled laboratory fermentations on 10-L scale can be performed to simulate the food environment in vitro , which permits detailed and kinetic analysis of the behavior of interesting food isolates, as to understand the relationship between population growth, microbial interactions, and metabolite production. Such insights can be used to better understand the impact of the food microbiota, either the natural species diversity or the applied starter cultures, and of the applied process technology on the characteristics of the end-product, such as fermented sausages, sourdough-based baked goods, fermented vegetables, and chocolate, leading to fermented foods of superior quality (e.g. prolonged shelf-life or enhanced flavour) that can be prepared in an optimised, standardised way.
55 Structure-function analyses and specificity studies of glycoside hydrolase components of the Trichoderma reesei genome
Department: Biochemistry and Microbiology, WE10
(Co) Promoter(s): Prof. Dr. Nico Callewaert, Dr. Kathleen Piens or Dr. Patricia Ntarima
Address + Phone number (Co) Promoter(s): Unit for Molecular Glycobiology, K.L. Ledeganckstraat 35, 9000 Gent, Floor 6 Tel. 09/264. 52.70.
Focus : BSB
Short description of the subject: Cellulose and hemicellulose (mostly xylan), together with lignin, are the major polymeric constituents of plant cell walls and form the largest reservoir of fixed carbon in nature. The enzymatic hydrolysis of polymeric substances by extracellular enzymes, such as cellulases, hemicellulases and laccases, is preferred to chemical depolymerisation to avoid the production of toxic by-products and waste that are expensive to treat. The monosaccharides released through enzymatic hydrolysis can subsequently be microbially converted to commercial commodities, such as bio-ethanol (fuel extender). The soft-rot fungus Hypocrea jecorina ( Trichoderma reesei ) is one of the most efficient and best characterized cellulolytic organisms. It secretes both endo- and exo-glycanases which work together to degrade cellulose and hemicellulose components of the plant cell wall. The Unit of Molecular Glycobiology is involved in a project concerning the characterization of H. jecorina glycoside hydrolases and the analysis of new variants (mutant enzymes) with the aim to obtain better performing enzyme coctails for biomass degradation. The project is suited for a student interested in gaining hands-on experience with the science which is behind the field of bio-fuels.
Aim: Characterization of H. jecorina enzymes involved in biomass degradation.
Techniques and methods: - Enzyme purification - Assay development - Kinetic assays using spectrophotometry and HPLC methods (analysis of the products formed (i.e. sugars)) - Binding studies (e.g. with isothermal calorimetry) - Analysis of the 3-D structure of the target enzyme (if available)
56 Identification and characterization of MALT1 paracaspase interacting proteins and substrates relevant in autoimmunity and cancer
Department: UGent Department for Biomedical Molecular Biology; VIB Department for Molecular Biomedical Research
Promoter: Prof. Dr. Rudi Beyaert
Address + Phone number Promoter: FSVM VIB-UGent research building, Technologiepark 927, 9052 Zwijnaarde tel. 09 33 13770; [email protected]
Focus : BIB, BSB
Short description of the subject: MALT1 is a protein that belongs to a novel family of proteases consisting of paracaspases and metacaspases. MALT1 plays a key role in antigen receptor-induced signalling and is indispensable for T and B cell activation and proliferation. Moreover, enhanced expression of MALT1 or aberrant expression of a API2-MALT1 fusion protein has been linked to B cell lymphoma. These data suggest that therapeutic targeting of MALT1 could be of use in the case of several autoimmune diseases, organ transplantation and cancer. We could recently identify the NF-κB inhibitory protein A20 as a first substrate of MALT1 (Coornaert et al., Nature Immunology 9:263-71, 2008). Ongoing research Focus es on the identification of additional MALT1 substrates and other interacting proteins using several proteomic approaches (mass spectrometry, yeast two-hybrid, MAPPIT). Such knowledge should contribute to the understanding of the function and regulation of MALT1 in several physiological and pathological conditions.
Aim: The aim of this master project is to participate in our research on the function and regulation of MALT1 in inflammatory and immune cell signaling. This work is embedded in several national and international collaborations to which the student will also be exposed. More specifically, the student will contribute to the identification and further characterization of MALT1 interacting proteins and novel substrates. If successful, the results of this study will be published in an international scientific journal.
Techniques and methods: Yeast two-hybrid screening, cell culture, cell transfection, DNA cloning and PCR, co- immunoprecipitation, western blotting, gene reporter assays, ELISA, in vitro transcription/translation, gene mutagenesis
57 Validation of NF-kB signaling proteins as potential therapeutic targets in human disease using genetically modified mice*
Department: UGent department for Biomedical Molecular Biology; VIB department for Molecular Biomedical Research
Promoter(s): Prof. Dr. Rudi Beyaert and Dr. Geert van Loo
Address + Phone number promoter(s): FSVM VIB-UGent research building, Technologiepark 927, 9052 Zwijnaarde R. Beyaert (tel. 09 33 13770; [email protected] ) G. van Loo (tel. 09 33 13 774; [email protected] )
Focus : BIB
Short description of the subject: Nuclear factor-κB (NF-κB)-dependent gene expression plays a crucial role in numerous cellular processes, and defects in their regulation contribute to a variety of human diseases including inflammatory and autoimmune diseases, neurological disorders and cancer. By combining conditional gene targeting of specific NF-κB signalling and regulatory molecules in mice with different mouse models for human disease, we aim to understand the specific function of these molecules in disease pathogenesis and their potential as novel therapeutic targets.
Aim: The aim of this master project is to participate in our research on NF-κB signaling, which is embedded in several national and international collaborations. The student will contribute to the characterization of specific genetically modified mice as well as their analysis in mouse models of human disease (e.g. asthma, Crohn’s disease, multiple sclerosis). If successful, the results of this study will be published in an international scientific journal.
Techniques and methods: Mouse handling and experimental models, genotyping PCR, qPCR, Western blotting, Southern blotting, cell and tissue culture, microscopy, histology, flow cytometry
58 Molecular mechanisms of inflammatory shock
Department: Biomedical Molecular Biology
(Co) Promoter(s): Prof. Dr. Peter Brouckaert/Dr. Anje Cauwels
Address + Phone number (Co) Promoter(s): DMBR, Technologiepark 927, 9052 Gent; [email protected] , 09 33 13 710; [email protected] , 093313712
Focus : BIB
Short description of the subject: Inflammatory shock (sepsis, anaphylaxis) is the first cause of death in intensive care units. The prevailing paradigm is that the cardiovascular collapse is caused by excessive NO production by NOS enzymes, that through activation of cytosolic guanylate cyclase relaxes smooth muscle cells and eventually results in severe hypotension. This led to the treatment of septic shock patients with NOS inhibitors. However, a large phase III trial had to be terminated because of excess mortality in the treatment group. Our current results show that the aforementioned NO paradigm is not correct: NO is not sufficient to cause the hypotension and parallel pathways exist. In this project we are identifying the factors that are necessary in addition to NO, the parallel pathways involved and downstream targets in the pathway (sGC, ion channels). This is done by mouse transgenic technology, pharmacological interventions, (immuno)histochemistry and protein interaction technology.
Aim: Depending on the state of the program next year, the student will be involved in the testing of one or more hypothesis
Techniques and methods: Manipulation of mice, measuring temperature and blood pressure, collecting blood and organs for later analysis; ex vivo determination of serum parameters, immunohistochemistry, western blotting, tissue culture, RT-QPCR. It may involve genetic engineering (construct work).
59 Identifying mammalian pathogenicity genes from influenza A virus*
Department: Biomedical Molecular Biology (WE14)
(Co) Promoter(s): Xavier Saelens and Marina De Filette
Address + Phone number (Co) Promoter(s): Technologiepark 927, B-9052 Zwijnaarde, 09 33 13 620
Focus : MIB, BIB
Short description of the subject:
The pathogenicity of influenza viruses differs according to the strain. Little is known about the influenza genes that control pathogenicity and about how influenza gene products determine differences in pathogenicity and adaptation to new hosts. In the Department of Molecular Biology, we have recently isolated an H3N3 influenza strain that is highly pathogenic in mouse. Our aim is to understand the genetic and molecular determinants that control the increased virulence of this mouse-adapted H3N3 strain.
Aim: The aim of the Master Dissertation project is to clone and functionally characterize the genome of a highly pathogenic H3N3 virus. By comparing the genes and gene combinations derived from the parental low-pathogenic H3N3 and its highly pathogenic descendant, novel critical determinants of influenza pathogenicity will be identified.
Techniques and methods: Influenza virus growth in cell culture and in vivo RT-PCR and cloning Production and characterization of influenza virus by reverse genetics Influenza virus replication assays
60 Analysis of the function and regulation of the delta-protocadherins
Department: Molecular Biomedical Research
(Co) Promoter(s): Dr. Karl Vandepoele + Prof. Dr. Frans Van Roy
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 Zwijnaarde 09/33.13.753 (Dr. Karl Vandepoele) 09/33.13.601 (Prof. Dr. Frans Van Roy)
Focus : BIB
Short description of the subject:
Protocadherins (PCDHs) are a family of transmembrane proteins that play a role in the formation of the central nervous system. They resemble classical cadherins, which play a crucial role in cell-cell adhesion during embryonic development and in tissue homeostasis. The protocadherins can be subdivided in clustered and non-clustered families, of which the majority of the non-clustered protocadherins belong to the subgroup of the delta- protocadherins. These are characterized by several conserved motifs in their cytoplasmic domain, for which a number of interacting proteins have been identified in our group. Some delta-protocadherins were reported to mediate weak cell-cell adhesion in vitro and cell sorting in vivo . In addition, individual delta-protocadherins might play important roles in signaling pathways. A role in the development of the human brain has been recently demonstrated by the implication of PCDH11X in the development of late-onset Alzheimer’s disease and by mutations in PCDH19 that cause female-restricted epilepsy and cognitive impairment. For other delta-protocadherins, a tumor suppressive role has been demonstrated. For instance, PCDH10 is frequently silenced by methylation in several tumor types, and upon re-expression of PCDH10, tumor cells are growth-retarded and less malignant. In our group, we are looking for new interaction partners of delta-protocadherins that can show us how these intriguing but still enigmatic membrane proteins perform their functions in the cells. Additionally, we are interested in the regulation of the delta- protocadherin genes, both at the level of transcription and of translation.
Aim: to provide more insight into the regulation and function of the delta-protocadherins
Techniques and methods: cell culture, (quantitative) RT-PCR, Western blotting, immunohistochemistry, plasmid construction, co-immunoprecipitation, mRNA and micro- RNA analysis, bio-informatics
61 Role of alpha-catenin proteins in the mouse
Department: Department of Molecular Biomedical Research
Promoter: Dr. Jolanda van Hengel
Address + Phone number Promoter: Molecular Cell Biology Unit, DMBR, Technologiepark 927, 9052 Zwijnaarde, Gent, Tel: 09.3313755, Jolanda.van [email protected]
Focus : BIB
Short description of the subject: Cadherins are calcium-dependent adhesion molecules that play a key role in initiating and maintaining intercellular contacts. The cytoplasmic region of cadherin binds β- catenin, which in turn associates with alpha-catenin . Alpha-catenins form a small but intriguing gene/protein family. The prototypic molecule alpha-E-catenin is well-known for its stabilizing role in adherens junctions and its anti-invasion activity. We discovered two more members of the family: alpha-T-catenin and alpha-catulin. Alpha-T-catenin is expressed mainly in testis, heart and brain. In cardiomyocytes, alpha-T-catenin is co-expressed with alpha-E-catenin at intercalated discs. We proposed that it has a specific strengthening function. A role for alpha-T-catenin in Alzheimer’s disease is not excluded. Alpha-catulin was identified as a player in the Rho signaling pathway, serving as a scaffold protein for Lbc, a member of the Dbl family of Rho guanine nucleotide exchange factors. Rho family GTPases play important roles during organization of the actin cytoskeleton and formation of focal adhesions. The mRNA and protein of α-catulin are ubiquitous.
Aim: We are scrutinizing the role of alpha-T-catenin and alpha-catulin by a variety of approaches, including organ cultures and analysis of transgenic mice.
Techniques and methods: Southern blotting, Western blotting, PCR, quantitative RT-PCR, mouse breedings, histology, immunohistochemistry, tissue culture, primary cultures, plasmid constructions.
62 Characterization of primary target genes of the canonical Wnt pathway in Xenopus embryos
Department: Department of Molecular Biomedical Research
(Co) Promoter(s): Prof. Kris Vleminckx
Address + Phone number (Co) Promoter(s): DMBR, Technologiepark 927 ([email protected] , tel. 09/3313720)
Focus : BIB
Short description of the subject: The canonical Wnt pathway plays a critical and evolutionary conserved role during embryonic development. In adult life, the pathway is a crucial regulator of stem cells (Reya and Clevers, Nature 2005). Deregulated constitutive activation of the Wnt pathway is associated with the development of cancer in particular organ systems and the pathway is seen as an important target for therapeutical intervention (Clevers, Cancer Cell 2004). We are identifying novel Wnt target genes via microarray analysis. To identify organ-specific target genes it is absolutely necessary to perform such an expression analysis in complex organs or whole organisms. In addition, for the discovery of primary target genes, the employment of inducible systems in transgenic organisms is essential to minimize interference with secondary and tertiary induced genes. The Xenopus model organism is well positioned for this type of analysis. Since the canonical Wnt pathway is evolutionarily well-conserved, studies performed in one organism can be extrapolated to other organisms. We have established an experimental platform using transgenic lines of Xenopus that allows us to identify primary Wnt target genes. Surprisingly, we found that within the embryo these genes can only be activated by the Wnt pathway in a restricted spatio- temporal window of expression (i.e. specific developmental stage and tissue/organ). It means that gene X can be induced by the Wnt pathway in organ A but not in organ B. Interestingly, some organs (most notably the colon) are more susceptible to the development of cancer by the oncogenic mutation of the Wnt pathway than others. We speculate that this is due to the activation of a specific set of target genes in these organs.
Aim: The goal of this thesis project is the analysis of the expression pattern of new primary Wnt target genes that were identified via microarrays and in silico data mining. This will be done via in situ hybrisation and/or reverse transcriptase PCR on embryos in different developmental stages. In addition it will be necessary to perform a functional characterization of these novel target genes. This will be done in gain-of-function (RNA injections or transgenesis) and loss-of-function (morpholino injections) experiments.
Techniques and methods: in situ hybridization, RT-PCR analysis, RNA- and morpholino injections, transgenesis in Xenopus , in silico data mining
63 Studying the role of matrix metalloproteinases (MMPs) in endotoxemia and immunity.
Department: Department for Molecular Biomedical Research (DMBR) - Molecular Mouse Genetics
(Co) Promoter(s): Prof. Dr. Claude Libert – Dr. Roos Vandenbroucke
Address + Phone number (Co) Promoter(s): Technologiepark 927 9052 Zwijnaarde Tel. +32-(0)9-331.37.02
Focus : BIB
Short description of the subject: The immune system is a collection of mechanisms that protects the host from pathogens. Unfortunately, our immune system is not always capable of effectively controlling an infection, a situation which can lead to an exaggerated, uncontrollable systemic inflammation, often with a fatal ending, a condition called ‘sepsis’. Despite improvements in care and therapy, sepsis still remains the most common cause of death in intensive care units. In mice we can mimic sepsis by ‘endotoxemia’, i.e. injection of LPS, the major cell wall component of Gram-negative bacteria. We recently observed that several matrix metalloproteinases (MMPs; proteases well known for their extracellular matrix degrading capacities) play an important role in endotoxemia, as several MMP specific knockout mice, i.e. mice lacking a specific MMP gene, survive after LPS challenge, in contrast to the wild type mice.
Aim: The aim of this project is to investigate the role of MMP-7 and -13 in more detail to explain the mechanism of LPS resistance of the knockout mice.
Techniques and methods: mouse work, cell culture, RNAi, miRNA analysis, bioassays, FACS analysis, histology, RT- PCR, QPCR, Western blot analysis
64 Apoptosis, necrosis and autophagy: a comparative study of the impact dying cells on innate immunity*
Department: Department for Molecular Biomedical Research, VIB-UGent Promoter(s): Dr. Dmitri V. Krysko and Prof. Dr. Peter Vandenabeele
Address + Phone number promoter(s):
Dr. Dmitri V. Krysko VIB - Ghent University 'Fiers-Schell-Van Montagu' building; Technologiepark 927 B-9052 Ghent (Zwijnaarde), Belgium Tel: +32-(0)9-331.37.64; E-mail: [email protected]
Prof. Dr. Peter Vandenabeele
Tel: +32-(0)9-331.37.60; E-mail: [email protected]
Focus : BIB
Short description of the subject: Activation of the innate immune system is the first line of defense against infection, localized injury, or trauma. The response of the immune system finally leads to elimination of the infectious agent and restoration of homeostasis. However, in the human body close to 500 x 10 9 cells die each day, and they are either shed off directly from body surfaces or continuously removed by a remarkably efficient phagocytic system without causing inflammation and scar formation. Impairment of the clearance of dying cells by antigen presenting cells (APC) or the presence of 'danger' signals may modify the balance between induction of tolerance and activation of T cells, leading to cancer and autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus. In mammals, several forms of cell death can occur, including type I (apoptosis), type II (cell death associated with autophagy) and type III (necrosis). Recent studies indicate that each cell death type may generate a specific pattern of damage- or death-associated molecular patterns (DAMPs). These patterns can be recognized by extracellular and intracellular receptors, through which they communicate with the immune system and exert specific immunomodulatory effects. An understanding of the underlying immunostimulatory effects of different types of dying cells may lead to the development of cancer vaccines to stimulate the anti-neoplastic immune responses in vivo , while understanding of the immunosuppressive effects of dying cells may be important to finding new strategies for treatment of autoimmune and inflammatory diseases, in which cell death plays an important role.
Aim: (1) Comparative study of immunomodulatory properties of apoptotic, necrotic and autophagic cells in vitro and in vivo and (2) analysis of mechanisms of recognition, interaction and sensing of different types of dying cells by the innate immune system in vitro and in vivo
Techniques and methods: cell culture (cell lines; isolation and culture of primary mouse peritoneal and bone marrow derived macrophages and dendritic cells); flow fluorocytometry; cytokine measurements by CBA Flex set and Luminex: Bio-Plex systems; Western blotting; Confocal microscopy; Cell death assays (LDH, MTT, Annexin V/ Sytox Red, caspase activation); in vitro and in vivo phagocytosis assays; in vivo cell attraction assays
65 Role of phosphorylation of RIP1K in cell death and NF-kB activation.
Department: Dept. for Molecular Biomedical Research (DMBR)
(Co) Promoter(s): Prof. dr. P. Vandenabeele/ dr. F. Van Herreweghe
Address + Phone number (Co) Promoter(s): VIB Department for Molecular Biomedical Research, UGent 'Fiers-Schell-Van Montagu' building Technologiepark 927 B-9052 Ghent (Zwijnaarde), Belgium 09/3313760 (PV) – 13762 (FVH)
Focus : BIB
Short description of the subject: Tumour Necrosis Factor alpha (TNF) exerts it proinflammatory role mainly through activation of NF-kB but in some cell lines TNF triggers also a cell death cascade. Both pathways are however dependent on the TNF-receptor associated RIP1 kinase. Upon TNF- receptor triggering this kinase undergoes different types of posttranslational modifications (PTMs), including phosphorylation, that are rapid and transient. In our research group, we study the role of RIPK1 in TNF-induced necrosis. In this particular project we will further study the role of phosphorylation of RIP1. Questions that will be addressed: which molecular mechanism drives this transient phosphorylation and how is it controlled, can we interfere with this mechanism and if so, what is the effect on RIP1 phosphorylation and at large, necrosis and NF-kB activation. What is the role of phosphorylation on the other PTMs of RIP1? Is it possible that some phosphorylation sites are dispensable for NF-kB activation but absolutely required for induction of necrosis?
Aim: To conduct experiments that can provide answers to the questions above. Priority will be given to work further on the knowledge/working hypothesis already obtained at that time, but there is possibility to explore the impact of other mechanisms, depending on the interest of the student involved.
Techniques and methods: Cell culture (including viral transduction/transfection), cell death assay, western blotting, immunodetection, immunoprecipitation, FACS analysis, PCR-based mutagenesis, cloning (including Gateway platform), siRNA-technology. In vivo work is possible.
66 Polyelectrolyte microcapsules: multifunctional antigen delivery platforms?
Department: Biomedical Molecular Biology
(Co) Promoter(s): Prof. Dr. Johan Grooten; Co-Promoter: Dr. Stefaan De Koker
Address + Phone number (Co) Promoter(s): Technologiepark 927 Zwijnaarde Tel. Promotor: 091333650; Tel. Co-promotor: 0913654
Focus : BIB
Short description of the subject: The development of vaccines to prevent infectious diseases constitutes without doubt one of the major accomplishments of human medicine. However, for several important pathogens that affect millions of people today – including HIV, malaria and Mycobacterium tuberculosis- no effective vaccines are available yet. Indeed, developing new vaccines that are able to protect against these insidious pathogens poses a major challenge. Although creating new vaccines starts with the identification of putative protective antigens, mere injection of these antigens is seldom enough to evoke strong immune responses. Micro-organisms are indeed not solely composed of antigens, but also contain certain microbial associated molecular patterns (cell wall components, viral dsRNA, …) that are recognized by the innate immune system, which subsequently initiates an antimicrobial and inflammatory response. These initial interactions between pathogen and innate immune systems also largely determine the shape of the adaptive immune response, a feature not to be underestimated given the need for different types of immune defense to protect against distinct pathogens types (e.g. a virus versus a fungus). In addition, the immune system has evolved to recognize viruses and bacteria which are of a particulate nature. These particulates are efficiently phagocytosed by dendritic cells (DCs), the main initiators of adaptive immune responses, while soluble antigens are only poorly taken up by DCn and more prone to degradation, resulting in weaker immune responses. Briefly summarized, the key components for a successful vaccine design are: 1. protective antigens 2. immunostimulating components activating the innate immune system 3. a delivery system that ensure optimal uptake and presentation of the antigen by DCs. In collaboration with the Lab of Pharmaceutical Analysis and General Biochemistry, a new type of microcapsule was developed and tested in the Lab of Molecular Immunology. These microcapsules not only allowed an efficient targeting of the antigen towards DCs, but also contained inherent immune activating properties resulting in the generation of a potent immune response. Moreover, the technique applied to produce these microcapsules also allows the easy modification of the microcapsules’ surface with other immune activators (mycolic acid, dsRNA, unmethylated DNA), which might allow us to steer the immune response in any desired way.
Aim: 1. Unraveling the mechanisms underlying the immune activating properties of the microcapsules 2. Can we steer the adaptive immune response by changing the microcapsules’ composition and/or incorporating microbial derived components in the microcapsules’ surface? 3. Analysis of the immune response following microcapsule based immunization with a clinically relevant antigen (Mycobacterium tuberculosis Ag85A/B)
Techniques and methods: - Cell culture, RT-PCR, Elisa, Elispot, Flow-cytometry/multiplex cytokine analysis
67 Deregulation of inflammatory and angiogenic signaling in human colon tumors
Department: Biomedical Molecular Biology
(Co) Promoter(s): Prof. J. Grooten, supervisor: Sarah Pringels
Address + Phone number (Co) Promoter(s): Lab of Molecular Immunology Technologiepark 927 9052 Zwijnaarde. 09 3313650 [email protected] [email protected]
Focus : BIB
Short description of the subject: Prostaglandins and leukotrienes are derived from arachidonic acid through the specific enzymes cycloöxygenase and lipoxygenase. The lipid mediators play an import role in inflammation, including promoting of angiogenesis through the induction of VEGF. These pathways also contribute to tumor vascularization and therefore constitute important targets for cancer therapy. By RT-qPCR of human colorectal carcinoma resections, we analyzed the mRNA expression levels of key enzymes involved in prostaglandin and leukotriene synthesis (COX1, COX2 and 5LOX) along with the VEGF-family members (VEGFA, -B, -C, - D and PlGF). This revealed (i) an aberrant VEGF -expression pattern in a large majority of tumors, and (ii) a contribution of COX2 and 5LOX to this aberrant VEGF -expression.
Aim: In a next step we want to verify to what extend epigenetic changes and/or micro-RNAs contribute to the aberrant VEGF -expression patterns in respons to prostaglandins and leukotrienes in human colon cancer.
Techniques and methods: Two or more of the following techniques will be used: tissue culture, RT-qPCR, western blot, immunohistochemistry and specific activity-assays.
68 Functional characterization of alveolar macrophages in a mouse model of allergic asthma
Department: Department for Molecular Biomedical Research
(Co) Promoter(s): Prof. Dr. Grooten Supervisor: Drs. Thomas Naessens
Address + Phone number (Co) Promoter(s): Molecular Immunology Lab (FSVM-building) Technologiepark 927 9052 Zwijnaarde Tel: 09/33.13.650 E-mail: [email protected] [email protected]
Focus : BIB
Short description of the subject: Asthma is a chronic inflammatory disorder of the airways characterised by recurrent episodes of airway obstruction and wheezing. In 80% of the cases, asthma is diagnosed as an allergic disease. After systemic sensitization for an allergen, a renewed exposure to the allergen leads to an infiltration of eosinophils, monocytes and CD4 + T-cells in the lungs. Resident alveolar macrophages (rAM), which represent the predominant innate immune cell population in the alveoli of healthy individuals, play also an important (regulatory) role in the pathogenesis of allergic asthma. In the Molecular Immunology lab, alveolar macrophage activation, function and dynamics during the onset and propagation of the allergic bronchial inflammation is intensively studied in a mouse model for allergic asthma. Recently, we showed that these rAM disappear from the alveoli during the course of the allergic inflammation. As a consequence a new ‘secondary’ rAM population resides the airways after the clearance of the inflammation. These ‘secondary’ rAM are functionally different from the ‘primary’ rAM present in the alveoli before the onset of inflammation. This was shown by an in vitro and in vivo challenge of these cells with the Gram negative bacterial component lipopolysaccharide (LPS). A striking feature of the ‘secondary’ rAM is their capability of interferon-β (IFN- β; an important cytokine in bacterial and viral infections) production which is absent in ‘primary’ rAM. As a consequence ‘secondary’ rAM may contribute to the higher sensitivity of asthmatic lungs for bacterial infections and new encountered allergens.
Aim: To further characterise the ‘secondary’ rAM and to explore the pathological consequences of their different functionality.
Techniques and methods: Flow cytometry, RNA-isolation, RT-PCR, qPCR, tissue culture, laboratory animal practices, microscopy, ELISA
69 Study of EMT during malignant progression of human breast cancer
Department: Department Molecular Biomedical Research
(Co) Promotor(s): G. Berx
G. Berx Moleculaire en Cellulaire Oncologie (UMCO) UGent-VIB Research Building FSVM Technologiepark 927 9052 Gent Tel. 09.3313740, [email protected] )
Practical Guidance: Drs. Marianthi Tatari Tel. 09.3313742, [email protected])
Focus : BIB
Short description of the subject: Malignant epithelial cancers are recognized by loss of intercellular adhesion and cellular differentiation. E-cadherin is a major epithelial cell-cell adhesion molecule that functions as an invasion-tumor suppressor protein. Many human tumors show loss of E-cadherin expression which seems to be an important step during malignant progression of e.g. breast cancer. Different mechanisms that explain loss of E-cadherin expression are known such as mutations, loss of heterozygosity or active transcriptional repression. Different transcriptionfactors belonging to the Snail and ZEB family repress E-cadherin expression through binding with the E-cadherin promoter. These transcriptionfactors play an important role in the regulation of epithelial mesenchymal transition (EMT). EMT is a process where epithelial cells undergo a transition towards a mesenchymal phenotype, which goes hand in hand with loss of intercellular adhesion and gain of cell motility. EMT seems to be a major determinant of the malignant behavior of breast cancer.
Aim: With the use of our cellular and mouse EMT model systems we will study in more detail the role of miRNAs on breast tumor progression.
MicroRNAs (miRNAs) are endogenous RNAs that can play important roles in animals and plants by targeting mRNAs for cleavage or translational repression. Mounting evidence shows that miRNAs are mutated or aberrantly expressed in human cancer, suggesting that some miRNAs act as tumor suppressors or oncogenes. MicroRNAs appear to be master regulators of EMT- inducer transcription factors like ZEB transcriptionfactors. The goal is to link the activity of several miRNAs to critical cancer genes, particularly components of the EMT pathway and to study their involvement in the control of normal and cancer cell proliferation and invasion.
Techniques and methods: The student will get acquainted with different techniques: as general molecular biologic techniques as DNA cloning, immunofluorescent analysis, tissue culture techniques as 3D growth mammary gland cells, transfection, lentiviral transduction, conditional miRNA expression, shRNA knock-down and specific technology for miRNA expression validation as real-time PCR, gene-promotor studies.
70 Heterologous expression of G protein-coupled receptors in yeast adapted to the expression of eukaryotic membrane proteins
Department: Unit for Molecular Glycobiology VIB Department for Molecular Biomedical Research, UGent. Department of Biochemistry and Microbiology, UGent
(Co) Promoter(s): Prof. Dr. Nico Callewaert, Dr. Noura Bensalem
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 GHENT-ZWIJNAARDE, Belgium, + 32 9 331 36 30
Focus : BSB, BIB, MIB
Short description of the subject: Membrane proteins play an important role in many biological processes such as ion transport, molecule recognition, and energy transport. Very little although is known about the structure-function relationships of these proteins. One reason is that many problems are encountered when trying to produce these proteins in a pure, homogenous and stable form to allow biophysical characterization. In the lab, the last two years a lot of effort was made to create a technology platform for the expression of eukaryotic membrane proteins. The selected expression systems are the yeast Pichia pastoris and Yarrowia lipolytica . Expression systems will be evaluated with a number of selected membrane proteins.
Aim: Validate the engineered yeast strains for the production of membrane proteins by overexpression of a selected set of membrane proteins
Techniques and methods: Plasmid construction, PCR, DNA isolation from yeast, isolation eukaryotic genes from cDNA bank, Southern blotting, Western Blotting, SDS-PAGE, small scale protein expression of heterologous proteins in yeast, preparation competent cells and transformation of yeast ( Y. lipolytica and P. pastoris )
71 Production of lysosomal enzymes with human-like glycosylation in yeast
Department: Unit for Molecular Glycobiology VIB Department for Molecular Biomedical Research UGent Department of Biochemistry and Microbiology
(Co) Promoter(s): Prof. Dr. Nico Callewaert, Dr. Petra Tiels
Address + Phone number (Co) Promoter(s): FSVM building Technologiepark 927 9052 Zwijnaarde
Focus : BIB-BSB-MIB
Short description of the subject: Lysosomal storage diseases are a family of genetic disorders. Most of these disorders are caused by a mutation in genes that code for enzymes involved in the breakdown of glycan structures in the lysosomes, leading to storage of the substrate of the enzyme. Some of these lysosomal storage diseases are treated with enzyme replacement therapy (ERT), where the recombinantly produced enzymes are administered intravenously. For the moment the enzymes are produced in mammalian cells leading to very expensive treatments (approximately $250,000 US a year/patient for Fabry disease). To lower this cost, we are performing biotechnological research to allow production of these enzymes in yeasts. In humans, these lysosomal enzymes have a specific type of glycosylation to target them to the lysosomes. To produce this kind of glycosylation in yeasts, pathway engineering is needed and promising results have already been obtained in our lab. In this thesis, you will produce one such enzyme with the correct glycosylation, purify the enzyme and then test it in vitro in mammalian cells in comparison with the current clinical preparation, to look for uptake by lysosomes and intracellular enzymatic activity.
Aim: Recombinant production of lysosomal enzymes with human-like glycosylation in yeast
Techniques and methods: Cloning Yeast work (transformation, expression) Enzyme purification In vitro enzymatic assays Uptake studies in mammalian cells
72 Analysing the glycoprofile of low abundant serum proteins: a step towards more specific cancer diagnostics.
Department: Unit for Molecular Glycobiology VIB Department for Molecular Biomedical Research and UGhent Department of Biochemistry and Microbiology
(Co) Promoter(s): Prof. Dr. Nico Callewaert, Dr. P. Ameloot
Address + Phone number (Co) Promoter(s): Technologiepark 927, 9052 GHENT-ZWIJNAARDE, Belgium, + 32 9 331 36 30
Focus : BSB, BIB
Short description of the subject: Posttranslational modification of serum proteins has become an important area in biomarker research. Of particular interest is the study of glycoproteins, as specific glycosylation patterns are produced by cancer cells. In this project you will help to develop a novel, clinically relevant technology to detect specific glycosylation patterns on tumor biomarkers. Hereto, we will make use of combinations of immunotrapping and glycosylation recognition, using lectin or glyco-antibody binding. This work has two topics: in a first step, the methodology will be optimised on recombinantly produced or highly purified tumor marker proteins in order to characterise in detail the robustness and specificity of the different method steps. Subsequently, the optimised procedures will be used to study patient samples.
Techniques and methods: Protein analysis: SDS-PAGE, western blot immunodetection, immunoprecipitation, ELISA, in vitro enzymatic assays ; Glyco-analysis: preparation of protein N-glycans and analysis by DSA-FACE, lectin binding, glyco-antibody binding ; Nucleic acid analysis: Quantitaive PCR,…
73 Role of VEGF and EMT modulators in vascular and hematopoietic cell differentiation*
Department: DMBR
(Co) Promoter(s): Dr. Jody J. Haigh (promoter) and Dr. Steven Goossens (co-promoter)
Address + Phone number (Co) Promoter(s): Technologiepark 927, Zwijnaarde, 09-33-13- 730
Focus : BIB
Short description of the subject: The Vascular Cell Biology unit at the DMBR/UGent/VIB is currently using cutting-edge mouse embryonic stem (ES) cell based technologies and the mouse as model genetic systems to study gene function in vitro and in vivo . These studies are centered on the VEGF ligand/VEGF receptor system and their roles in the normal development of the bone, hematopoietic and nervous system. Alterations in this signaling pathway have been causally linked to a wide spectrum of human diseases ranging from heart disease (arthrosclerosis), neural degenerative diseases (such as ALS, Alzheimer’s), Osteoporosis (bone loss), Diabetes, and Cancer. During the last several years we have developed several interesting ES cell and mouse based models that will allow us to understand the role of this key signaling pathway during both normal vascular/hematopoietic development and disease processes. Recently we have initiated studies concerning the ability of two families of transcriptional repressors; the Snail family (Slug and Snail) and the Zeb family (Sip1 and deltaEF1) in modulating cardiovascular/hematopoietic development and homeostasis. Here we have documented several intriguing phenotypic alterations including a block in hematopoiesis resulting in intracerebral bleedings and/or drastic fetal anemia. We are seeking highly motivated candidates to participate in the analysis of these phenotypes that have relevance to human disease. If successful, applicants will be co- authors on several ongoing projects that are expected to result in internationally recognized publications and will be encouraged to continue their studies at the Doctoral level with support from the IWT and FWO.
Aim: Understanding the role of VEGF and EMT modulators in vascular and hematopoietic cell differentiation
Techniques and methods: Students will learn in vitro hematopoietic differentiation assays, FACS analysis, isolate primitive hematopoietic stem cells from bone marrow, Q-RT-PCR, Western blot, cell culture, immunohistochemistry and mouse works (breeding, genotyping, bleeding)
74 Analysis of the function of RIP4 in skin differentiation.
Department : Department for Molecular Biomedical Research
(Co) Promoter(s) : Prof. Wim Declercq and Dr. Saskia Lippens (guidance: Drs. Philippe De Groote)
Address + Phone number (co) promoter(s) : FSVM building, Technologiepark 927, 9052 Gent-Zwijnaarde. W.D., tel. +32 (0)9 33 13660, [email protected] ; S.L., tel +32 (0)9 33 13662, [email protected].
Focus : BIB
Short description of the subject : RIP4 belongs to a family of Ser/Thr kinases, the RIP kinases, generally involved in activation of the transcription factors NF-κB and AP-1 (Meylan and Tschopp, 2005). NF- κB is mainly involved in upregulation of cell survival genes, while AP-1 plays a major role in cell proliferation. The RIP4 kinase domain can activate NF-κB and JNK, a kinase of the AP-1 pathway (Meylan et al., 2002; Moran et al., 2003). The RIP4 C-terminus contains 11 ankyrin repeats, which are separated from the kinase domain by an intermediate domain. Genetic deletion of Receptor Interacting Protein 4 (RIP4) in the mouse results in abnormal skin formation (Holland et al., 2002). The skin of these mice shows different characteristics of psoriatic skin, like hyperplasia, the presence of a parakeratotic outer layer in which, in contrast to a normal cornified layer, the nuclei are still present. Several genes involved in epidermal differentiation and inflammation are deregulated (Holland et al., 2002). Recently, we have identified amino acid residues that are important for RIP4 activity. In addition we have shown that overexpression of RIP4 strongly induces keratinocyte differentiation. Both the NF −κ B and AP-1 pathways play an important role in skin differentiation (Bell et al., 2003; Zenz and Wagner, 2006). Different skin disorders show an altered expression pattern of NF-κB proteins and target genes. Genetic deletion of NF-κB signal transducers, like IKK α, IKK β, IKK γ and TAK1 results in a disturbed skin homeostasis. However, the signaling pathways governing RIP4 activity are currently unknown. We have performed a yeast two-hybrid screen with RIP4 to identify RIP4 regulatory proteins. We identified several interesting proteins and will now further study the role of these proteins in RIP4 signaling pathways.
Aim : With this research we want to gain new insights into the RIP4 signaling pathways, especially in keratinocytes.
Techniques and methods : PCR, processing and analysis of tissue (mainly skin), immmunohistochemistry, western blotting, cell culture, isolation of keratinocytes, transfection techniques, RNAi technology.
75 In silico prediction of functional microRNA targets
Department : Department for Molecular Biomedical Research
(Co) Promoter(s) : Dr. Pieter De Bleser, Prof. Dr. Frans Van Roy
Address + Phone number (co) promoter(s): Bioinformatics Core, Department for Molecular Biomedical Research (DMBR), VIB, Department of Biomedical Molecular Biology, Ghent University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium Tel : +32-(0)9-33-13.693 email: [email protected]
Focus : BIS, BSB, BIB, MIB, PLB
Short description of the subject: MicroRNAs (miRNAs) are small noncoding RNAs typically between 19-25 nucleotides long and widely found in animals and plants. Many miRNAs are highly conserved across species and exert post-transcriptional control by base-pairing interactions with an mRNA target, causing degradation of the target transcript or translational repression. Computer- based miRNA target prediction is considered indispensable in miRNA research and a number of algorithms have been developed to this purpose. The identification of miRNA targets in animals is particularly difficult as these miRNAs bind only with partial complementarity to their targets, contrasting the situation in plants. At current, the different available algorithms produce non-overlapping lists of putative miRNA targets indicating a lot of false positive predictions, despite the fact they all rely on (a combination of) only four basic rules: (1) the complementarity of the miRNA to the 3'UTR of its target transcript, (2) the thermodynamic stability of the miRNA-mRNA duplexes, (3) the phylogenetic conservation of the target site and (4) whether the putative target sites are accessible, i.e. located in parts of the 3'UTR that lack secondary structure. Using data sets of 3'UTR sequences positionally aligned around experimentally validated miRNA target sites, we will calculate the global RNA tertiary structure using NAST (Nucleic Acid Simulation Toolkit), a knowledge-based coarse-grained tool for modeling RNA structures ( Jonikas et al, RNA 2009 ). For each of these sequences, geometric features such as curvature and torsion will be calculated along the structural axis. Next, the procedure will be repeated on a data set of experimentally validated, non-functional miRNA target sites. Pair-wise statistical analysis of the averaged curvature and torsion at each position between the positive and negative data sets might reveal the geometrical features able to distinguish between functional and non-functional miRNA target sites. These features will then be used to implement a target-prediction method using a machine-learning approach.
Aim: see title
Techniques and methods : Bioinformatics methods, Perl scripting, Computational Biology
76 Tissue specificity of hormonal responses
Department: Physiology, WE15
(Co) Promoter(s): Prof. Dr. Dominique Van Der Straeten; Dr. F. Vandenbussche
Address + Phone number (Co) Promoter(s): K. L. Ledeganckstraat 35, 9000 Gent; 092645185, 5186
Focus : PLB
Short description of the subject: Plant architecture is determined by external and internal factors. Plant hormones are an internal prerequisite for plants to adapt or maintain their shape. Hormonal responses in Arabidopsis elongation processes are known to be regulated by specific cell types. This is the case for auxin, brassinosteroids and gibberellins. For ethylene, this remains to be determined. Tissue-specific promoters driving the expression of EIN3 binding F-box proteins (EBF1 and EBF2; negative regulators of the ethylene response) will be used to interfere in a cell type specific manner with the ethylene signal. In order to dissect which cell types play a role in ethylene response, promoters specific to a unique cell type or to a combination of cell types will be used. Interactions with other hormones will be assayed based on the already known cell type dependence for these hormones.
Aim: Obtain a profound insight in tissue or cell type dependence of ethylene responses.
Techniques and methods: Tissue culture techniques; macroscopic biometrics; Bright field, fluorescence & confocal microscopy; Reporter gene analysis (e.g. GUS, GFP); DNA and RNA analysis; (RT-PCR); crossing and genetic analysis
77 Global change effects on plant growth: a molecular-physiological study of hormonal dependence of UV-B responses
Department: Physiology, WE15
(Co) Promoter(s): Prof. Dr. Dominique Van Der Straeten; Dr. F. Vandenbussche
Address + Phone number (Co) Promoter(s): K. L. Ledeganckstraat 35, 9000 Gent; 092645185, 5186
Focus : PLB
Short description of the subject: Ultraviolet B radiation is the most harmful radiation of the solar spectrum that can reach the earth’s surface. Plants, being sessile organisms have developed mechanisms to protect themselves from UV-B damage, but they also use this radiation for regulating their architecture. In Arabidopsis, the latter includes photomorphogenic responses and a reduction of apical dominance. These responses are known to be highly dependent on plant hormones. A first link between UV-B and plant hormones has been found from a micro-array study. This aside, evidence is poor on how UV-B and plant hormones interact and the mechanisms are far from clear.
Aim: Determine the degree and nature of hormonal dependence of the UV-B effects on plant architecture. The main Focus will be on ethylene and auxins.
Techniques and methods: Tissue culture techniques; macroscopic biometrics; Bright field, fluorescence & confocal microscopy; Reporter gene analysis (e.g. GUS, GFP); DNA and RNA analysis; (RT)-PCR; pigment extraction and spectroscopy.
78 Photomorphogenic regulation of hormone pathways during seedling development
Department: Physiology, WE15
(Co) Promoter(s) : Dominique Van Der Straeten, Filip Vandenbussche
Address + Phone number (co) promoter(s): KL Ledeganckstraat 35, Gent [email protected] 09 264 5185 [email protected] 09 264 5186
Focus : PLB
Short description of the subject: After underground germination, dicot plants obtain a skotomorphogenic growth pattern that includes the development of an apical hook in the hypocotyl. This hook protects the meristem and the cotyledons from damage when growing through soil. The establishment of the apical hook in Arabidopsis depends on the presence of the growth regulators auxin, brassinosteroids and ethylene. Emerging from soil and exposure to light induces photomorphogenic responses; the hook opens and cotyledons unfold, and the seedling starts photosynthesis. Details on hormonal interactions in this process are currently being unraveled. However, the mechanisms of the influence of light on these processes has remained largely elusive.
Aim: The aim of this project is to unravel how different wavelenghts of the solar spectrum influence hormonal processes during hook opening. We will focus on the influence of blue light on auxin and ethylene metabolism. Auxin synthesis and transport processes within the hook will be studied, using a collection of auxin biosynthesis, perception, signal transduction and transport mutants, as well as reporter fusions (both with GUS and GFP). Likewise, ethylene synthesis and signalling will be tackled.
Techniques and methods: Reporter gene analysis; bright field, fluorescence and confocal microscopy; real time RT- PCR; time lapse imaging; general plant molecular techniques
79 SUNSCREENS: Protecting plants from UV-B
Department: Physiology, WE15
(Co) Promoter(s) : Prof. Dr. Dominique Van Der Straeten, Dr. Filip Vandenbussche
Address + Phone number (co) promoter(s): K.L. Ledeganckstraat 35, Gent [email protected] 09 264 5185 [email protected] 09 264 5186
Focus : PLB
Short description of the subject: Ultraviolet B radiation is the most harmful radiation of the solar spectrum that can reach the earth’s surface. Plants have adapted to a sessile way of living by various photoprotective mechanisms. Nevertheless, both ambient and enhanced UV-B radiation can cause (undesired) changes in plant architecture and metabolism. These changes arise from photomorphogenic and stress physiological processes.
Aim: The proposed study aims at identifying compounds and pathways that can be used for regulating adverse effects of UV-B. To this end, selected natural and synthetic chemicals will be supplied to Arabidopsis seedlings and evaluated for their UV-B protective action. In addition, plants with enhanced content of anti-oxidants or vitamins (tocopherols, ascorbate, folates) or UV-B screening compounds (phenylpropanoids and flavonoids) will equally be tested for differences in their responses. Marker genes for UV-B response will also be assessed in these different mutant and transgenic backgrounds. In this way we hope to characterize the role and beneficial action of these compounds in UV-B response.
Techniques and methods: Macroscopic biometry; time lapse imaging; reporter gene analysis; bright field, fluorescence and confocal microscopy; pigment quantification; real time quantitative RT- PCR and general plant gene manipulation techniques.
80 Metabolic engineering of folate (Vit B9) biosynthesis in plants
Department: Physiology, WE15
(Co) Promoter(s): Prof. Dr. Dominique Van Der Straeten, Dr. Sergei Storozhenko
Address + Phone number (Co) Promoter(s): K. L. Ledeganckstraat 35, 9000 Gent; 092645185, 5186
Focus : PLB
Short description of the subject: Folates (vitamin B9) are crucial co-factors for enzymatic reactions that involve the transfer of one-carbon units (C1 metabolism). Folate deficiency in humans results in serious health problems such as neural tube defects (e.g. spina bifida) and megaloblastic anemia. Plant food is the main dietary source of folates for humans and animals. A number of important staple crops, particularly rice, contain extremely low folate amounts leading to chronic folate deficiency, especially in developing countries. Biofortification of rice by metabolic engineering was achieved in our group, resulting in up to 100 times higher folate levels in rice grains (Storozhenko et al., Nature Biotechnol. 25, 1277-1279, 2007). Folates are prone to (photo)oxidative damage. This instability might decrease folate levels in engineered seeds over extended storage time. Therefore, the goal of the project is to study folate stability in folate biofortified rice seeds and, eventually, carry out metabolic engineering aiming at folate stabilization. To cope with the intrinsic folate instability, plants have developed special enzymatic folate recycling mechanisms, several components of which have been recently characterized. We will transform folate biofortified rice plants with constructs overexpressing or downregulating (by RNAi) the corresponding folate recycling genes in seeds. Detailed molecular analysis of the transgenic plants will follow to investigate the effects of disturbing the folate recycling system on the folate biosynthesis gene expression, folate levels and stability in the seeds.
Aim: 1. Determination of folate stability in biofortified rice seeds; 2. Generation and analysis of transgenic rice with modified folate recycling capacity
Techniques and methods: Molecular cloning: plant transformation vector design and construction; Analysis of transgenic plants: PCR, Q-PCR, RT-PCR, Southern, northern and western blotting; Transcript profiling using microarrays and Q-PCR
81 Investigation of the involvement of the serotonin 5-HT7 receptor in Alzheimer’s disease
Dr. Kathleen Van Craenenbroeck and Prof. Dr. Guy Haegeman Department of Molecular Biology – LEGEST K.L. Ledeganckstraat 35 (11e hoogbouw) 09/264.51.35 or 09/264.51.66
Focus : BIB
Short description of the subject: Alzheimer’s disease (AD) is the most common neurodegenerative disorder of the central nervous system, characterized by progressive impairment of memory and cognition. It is the most frequent form of dementia found in the elderly. Two types of protein aggregates in the brain characterize AD: the intracellular neurofibrillary tangles (NTF), consisting of hyperphosphorylated tau, and the senile plaques, which are largely composed of the extracellular deposition of amyloid-β (Αβ ). Αβ is generated from precursor protein (APP), which belongs to a larger gene family including amyloid precursor like protein-1 and -2 (APLP1 and APLP2). A disturbed balance between several neurotransmitter systems has been implicated in the pathogenesis of AD. Amongst this, serotonin (5-HT) seems to play a pivotal role. Serotonin is an important neurotransmitter involved in diverse physiological processes such as sleep, sexual behavior, food intake, mood regulation and certain cardiovascular and gastro- intestinal functions. Serotonin binds to serotonin receptors, which belong to the family of G protein-coupled receptors (GPCRs). The most recently identified serotonin receptor is the 5- HT7 receptor. To investigate the function of the 5-HT7 receptor, we decided to search for proteins interacting with the C-terminal regions. Therefore, we performed a yeast two-hybrid screening with the C-terminal end of the human 5-HT7 receptor as a bait and an adult- human brain cDNA library as prey. This led to the identification of several interesting interaction partners, of which the human -amyloid precursor like protein 1 (APLP1) was one. Coimmunoprecipitation studies confirmed the interaction of the 5-HT7 receptor with APLP1 and also with APP and APLP2 in mammalian cells. These interactions indicate a novel link between the 5-HT7 receptor and Alzheimer’s Disease .
Aim: The aim of this project is to understand the functional role of the cross-talk between the “APP family of proteins” and the 5-HT7 receptor . We will investigate: II.1. The role of 5-HT7 receptor on the processing of the “APP family of proteins” II.2. The influence of the “APP family of proteins” on 5-HT7 receptor signaling II.3. The influence of the “APP family of proteins” on 5-HT7 receptor localization
Techniques and methods: Molecular biology techniques such as cloning, immunofluorescence Tissue culture techniques such as culturing mammalian cell lines, transfection Biochemical techniques such as coimmunoprecipitation, Western blot analysis, cAMP assay
82 Dimerization of the dopamine D4 receptor and the µ opioid receptor
Dr. Kathleen Van Craenenbroeck and Prof. Dr. Guy Haegeman Department of Molecular Biology – LEGEST K.L. Ledeganckstraat 35 (11e hoogbouw) 09/264.51.35 or 09/264.51.66
Focus : BIB
Short description of the subject: Morphine, like other drugs of abuse, activates the dopaminergic mesolimbic pathway. This pathway acts as a cerebral centre of pleasure and is stimulated by natural stimuli (such as food, drink, sex) or non-natural (substances of abuse) stimuli. A cross talk between the dopamine D4 receptor (which binds dopamine) and the µ opioid receptor (which binds morphine) has been demonstrated. Both the dopamine D4 receptor and the µ opioid receptor belong to the super-family of G protein-coupled receptors (GPCRs). All the members of this family are characterized by their seven transmembrane spanning structure with an extracellular amino-terminal and an intracellular carboxy-terminal domain. These receptors mediate the ligand-induced signal into the cell by serving as a GTP exchange factor for the heterotrimeric G protein family.
Aim: We want to investigate whether there is a direct interaction of the dopamine D4 receptor and the µ opioid receptor. II.1. Identifying oligomerization II.1.1. Previous studies show that the dopamine D4 receptor and the µ opioid receptor interact in the striatum and the substantia nigra. However it is not known whether this is a direct or an indirect interaction. The formation of heterodimers will be studied in vitro and in vivo by coimmunoprecipitation studies. II.1.2. Coimmunoprecipitation studies should be confirmed by assays in living cells. Therefore we will construct tagged receptors for FRET (fluorescence resonance energy transfer).
II.2. Functionality studies: what is the role of this interaction? II.3.1. We will investigate whether activation of the dopamine D4 receptor and/or the µ opioid receptor influences each others internalization. II.3.2. We will study the pharmacological significance of this interaction by measuring the differences in intracellular signaling (e.g. cAMP assay), when dopamine D4 receptor and/or the µ opioid receptor is stimulated.
Techniques and methods: Molecular biology techniques such as cloning, immunofluorescence,… Tissue culture techniques such as culturing mammalian cell lines, transfection,… Biochemical techniques such as coimmunoprecipitation, Western blot analysis, cAMP assay,…
83 Investigating the therapeutic advantage and molecular mechanisms of a nonsteroidal anti-inflammatory compound derived from a Namibian desert plant.
Department of Molecular Biology-LEGEST Dr. Karolien De Bosscher and Prof. Dr. Guy Haegeman K.L. Ledeganckstraat 35 9000 Gent Tel: 09/264.51.47 or 09/264.51.66
Focus: BIB
Short description of the subject: Glucocorticoids are steroidal hormones with a widespread use in the clinic, because of their efficient anti-inflammatory activity. However, side effects (diabetes, skin thinning, osteoporosis and even depression) overshadow their success in a range of chronic inflammatory diseases. Alternative ways to circumvent this problem include the use of non- steroidal compounds that are able to modulate the Glucocorticoid Receptor (GR) in a more specific way. Our lab has recently characterized a phytomodulator of GR, derived from a Namibian desert plant Salsola tuberculatiformis Botsch ., named CpdA. This compound blocks inflammation, but does not support the development of diabetes or osteoporosis. At a molecular level and in immune cells, glucocorticoids block the activity of two master transcription factors, driving cytokine expression and differentiation of T-helper (Th) cells, namely T-bet and GATA-3. The first one leads to a Th1 phenotype, whereas the latter one leads to a Th2 phenotype. Recently, also a new Th phenotype has been described, namely the Th17 phenotype. Th17-driven processes play an important role in auto-immune diseases. So far, it is not yet known, how the phytomodulator CpdA is able to influence the various T-helper cell-driven processes.
Aim: We want to investigate the molecular mechanisms by which a novel non-steroidal modulator of the Glucocorticoid Receptor is able to affect the T-helper cell differentiation processes
Techniques and methods: Cell culture techniques (cell lines and primary spleen cells), transfection, eukaryotic promoter studies and luciferase reporter gene assays, ELISA, co-immunoprecipitation and Western Blot analysis, chromatin immunoprecipitation analysis (ChIP), GFP-visualization and indirect immunofluorescence techniques.
84 Nutritional epigenetic regulation of inflammatory genes
Department: Physiology
(Co) Promoter(s): Prof.Dr. W. Vanden berghe
Address + Phone number (Co) Promoter(s): K.L. Ledeganckstraat 35, 9000 Gent [email protected] , 09/2646147
Focus : BIB, PLB, MIB
Short description of the subject: Epigenetics can be defined as all the meiotically and mitotically inherited changes in gene expression that are not encoded in the DNA sequence itself. Epigenetic modifications of chromatin and DNA have been recognized as important permissive and suppressive factors in controlling the expressed genome via gene transcription. Two major epigenetic mechanisms are the posttranslational modification of histone proteins in chromatin and the methylation of DNA itself, which are regulated by distinct, but coupled, pathways. Emerging evidence suggests a key role for epigenetics in human pathologies, including inflammatory, metabolic and neoplastic diseases. The epigenome is influenced by environmental factors throughout life. Nutritional factors (epicatechins, sulphorophanes, isoflavones, folates) can have profound effects on the expression of specific genes by epigenetic modification, and these may be passed on to subsequent generations. In this master project, we will compare the epigenetic effects of bioactive nutritional compounds with activities of histone deacetylase and dna methyltransferase inhibitors on inflammatory gene expression. The importance of the epigenome in the pathogenesis or prevention of common human diseases is likely to be as significant as that of traditional genetic mutations.
Aim: Investigate nutritional epigenomic regulation
Techniques and methods: cloning, reporter gene assays, DNA methylation assays, methylation specific QPCR, transfection, methylation sensitive isoschizomer CHART, chromatin immunoprecipation, immune-fluorescence microscopy
85 Study of crosstalk between neurotransmitter signals and the NFkB signaling cascade.
Department: Department of Physiology (WE15)
(Co) Promoter(s): Prof. Guy Haegeman, Dr. Sarah Gerlo
Address + Phone number (Co) Promoter(s): K.L. Ledeganckstraat 35, 11 th floor, 9000 Gent Prof. Haegeman: 092645166/ Dr. Gerlo: 092645135
Focus : BIB, BSB
Short description of the subject: The transcription factor NF-kB is one of the central mediators of inflammatory gene expression. NF-kB activation has been mainly studied in cells triggered with typical pro- inflammatory stimuli. However, in physiological settings cells are exposed to a multitude of environmental signals, that interact and are integrated at different levels of the signaling cascade. The G protein-coupled receptor (GPCR) superfamily is the largest class of transmembrane receptors in the human genome. GPCRs are involved in a multitude of physiological processes. Recently, GPCRs have also been implicated in inflammation and therefore represent major new targets for innovative drug discovery in the immune research field. As both GPCRs and NF-kB have been implicated in inflammation and cancer and are major targets for drug discovery, we have recently started up a new project, investigating crosstalk between GPCR-induced signaling cascades and the NF-kB pathway. The current Focus in this project is on modulation of NF-kB function by signals originating from the prototypical β2-adrenergic GPCR, that are mediated via the second messenger cAMP. We are investigating crosstalk both at the cellular level (in astrocyte, macrophage and muscle cell models) as well as at the physiological level, with special emphasis on crosstalk in the brain.
Aim: In this project we will investigate crosstalk between β2 adrenergic receptor signals and the NF kB signaling cascade.
Techniques and methods: ELISA, qPCR, (co)-immunoprecipitation (IP), western blotting, Chromatin IP, CHART-PCR, cloning, transfection, siRNA-based gene knock-down, immunohistochemistry, proteomics (mass spectrometry), in vivo rat/mouse experiments
86 Investigating the action mechanism of non-steroidal anti-inflammatory drugs
Department: WE15
(Co) Promoter(s): Prof. Dr. Guy Haegeman Dr. Linda Vermeulen
Address + Phone number (Co) Promoter(s): K. L. Ledeganckstraat 35 9000 Gent 09/264.51.66 (Prof. Haegeman) 09/264/51.35 (Dr. Vermeulen)
Focus : BIB
Short description of the subject: In this project, we will study the effect of non-steroidal anti-inflammatory drugs (like aspirin, ibuprofen,…) on the regulation of the transcriptional activity of NF-κB. NF-κB comprises a family of inducible transcription factors that serve as important regulators of immune and inflammatory responses. NF-κB deregulation is associated with immune and inflammatory diseases, as well as with cancer and some neurodegenerative diseases. The primary level of regulation of NF-κB activity is situated in the cytoplasm by liberating the factor from its inhibitor I κB, resulting in subsequent nuclear translocation and DNA binding. The transactivation capacity of NF-κB in the nucleus (that is the ability to recruit the transcriptional apparatus and stimulate target gene expression) is ensured by additional post-translational modification (i.e. phosphorylation, acetylation…) at multiple sites. We will investigate the effect of non-steroidal anti-inflammatory drugs on both the activation and post-transcriptional modification of NF-κB.
Aim: To reveal the effect of non-steroidal anti-inflammatory drugs on the transcriptional activity of NF-κB and gain more insight in the action mechanisms of these drugs.
Techniques and methods: Eukaryotic cell culture, ELISA, western blotting, RNA preparation, cDNA synthesis, real-time PCR
87 A combined computational-experimental approach to study structure-function relationships in a novel cytokine (IL-34).
Department: -Center for Molecular Modeling; Subatomic and Radiation Physics (WE05) -L-ProBE; Biochemistry and Microbiology (WE10) Promotors: -Ewald Pauwels ( [email protected] ); Center for Molecular Modeling -Savvas Savvides ( [email protected] ) ; L-ProBE Address + Ph. number: -Proeftuinstraat 86, 9000 Gent; +32 (0)9 264 65 76 -K.L. Ledeganckstraat 35, 9000 Gent; +32 (0)9 264 51 24; 0472 928 519
Focus : BSB, BIS, BIB
Cytokines mediate intracellular signaling by oligomerizing their respective receptors at the cell surface to trigger signaling cascades which contain the information necessary for correct and controlled cellular proliferation and differentiation. To facilitate such a process most cytokine receptors expose Ig-like extracellular domains, followed by single transmembrane domains, and end with cytosolic kinase domains. Colony-stimulating factor-1 (CSF-1, also known as macrophage-CSF) activates its cognate receptor CSF1R to regulate the survival, proliferation, differentiation and function of mononuclear phagocytes. Recently, a new cytokine termed IL-34 was identified as a second ligand to CSF-1R, but the biology and structure-function relationships of this cytokine remain largely unexplored. IL34 exhibits an intriguing amino acid sequence, in that it shares no sequence similarity to any other protein. Furthermore, the last 60 residues in the C-terminus of IL-34 are predicted to be inherently disordered , a feature often linked to protein-protein recognition and communication. In this project we propose an interdisciplinary research package that combines computational methods with biophysical methods under the umbrella of structural biology, to understand the molecular architecture and dynamics of this intriguing cytokine. In particular, a molecular modeling approach will be pursued to examine the conformational properties and stability of IL-34, which are ultimately the cornerstones of its function. In parallel, we will carry out analogous comparative studies on CSF1, which is the other ligand to the CSF1R. We hope to generate functional insights that can be correlated with results from other experimental avenues we are currently pursuing. Given the enormous biomedical relevance of the target cytokine-receptors complexes, our ultimate goal will be to design molecules with antagonistic/agonistic activity to facilitate classical and novel therapeutic avenues against numerous hematopoietic disorders, inflammatory disorders, and cancer.
Methods and Techniques • Computational methods: (To be carried out using the UGent Supercomputer Cluster) o Molecular dynamics (MD) o Replica Exchange Molecular Dynamics (REMD) o Ab-initio structure prediction (ROSETTA) o Protein Folding algorithms o Maximum-Likelihood structured-based sequence alignments • Biophysical studies: o Protein Folding/unfolding studies (Circular dichroism, Dynamic Light Scattering, Differential Scanning Calorimetry, Fluorescence)
88 Quantitative plant proteomics for understanding growth
Department: 1Department of Biochemistry and Microbiology 2Department of Plant Systems Biology
(Co) Promoter(s): Dr. Samy Memmi 1,2 (09/264.52.74), Prof. Bart Devreese 1 (09/264.52.73), Prof. Dirk Inzé 2 (09/331.38.06)
Address: L-ProBE Laboratory for Protein Biochemistry and Protein Engineering Ledeganckstraat 35 – 9000 Ghent VIB Department of Plant Systems Biology Technologiepark 927 – 9052 Zwijnaarde
Focus : BSB, PLB
Short description of the subject: As our world population continues to grow, the demand for more plant-derived products will also increase accordingly. It is therefore expected that biotechnological innovations will play a pivotal role in further sustaining our food and bioenergy needs, and enabling our plants to produce more biomass with a minimum input of water, fertilizers and agrochemicals. For this purpose, we need to understand how the molecular machinery integrates several processes for growth, such as photosynthesis, water and mineral uptake, and stress tolerance, in order to optimize plant productivity for future generations.
Aim: Through study of the model plant Arabidopsis thaliana we aim to identify genes and regulatory networks that define its intrinsic growth properties, and which could be used to optimize crop productivity. We already found several genes that, when ectopically expressed, form larger leaves. In this project, we intend to further characterize global gene expression in these ‘yield’ lines in order to identify which molecular processes are involved in a better growth.
Techniques and methods: Whole cellular and/or organellar protein extracts from different ‘yield’ lines will be differentially labeled in vitro or in vivo using stable isotopes and relative to a control. Subsequently, the complex protein mixtures will be analyzed using a range of HPLC fractionation techniques (SCX, RP) coupled to high resolution tandem mass spectrometry (LC-MS/MS). Integrated software platforms for processing mass spectrometric data will then allow us to identify the proteins and calculate their relative abundances. Finally, protein expression values are statistically evaluated to reveal interesting protein regulations relevant to growth.
89 Functional study of cell morphogenesis and cell wall biogenesis
Department: Biology and Plant Systems Biology
(Co) Promoter(s): Prof. Dr. Vyverman W. ([email protected]) and Prof. Dr. Inzé D. ([email protected])
Address + Phone number (Co) Promoter(s): Protistology & Aquatic ecology Krijgslaan 281/S8 B-9000 Ghent, Belgium 09 264 85 01
VIB - Ghent University 'Fiers-Schell-Van Montagu' building Technologiepark 927 B-9052 Ghent (Zwijnaarde), Belgium 09 331 38 00 Focus : PLB - MIB
Short description of the subject: The present knowledge of the molecular mechanisms regulating cell wall formation and morphogenesis in plant cells is rather limited. Given the difficulty to study these processes in plant tissues, unicellular organisms are an attractive alternative to identify key genes and their function. The desmid Micrasterias denticulata (Chlorophyta) is particularly suitable for this research thanks to its evolutionary relationship to land plants and its characteristic, well synchronizable morphogenesis. A Micrasterias cell is oval and consists of two semicells. Characteristic are the indentations in the cell wall, separating the cell body in lobes. After mitosis each semicell has to form a new semicell with the same elaborated morphology during a strictly phased process starting with the bulging of the cell plate. Next, indentations are formed, giving rise to a 3-, 5- and 9-lobed semicell before reaching maturity. Finally the primary pectinous cell wall is replaced by a secondary cellulose wall. By cDNA-AFLP analysis, a collection of genes upregulated/modulated during cell morphogenesis and cell wall formation has been built. Of interest are those transcripts presumably operating in cell pattern establishment and the synthesis of the wall polysaccharides pectin, xyloglucan, and cellulose, and those transcripts related to the physical properties of the growing cell wall. These candidate genes have to be further functionally characterized in vivo . First, full length cDNA sequences will be obtained by means of a cDNA library, using RACE-PCR and a probe hybridization technique. By means of classical cloning, those sequences will be built in into a vector in order to genetically transform Micrasterias by means of particle bombardment. On the one hand, phenotypes resulting from these overexpression studies will provide information concerning the corresponding protein. On the other hand, the GFP-fusion will allow localization of the gene product by means of confocal microscopy studies, yielding additional information about its molecular function.
Aim: Elucidating the molecular function of genes involved in cell morphogenesis and cell wall formation by means of genetic transformation.
Techniques and methods: cell culturing, PCR, RACE-PCR, cDNA-library screening, probe hybridization, plasmid preparation, classical cloning of genes, biolistic transformation, confocal microscopy.
90 Optimizing diatom cultivation for the production of high-value bioproducts and renewable energy
Department: Biology – Plant Systems Biology
Promoter: Prof. Dr. Wim Vyverman ( [email protected] ), Prof. Dr. Lieven De Veylder ( [email protected] ) Guidance:Marie Huysman ( [email protected] )
Address + Phone number (Co) Promoter(s):
Protistology & Aquatic ecology Krijgslaan 281/S8 B-9000 Ghent, Belgium 09 264 85 01
VIB - Ghent University Plant Systems Biology, Technologiepark 927, 9052 Gent Tel: 09 33 13961
Focus : PLB - MIB
Short description of the subject: Microalgae like diatoms offer unique opportunitities for the sustainable and commercial production of bioproducts like lipids, pigments, proteins and carbons for production of bioplastics, bio-energy and biofuels. However, mass cultivation techniques for diatoms are still in development and need first to be optimized in order to enter the “Blue Biotechnology” era. In this project we investigate the exact role of different interesting cell division genes in the model diatom Phaeodactylum tricornutum as well as the effect of external environmental signals on the progression of the cell cycle. New insights on the diatom cell cycle regulation will help us to further optimize the existing cultivation techniques as well as to engineer new transgenic lines with the most optimal cultivation properties for further use in the commercial production of bioproducts.
Aim: In this project we aim to functionally characterize several of the annotated diatom cell cycle genes in Phaeodactylum tricornutum and evaluate the effect of external signals (light, temperature, nutrients,…) on the cell division process.
Techniques and methods: Methods that will be used include among others epi-fluorescence microscopy, flow cytometry, quantitative real-time PCR, gateway cloning, transformation through microparticle bombardment, Western Blot and yeast-2-hybrid assays.
91 Environmental stress in intertidal biofilms: a comparative ecophysiological and molecular study in benthic diatoms
Department : Biology
Promoter : Prof. Dr. Koen Sabbe ( [email protected] ), Prof Dr Wim Vyverman ([email protected] )
Guidance:Bart Vanelslander ( [email protected] ), Marie Husyman ([email protected] )
Address + Phone number (co) promoter(s):
Protistology & Aquatic ecology Krijgslaan 281/S8 B-9000 Ghent, Belgium 09 264 85 11
Focus : MIB
Short description of the subject: Intertidal sediments are extreme environments, characterized by rapid changes and pronounced gradients in environmental conditions (e.g. irradiance and redox) as a result of the interplay of tides, weather and biology. Nevertheless, intertidal sediments belong to the most productive systems on Earth owing to primary production by microphytobenthos (MPB). Intertidal MPB mainly consists of highly diverse communities of diatoms, displaying marked changes in species and growth form composition along environmental gradients (salinity, sediment type, etc.). Two main growth forms can be distinguished. The epipelon comprises motile species (usually > 10 µm) that typically build dense biofilms. Epipsammic diatoms are smaller (< 10 µm), and are not or only slightly motile. They live attached to sand grains and dominate sandy sediments. Given the pronounced differences in growth form between both groups, it is to be expected that they will possess different, and, especially for the epipsammon, as yet largely unexplored ecophysiological and metabolic features and life strategies for dealing with life in highly changeable light and nutrient conditions
Aim: We aim at increasing our understanding of the functional adaptations of the two main MPB diatom growth forms (viz. epipelon and epipsammon) to life in intertidal sediments. To this end, we will combine ecophysiological experiments with molecular-genomic approaches to compare how epipelic and epipsammic diatoms respectively cope with variable light and nutrient conditions as key factors determining their growth performance.
Techniques and methods: Pulse Amplitude Modulated (PAM) Fluorometry, HPLC, quantitative real-time PCR
92 Dynamics of bacterial degradation in C-rich exudates of experimental phytoplankton blooms
Department: Biology
Promoter : Prof. Dr. Koen Sabbe ( [email protected] ), Prof Dr Eric Boschker (NIOO- CEME Yerseke, Nl) Guidance:Nicolas Van Oostende ( [email protected] )
Address + Phone number (co) promoter(s):
Protistology & Aquatic ecology Krijgslaan 281/S8 B-9000 Ghent, Belgium 09 264 85 11
Focus : MIB
Short description of the subject: Microalgae such as coccolithophores and diatoms are important primary producers in the world’s oceans, and as such play a crucial role in fluxes of energy and matter in these ecosystems. Nutrient limitation during massive blooms can lead to the activation of photosynthetic overflow mechanisms which result in the excretion of extracellular polymeric saccharides (EPS). Despite the fact that this pool of dissolved organic C is of the same order of magnitude as the amount of C in the atmosphere, little is as yet known about its fate. Both mineralisation and deposition plus storage in bottom sediments occur but have not yet been properly quantified. An additional complicating factor is the fact that EPS composition differs between phytoplankton groups. This affects the structure and activity of the colonizing bacterial communities, and hence polymer aggregation and sinking velocity. For example, coccolithophores mainly produce acid polymers which strongly promote EPS aggregation. In addition, bacterial successions may impact EPS degradation.
Aim: We will investigate the production and consumption of sugars in cultures of the coccolithophore Emiliana huxleyi and selected diatoms. EPS dynamics will be quantified using colorimetric methods; the sugar composition will be studied using HPLC-IRMS. The dynamics of the bacterial community will be monitored using molecular fingerprint techniques (e.g. DGGE). This will allow assessing which bacterial groups may be involved in which specific processes. The uptake and transport of the stable isotope 13 C will be followed through the microalgal compartment, the EPS fraction and the bacteria. The results will on the one hand allow to identify and quantify specific C-flux pathways, and on the other to determine the role of specific bacterial groups in the mineralisation of organic C in phytoplankton blooms.
Techniques and methods: HPLC-IRMS, DGGE, stable isotope labelling
93 Identifying organisms and determining diversity in the 21 st century: towards a molecular characterisation of marine nematode communities using microarray technology
Department: Biology Department (WE11)
(Co) Promoter(s): Dr. Sofie Derycke (promoter), Prof. Dr. Tom Moens (copromotor)
Address + Phone number (Co) Promoter(s): Marine Biology Lab, Krijgslaan 281/S8 Tel. 09 264 85 25 (SD) or 85 24 (TM)
Focus : BIS
Short description of the subject: Marine sediments contain high species diversity and high densities of microscopic organisms, and are typically dominated by nematodes. The high species diversity on a local scale and their ease of use in the lab render nematode communities ideal model systems to investigate the relationship between biodiversity and ecosystem functioning. However, this requires a correct and rapid species identification, which is highly challenging in free-living marine nematodes as many diagnostic characters are difficult to detect and may be flawed by substantial intraspecific variability. Moreover, genetic studies in three marine nematode species have revealed the presence of cryptic diversity, rendering the morphological identification of nematodes even more difficult. Some nematode species and genera can also be used as bioindicators for different kinds of disturbances, but this requires, next to correct species identification, also a rapid processing of samples, which is now highly problematic as samples need to be washed, sorted and counted, and microscope slides have to be made prior to species identification. The problems encountered for nematode communities are also well known to microbial communities, for which efficient molecular tools like microarrays have been developed. Instead of morphological identification of the organisms in the samples, total DNA is extracted and further processed to hybridise with species or genus specific molecular probes.
Aim: to develop and optimalise a microarray for the rapid detection and identification of marine nematode communities, enabling us 1/ to make a correct estimate of the local diversity of Nematoda; 2/ to provide a quick characterization of total nematode communities and 3/ to rapidly detect key species/genera.
Techniques and methods: DNA-extraction, PCR, sequencing, microarray technology, realtime PCR
94 Development van a web based system for the (graphical) interpretation and analysis of high throughput arrayCGH, haplotyping and sequencing data
Department : Center for Medical Genetics, Ghent University Hospital
(Co) Promoter(s) : Dr. ir. Björn Menten / Prof. dr. Frank Speleman
Address + Phone number (co) promoter(s): Center for Medical Genetics Ghent University Hospital De Pintelaan 185 9000 Ghent +32-(0)9 332 52 84 [email protected]
Focus : BIS, BIB
Short description of the subject: Submicroscopic deletions and duplications in the human genome can be responsible for several kinds of malignancies, as for congenital abnormalities and mental retardation in children. Array comparative genomic hybridisation (arrayCGH) is a technique developed for the detection of submicroscopial deletions and duplications in the human genome with an unprecedented resolution. The microarray technology is an emerging evolution in genetic research and allows the simultaneous interrogation of thousands to millions of nucleic acid sequences. During each experiment, vast amounts of data are generated, linked to a multitude of sequence, protein and literature information. In the past, a MySQL database was developed at the Center for Medical Genetics, linked to a PHP-based web application for the graphical visualization of all microarray data ( http://medgen.ugent.be/arrayCGHbase/ ).
Aim: The development of several modules to further explore microarray data, link it to haplotype and sequence information. The main aim is the development of tools to help in interpretation of the large amounts of data and extract the clinical significance of observed microarray aberrations.
Techniques and methods: Notions of database technology (MySQL), HTML, PHP, Perl or R knowledge are helpful, but are not necessary.
95 Evaluation of PHF6 as a tumor suppressor in hematological malignancies
Department: Center for Medical Genetics, Ghent University Hospital
(Co) Promoter(s): G. Berx and B. Poppe
Address + Phone number (Co) Promoter(s): G. Berx UGent-VIB Research Building FSVM Technologiepark 927 9052 Gent
B. Poppe Center for Medical Genetics Ghent University Hospital De Pintelaan 185 9000 Gent
Focus : BIB
Short description of the subject: We recently identified PHF6 as a novel tumor suppressor in T-cell acute lymphoblastic leukemia. In the current project, an extensive screening of PHF6 will be performed in T- ALL and other hematological malignancies. The results of the screening will be correlated to phenotypical characteristics of the neoplasms and to clinical outcome, in order to identify the clinical relevance of the presence of PHF6 mutations.
Aim: To identify the biological and clinical relevance of acquired mutations in PHF6 in hematological malignancies
Techniques and methods: karyotyping FISH arrayCGH direct sequencing high resolution melting curve analysis (HRM-CA)
96 Cross-sectional study of non-O157 STEC on beef cattle farms
Department: Veterinary Public Health and Food Safety
(Co) Promoter(s): Prof. Dr. L. De Zutter and Prof. Dr. K. Houf
Address + Phone number (Co) Promoter(s): Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke – 09 264 74 51
Focus : MIB
Short description of the subject: Shiga-toxin producing Escherichia coli have been described as the cause of severe food-related clinical cases, such as hemorrhagic colitis and hemolytic uremic syndrome. Attention is especially given to the well-known STEC O157 strains, however the non- O157 STEC strains, mainly O26, O103, O111 and O145 have been increasingly isolated from clinical cases.
Aim: This study aims to perform a cross-sectional study on selected (Sanitel Databank and previous research) cattle farms. Blood and feces will be collected and screened immunological and bacteriological. The main aim is to determine if there is a correlation between the immunological and bacteriological screening results and to gain a clear insight into the prevalence of the non-O157 STEC in beef cattle.
Techniques and methods: The feces will be enriched selectively. Immunomagnetic separation will be applied after 24 hours of incubation of the enrichment broth additional to the direct plating after 6 and 24 hours. Suspected colonies on the differential medium will be transferred to serogroup- specific confirmation media. Isolates with a suspected morphology on both media will be confirmed using PCR methods. The serum fraction will be collected from the blood samples. We will apply ELISA to detect antibodies against certain STEC virulence factors, such as intimin, EspA, etc.
97 Association of foodborne pathogens with free-living protozoa
Department: Department Veterinary Public Health and Food Safety (DI06), Faculty of Veterinary Medicine
(Co) Promoter(s): Prof. Dr. Kurt Houf (Vakgroep Veterinaire Volksgezondheid en Voedselveiligheid (DI06), Salisburylaan 133, 9820 Merelbeke ( [email protected] , tel.: 09/ 264.74.51))
Focus: MIB, BSB
Short description of the subject: Protozoa feed on bacteria, microalgae, and particulate or dissolved matter. In turn, they serve as food for other protozoa and metazoa. Besides the prey-predator relationship, there is a particular association of bacteria with protozoa, namely, survival and/or replication of a bacterium within a protozoan. First, some bacteria commonly described as obligate endosymbionts live intracellularly in the cytoplasm or the macronucleus and often cannot be cultivated outside the protozoan host. Second, in the last two decades, more attention has been paid to bacteria which were not expected to have an intracellular protozoan life cycle. Internalization of human pathogens ( Helicobacter pylori , Mycobacterium bovis ) and foodborne pathogens ( Campylobacter jejuni , Escherichia coli O157:H7, Listeria monocytogenes , Salmonella , Staphylococcus aureus ) in protozoa such as Acanthamoeba castellanii , Acanthamoeba polyphaga , Acanthamoeba rhysodes , and Tetrahymena pyriformis has been demonstrated. The mechanism of the bacterium- protozoan interaction has been intensively studied for Legionella pneumophila . The facultative intracellular lifestyle of bacterial (foodborne) pathogens in protozoa is of special concern for several reasons: (i) some protozoa, such as A. castellanii , A. polyphaga , Glaucoma sp., and Tetrahymena spp., produce small vesicles which can contain living bacteria and these vesicles might be inhaled or can contaminate the environment; (ii) bacteria surviving within protozoa or protozoan cysts resist unfavorable conditions, such as desiccation and exposure to disinfectants; and (iii) an increase in antimicrobial resistance and virulence of bacterial pathogens after passage through protozoa has been demonstrated. The association of foodborne pathogens with free- living protozoa is of particular interest because it might explain how, besides well-known strategies such as biofilm formation, some foodborne pathogens persist in food- processing areas despite daily cleaning and disinfection.
Aim: The specific objective is to determine whether free-living protozoa internalize viable cells and thereby affect survival of foodborne pathogens in the environment.
Techniques and methods: Co-cultures of protozoa (reference strains of culture collections and environmental isolates) and GFP-labeled pathogens will be used to determine whether internalization takes place. In a further phase, the role of virulence genes will be investigated.
98 Isolation, identification and characterization of Yersinia enterocolitica from Belgian slaughter pigs
Department: Veterinary Public Health and Food Safety
(Co) Promoter(s): Prof. Dr. L. De Zutter and Prof. Dr. K. Houf
Address + Phone number (Co) Promoter(s): Faculty of Veterinary Medicine, Salisburylaan 133, 9820 Merelbeke – 09 264 74 51
Focus : MIB
Short description of the subject: Yersinia enterocolitica is a foodborne pathogen that causes various clinical symptoms in humans, especially in young children. Pigs are probably the main reservoir of this pathogen since pigs are the only animals in which pathogenic Y. enterocolitica are frequently detected. Case-control studies indicate that humans are infected by the consumption of contaminated pork. However, the epidemiology of pathogenic Y. enterocolitica is still not fully elucidated and data about the presence of Y. enterocolitica in Flemish pigs are limited.
Aim: The aim of the study is to gain better insight in the epidemiology of pathogenic Y. enterocolitica in the pigs and their role of a infection reservoir to humans. The distribution of genetic types in different pig organs and on pig carcasses will be determined and possible contamination sources of pig carcasses will be identified. Furthermore, isolates from pigs will be further compared to human isolates.
Techniques and methods: The presence of Y. enterocolitica will be monitored in different pig organs and carcasses at slaughter. Isolates will be identified and further characterized below species level by molecular based techniques (PCR-based and PFGE). Moreover, human isolates obtained from clinical cases of yersiniosis during the same period will be genotyped using PFGE and compared to the pig isolates.
99 Localisation of the emerging pathogen Arcobacter in the intestinal tract of pigs and their interaction with intestinal epithelial cells in vitro
Department: Department of Veterinary Public Health and Food Safety (DI06)
(Co) Promoter(s): Prof. Dr. Kurt Houf
Address + Phone number (Co) Promoter(s): Faculty of veterinary Medicine, Salisburylaan 133, 9820 Merelbeke; tel: 09.264.74.51; email: [email protected])
Focus : MIB, BIB
Short description of the subject: Arcobacters, formerly known as ‘aerotolerant campylobacters’, differ from the closely related campylobacters by their ability to grow at temperatures below 30°C and their aerotolerance. To date, Arcobacter spp. have been detected in animals, food products of animal origin as well as in surface water. Contaminated drinking water has been identified as the major infection source for humans in developing countries, though the manipulation, consumption and cross-contamination of raw and undercooked meat products are more likely infection routes in industrialized countries. Clinical symptoms are similar to campylobacteriosis, but a higher frequency of persistent and watery diarrhea has been reported. Epidemiologic studies revealed that pigs are an important reservoir for the emerging pathogen Arcobacter. Their frequent presence in healthy pigs may lead faecal contamination of porcine carcasses during slaughter and pork and transfer of arcobacters in the environment which can become a significant problem with respect to public health. Although to this date some virulence factors in arcobacters have been identified, the mechanisms of their pathogenicity and potential virulence factors are however not elucidated yet.
Aim: The study aims to localize Arcobacter spp. in the intestinal tract of pigs (duodenum, jejunum, ileum, cecum, colon, rectum) and to study their interaction on the intestinal tissues. Subsequently, the interaction (adhesion, invasion, translocation, survival, cytotoxic effect) of Arcobacter isolates and porcine intestinal epithelial cells will be investigated.
Techniques and methods: Arcobacters will be isolated from the pigs’ intestinal tract and further analyzed by molecular based methods (identification, characterization, presence of virulence factors, …). The interaction with the intestinal tissues will be studied (immuno)histologically and adhesion and invasion will be tested on primary cell lines.
MASTERPROEVEN 2 de Master BIOTECHNOLOGIE EN BIOCHEMIE 2009-2010
Nr FOCUS TITEL PROMOTOR / BEGELEIDING
Onderwerpen aangeboden door de vakgroep Plantenbiotechnologie en Genetica (WE09)
1 PLB Molecular analysis of PIN exocytosis Prof. J. Friml, Dr. S. Vanneste
2 PLB, BIS Oxidative stress signal transduction in plants Prof. F. Van Breusegem
3 PLB, BSB Functional evaluation of Arabidopsis metacaspases targets Prof. F. Van Breusegem PLB, BSB, 4 Molecular function of plant Elongator Dr. M. Van Lijsebettens BIS PLB, BSB, Dr. M. Van Lijsebettens, Dr. K. 5 Molecular network of histone monoubiquitination1 and chromatin in transcriptional regulation BIS Himanen Functional analysis of maize growth regulatory genes under limiting environmental 6 PLB Prof. D. Inzé, Prof. G. Beemster conditions Dr. K. Vandepoele, Prof. Y. Van 7 BIS, PLB Detection and evolution of expression modules in eukaryotic species de Peer 8 PLB Identitification and characterization of bioactive chemicals that alter PIN polar localization Prof. J. Friml, Dr. S. Robert Identification of differentially expressed genes underlying growth response variation in 9 BIS Dr. M. Vuylsteke pepper 10 PLB Mapping the Seminavis robusta MT locus Dr. M. Vuylsteke
11 BSB, PLB Biosynsthesis of novel plant-derived molecules with pharmaceutical activity Dr. A. Goossens, Prof. D. Inzé
12 PLB Engineering of jasmonate signaling to improve plant growth and defense Dr. A. Goossens, Prof. D. Inzé Prof. L. De Veylder, Dr. T. 13 PLB Identification of novel endocycle regulators through the use of chemical genomics Lammens 14 PLB Identification and characterization of novel DNA stress-inducing genes Prof. L. De Veylder 1 Identification and analysis of new genes involved in division plane determination in plant Prof. G. De Jaeger, Dr. D. Van 15 PLB, BSB cells Damme 16 PLB Tracking the endocytic routes of brassinosteroid receptor complex in Arabidopsis Dr. J. Russinova Prof. S. Goormachtig, Prof. M. 17 PLB, MIB MtCLE peptide receptors in Medicago truncatula Holsters Prof. S. Goormachtig, Prof. M. 18 PLB, MIB Nodule meristem development in Medicago truncatula Holsters Prof. D. Inzé, Prof. Y. Van de 19 BIS Data mining and integration to uncover the molecular mechanisms underlying growth Peer, Dr. S. De Bodt 20 PLB Systems biology of drought tolerance in Arabidopsis Prof. D. Inzé, Dr. A. Skirycs
21 PLB Designing plant cell walls for a better conversion to biofuels Prof. W. Boerjan, Dr. R. Vanholme Dr. J. Russinova, Dr. M. 22 PLB Role of the multifunctional GSK-3 in plant cell division Zhiponova Functional characterization of genes involved in lateral root initiation in maize and 23 PLB Dr. T. Beeckman, Dr. B. Parizot Arabidopsis thaliana Unraveling the function of trehalose biosynthesis genes in root development and stress 24 PLB Dr. T. Beeckman tolerance Prof. A. Depicker, Dr. S. De Buck, 25 PLB, BSB Characterization of camel antibodies produced in transgenic Arabidopsis seeds Dr. A. De Paepe Prof. A. Depicker, Dr. S. De Buck, 26 PLB, BSB Evaluation of in planta produced camel antibodies as proteomic tools Dr. A. De Paepe 27 PLB The molecular basis of plant yield Prof. D. Inzé, Dr. N. Gonzalez Dr. P. Rouzé, Prof. Y. Van de 28 BIS Study of the introner-elements found in the Micromonas genomes Peer Looking deeper in genome annotation of the Chromosome-2 in three different Ostreococci Dr. P. Rouzé, Prof. Y. Van de 29 BIS (green alga) genomes Peer Dr. S. Rombauts, Prof. Y. Van de 30 BIS Functional annotation of fungal genomes Peer Dr. P. Rouzé, Prof. Y. Van de 31 BIS Looking for the origin of chromosomes 18/19 of the green alga Ostreococcus Peer
1 2 32 PLB, BIS Characterization of the GOLVEN peptide binding sites Dr. ir. P. Hilson
33 PLB, BIS Peptide signaling in plant roots Dr. ir. P. Hilson
34 PLB Hormonal interactions shaping root system architecture Dr. E. Benkova
Onderwerpen aangeboden door de vakgroep Biochemie en Microbiologie (WE10)
The involvement of persistence in multidrug tolerance and in biofilm formation of 35 BSB, MIB Prof. B. Devreese, Dr. L. De Smet Shewanella oneidensis A novel mass spectrometric tool for determining protein:protein and protein:ligand 36 BSB Prof. B. Devreese interactions 37 BSB, MIB Phosphoproteomic analysis of bacterial resistance to antibiotics and biofilm formation Prof. B. Devreese Dr. B. Vergauwen, Prof. B. 38 BSB Design of genetically encoded glutathione disulfide biosensors Devreese Development and automation of a new approach for selective enrichment of N-terminal 39 BSB Dr. B. Samyn, Prof. B. Devreese peptides from complex proteomic samples BIB, BSB, A Mycobacterium bovis BCG transposon insertion library as a resource for the study of the Prof. N. Callewaert, Dr. N. 40 MIB role of mycobacterial glycolipids in pathogenesis Festjens Dissection of the interaction between cytokines and the extracellular domains of their 41 BSB, BIB Prof. S. Savvides, Dr. J. Elegheert cognate receptors Structural biology of the malaria parasite Plasmodium falciparum : The intriguing interaction Prof. S. Savvides, Dr. K. 42 BSB, BIB of Adenylate Kinase with N-Myristoyl Transferase Moharana BSB, BIB, Investigating the role of atypical glutaredoxins in S-(de)glutathionylation in cellular oxidative Prof. S. Savvides, Dr. B. 43 MIB stress Vergauwen, Dr. G. Buysschaert BSB, BIB, Prof. S. Savvides, Dr. Ruben Van 44 Dissection of the Molecular basis of bacterial type II secretion in pathogenic bacteria MIB der Meeren Prof. A. Willems, Dr. M. 45 MIB, BIB Microbiological quality of fish and fishery products Heyndrickx, Dr. ir. G. Vlaemynck Prof. A. Willems, Dr. M. 46 MIB Molecular identification of spoilage microflora on common shrimp (Crangon crangon) Heyndrickx, Dr. ir. G. Vlaemynck Community AFLP as a new tool for the characterization of human intestinal tract microbiota Dr. G. Huys, Prof. Dr. P. 47 MIB, BIB in health and disease Vandamme
2 3 Development of a diagnostic tool for identification and detection of plantpathogenic 48 MIB Prof. P. De Vos clavibacter species 49 MIB Assessment of novel primer(s) for the amplification of nor and nir genes Prof. P. De Vos Prof. P. De Vos, Prof. Dr. P. 50 MIB Cultivation and identification of fast growing methane oxidizers Vandamme Prof. P. Vandamme, Dr. M. 51 MIB MRSA (methicillin resistant Staphylococcus aureus ) on Belgian pig farms Heyndrickx, Dr. G. Rasschaert Development of matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) Prof. P. Vandamme, Prof. Dr. A. 52 MIB, BSB mass spectrometry for the study of spoilage microorganisms in Belgian artisan Beers Van Landschoot 53 MIB, BIB Study of the predominant colon microbiota of cystic fibrosis patients Prof. P. Vandamme Linking the microbial diversity of traditional fermented foods to their artisan-type Prof. P. Vandamme, Prof. L. De 54 MIB characteristics Vuyst, Prof. Dr. F. Leroy Structure-function analyses and specificity studies of glycoside hydrolase components of the Prof. N. Callewaert, Dr. K. Piens, 55 BSB Trichoderma reesei genome Dr. P. Ntarima Onderwerpen aangeboden door de vakgroep Biomedische Moleculaire Biologie (WE14)
Identification and characterization of MALT1 paracaspase interacting proteins and 56 BIB, BSB Prof. R. Beyaert substrates relevant in autoimmunity and cancer Validation of NF-kB signaling proteins as potential therapeutic targets in human disease 57* BIB Prof. R. Beyaert, Dr. G. van Loo using genetically modified mice Prof. P. Brouckaert, Dr. Anje 58 BIB Molecular mechanisms of inflammatory shock Cauwels Prof. X. Saelens, Dr. Marina De 59* MIB, BIB Identifying mammalian pathogenecity genes from influenza A virus Filette Dr. K. Vandepoele, Prof. Dr. F. 60 BIB Analysis of the fuction and regulation of the delta-protocadherins Van Roy 61 BIB Role of alpha-catenin proteins in the mouse Dr. J. van Hengel
62 BIB Characterization of primary target genes of the canonical Wnt pathway in Xenopus embryos Prof. K. Vleminckx Prof. C. Libert, Dr. R. 63 BIB Studying the role of matrix metalloproteinases (MMPs) in endotoxemia and immunity Vandenbroucke
3 4 Apoptosis, necrosis and autophagy: a comparative study of the impact dying cells on innate Prof. P. Vandenabeele, Dr. Dmitri 64* BIB immunity Krysko Prof. P. Vandenabeele, Dr. F. Van 65 BIB Role of phosphorylation of RIP1K in cell death and NF-kB activation Herreweghe Prof. Dr. J. Grooten, Dr. S. De 66 BIB Polyelectrolyte microcapsules: multifunctional antigen delivery platforms ? Koker 67 BIB Deregulation of inflammatory and angiogenic signaling in human colon tumors Prof. J. Grooten
68 BIB Functional characterization of alveolar macrophages in a mouse model of allergic asthma Prof. J. Grooten
69 BIB Study of EMT during malignant progression of human breast cancer Dr. G. Berx BSB, BIB, Heterologous expression of G protein-coupled receptors in yeast adapted to the expression Prof. Dr. N. Callewaert, Dr. M. 70 MIB of eukaryotic membrane proteins Guerfal BIB, BSB, Prof. Dr. N. Callewaert, Dr. P. 71 Production of lysosomal enzymes with human-like glycosylation in yeast MIB Tiels Analysing the glycoprofile of low abundant serum proteins: a step towards more specific Prof. Dr. N. Callewaert, Dr. P. 72 BSB, BIB cancer diagnostics Ameloot 73* BIB Role of VEGF and EMT modulators in vascular and hematopoietic cell differentiation Dr. Jody Haigh, Dr. S. Goossens Prof. Dr. Wim Declercq, Dr. 74 BIB Analysis of the function of RIP4 in skin differentiation Saskia Lippens BIS, BSB, Dr. P. De Bleser, Prof. Dr. F. Van 75 In silico prediction of functional microRNA targets BIB, MIB, PLB Roy Onderwerpen aangeboden door de vakgroep Fysiologie (WE15)
Prof. D. Van Der Straeten, Dr. F. 76 PLB Tissue specificity of hormonal responses Vandenbussche Global change effects on plant growth: a molecular-physiological study of hormonal Prof. D. Van Der Straeten, Dr. F. 77 PLB dependence of UV-B responses Vandenbussche Prof. D. Van Der Straeten, Dr. F. 78 PLB Photomorphogenic regulation of hormone pathways during seedling development Vandenbussche Prof. D. Van Der Straeten, Dr. F. 79 PLB Sunscreens: Protecting plants form UV-B Vandenbussche
4 5 Prof. D. Van Der Straeten, Dr. S. 80 PLB Metabolic engineering of folate (Vit B9) biosynthesis in plants Storozhenko Prof. G. Haegeman, Dr. K. Van 81 BIB Investigation of the involvement of the serotonin 5-HT7 receptor in Alzheimer’s disease Craenenbrouck Prof. G. Haegeman, Dr. K. Van 82 BIB Dimerization of the dopamine D4 receptor and the µ opioid receptor Craenenbrouck Investigating the therapeutic advantage and molecular mechanisms of a nonsteriodal anti- Prof. G. Haegeman, Dr. K. De 83 BIB inflammatory compound derived from a Namibian desert plant Bosscher 84 BIB, PLB, MIB Nutritional epigenetic regulation of inflammatory genes Prof. W. Vanden Berghe
85 BIB, BSB Study of crosstalk between neurotransmitter signals and the NFkB signaling cascade. Prof. G. Haegeman, Dr. S. Gerlo Prof. Dr. G. Haegeman, Dr. L. 86 BIB Investigating the action mechanisms of non-steroidal anti-inflammatory drugs Vermeulen
Onderwerpen aangeboden door 2 vakgroepen: WE05 en WE10
A combined computational-experimental approach to study structure-function relationships Prof. E. Pauwels, Prof. Dr. S. 87 BSB, BIS, BIB in a novel cytokine (IL-34) Savvides
Onderwerpen aangeboden door 2 vakgroepen: WE09 en WE10 Dr. S. Memmi, Prof. B. Devreese, 88 BSB, PLB Quantitative plant proteomics for understanding growth Prof. D. Inzé
Onderwerpen aangeboden door 2 vakgroepen: WE11 en WE09
89 PLB, MIB Functional study of cell morphogenesis and cell wall biogenesis Prof. W. Vyverman, Prof. D. Inzé Optimizing diatom cultivation for the production of high-value bioproducts and renewable Prof. W. Vyverman, Prof. L. De 90 PLB, MIB energy Veylder
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Onderwerpen aangeboden door andere vakgroepen: WE11 Environmental stress in intertidal biofilms: a comparative ecophysiological and molecular Prof. K. Sabbe, Prof. W. 91 MIB study of benthic diatoms Vyverman Prof. K. Sabbe, Nicolas Van 92 MIB Dynamics of bacterial degradation in C-rich exudates of experimental phytoplankton blooms Oostende Identifying organisms and determining diversity in the 21 st century: towards a molecular 93 BIS Dr. S. Derycke, Prof. Dr. T. Moens characterization of marine nematode communities using microarray technology Onderwerpen aangeboden door andere vakgroepen: vakgroep Pediatrie en Genetica (GE02) Development of (graphical) web applications for the processing and interpretation of Dr. ir. B. Menten, Prof. Dr. F. 94 BIS, BIB arrayCGH data Speleman 95 BIB Evaluation of PHF6 as a tumor suppressor in hematological malignancies Prof. Dr. B. Poppe, Dr. G. Berx
Onderwerpen aangeboden door andere vakgroepen: vakgroep Veterinaire Volksgezondheid en Voedselveiligheid (DI06) Prof. L. De Zutter, Prof. Dr. K. 96 MIB Cross-sectional study of non-O157 STEC on beef cattle farms Houf 97 MIB, BSB Association of foodborne pathogens with free-living protozoa Prof. K. Houf Isolation, identification and characterization of Yersinia enterocolitica from Belgian slaughter Prof. L. De Zutter, Prof. Dr. K. 98 MIB pigs Houf Localisation of the emerging pathogen Arcobacter in the intestinal tract of pigs and their 99 MIB, BIB Prof. K. Houf interaction with intestinal epithelial cells in vitro
* Volgen van de cursus Proefdierkunde is verplicht Gelieve uw keuze (1 tem 5) kenbaar te maken via de website van de opleiding Biochemie en Biotechnologie voor 6 juli 2009 18:00.
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