DOCSLIB.ORG

Masterarbeit

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Masterarbeit MASTERARBEIT Titel der Masterarbeit A zebrafish model of Letm1 and Letm2 deficiency verfasst von Stefan Hajny, BSc angestrebter akademischer Grad Master of Science (MSc) Wien, 2015 Studienkennzahl lt. Studienblatt: A 066 834 Studienrichtung lt. Studienblatt: Masterstudium Molekulare Biologie (UG2002) Betreut von: Priv. Doz. Dr. Karin Novikovsky Table of contents Acknowledgements ............................................................................................................... I Abbreviations ...................................................................................................................... II 1. Introduction .................................................................................................................... 1 1.1. Wolf Hirschhorn Syndrome .............................................................................................................................. 1 1.2. LETM1, an important protein for mitochondrial potassium homeostasis ....................................................... 1 1.3. The KHE is an indispensable complex to maintain mitochondrial volume and pH homeostasis ..................... 3 1.4. The elusive role of LETM2, a protein closely related to LETM1 ....................................................................... 5 1.5. The zebrafish as a model organism .................................................................................................................. 6 1.6. Genomic engineering approaches ................................................................................................................... 7 1.6.1 TALEN ......................................................................................................................................................... 7 1.6.2 The CRISPR/Cas9 system ............................................................................................................................ 9 1.6.3 Multisite Gateway Cloning ....................................................................................................................... 10 1.7. Studying different aspects of LETM1 and LETM2 in vivo and in vitro ............................................................ 13 2. Material and Methods .................................................................................................... 14 2.1. Animal handling ............................................................................................................................................. 14 2.1.1 Husbandry ................................................................................................................................................ 14 2.1.2 Breeding and nutrition ............................................................................................................................. 14 2.1.3 Microinjections......................................................................................................................................... 14 2.1.4 Genotyping ............................................................................................................................................... 15 2.1.5 Euthanasia ............................................................................................................................................... 16 2.2. General procedures ....................................................................................................................................... 17 2.2.1 Restriction digest ..................................................................................................................................... 17 2.2.2 Agarose gel electrophoresis ..................................................................................................................... 17 2.2.3 Agarose gel extraction ............................................................................................................................. 17 2.2.4 Bacterial transformation and Inoculation ................................................................................................ 17 2.2.5 DNA Isolation ........................................................................................................................................... 18 2.2.6 RNA isolation and cDNA synthesis ........................................................................................................... 19 2.2.7 PCR Protocols and primer ........................................................................................................................ 20 2.2.8 Sequencing ............................................................................................................................................... 22 2.3. Genetic modifications .................................................................................................................................... 22 2.3.1 Gateway Cloning ...................................................................................................................................... 22 2.3.2 Generation of zebrafish, overexpressing letm1 or letm2 ......................................................................... 26 2.3.3 TALEN induced knock-out ........................................................................................................................ 26 2.3.4 TALEN mediated knock-in ........................................................................................................................ 31 2.3.5 CRISPR/Cas9 induced knock-out .............................................................................................................. 32 2.4. In situ hybridization ....................................................................................................................................... 36 2.4.1 Preparation of probes .............................................................................................................................. 36 2.4.2 Whole mount in situ hybridization ........................................................................................................... 37 2.4.3 Brain sections ........................................................................................................................................... 39 2.5. Quantitative gene expression analysis ........................................................................................................... 40 2.5.1 qPCR analysis ........................................................................................................................................... 40 2.6. Localization studies in HeLa cells ................................................................................................................... 41 2.7. Locomotor behavior ....................................................................................................................................... 41 2.8. Bioinformatical approaches and data evaluation .......................................................................................... 42 2.8.1 Phylogenetic analysis ............................................................................................................................... 42 2.8.2 Software ................................................................................................................................................... 44 2.9. Utilized buffers and commercial kits.............................................................................................................. 45 3. Results ........................................................................................................................... 48 3.1. The gene duplication of LETM1 generated an evolutionary conserved protein family ................................. 48 3.2. Letm1 and letm2 are ubiquitously expressed in larval and adult zebrafish tissues ....................................... 52 3.2.1 Zebrafish larvae display a ubiquitous letm1 and letm2 distribution ........................................................ 52 3.2.2 Letm1 and letm2 are ubiquitously expressed in the adult zebrafish brain .............................................. 53 3.2.3 Letm1 and letm2 are ubiquitously expressed in zebrafish and human cells ............................................ 54 3.3. Letm1 and Letm2 are highly conserved, mitochondrial associated proteins ................................................ 57 3.4. Establishment of a vital, haploinsufficient letm1 zebrafish line .................................................................... 59 3.5. Generation of a CRISPR/Cas9 mediated letm2 knock-out strain yield first viable larvae .............................. 60 3.6. Letm1 haploinsufficient mutants reveal an unchanged locomotor behavior ................................................ 61 4. Discussion ...................................................................................................................... 65 4.1. LETM1 and LETM2 are highly conserved proteins throughout the eukaryotic kingdom ............................... 65 4.2. The ubiquitously abundant LETM proteins may play a crucial role during development ............................. 66 4.3. Establishment of novel letm1 and letm2 zebrafish lines ............................................................................... 68 4.3.1 The generated letm1 haploinsufficient zebrafish strain will serve as an indispensable model to investigate several aspects of the mitochondrial protein .......................................................................
Load more

Recommended publications
  • Mouse Letmd1 Conditional Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Letmd1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Letmd1 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Letmd1 gene (NCBI Reference Sequence: NM_134093 ; Ensembl: ENSMUSG00000037353 ) is located on Mouse chromosome 15. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000037001). Exon 5~7 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Letmd1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-81M8 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 5 starts from about 43.89% of the coding region. The knockout of Exon 5~7 will result in frameshift of the gene. The size of intron 4 for 5'-loxP site insertion: 2391 bp, and the size of intron 7 for 3'-loxP site insertion: 1135 bp. The size of effective cKO region: ~1132 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 5 6 7 8 9 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Letmd1 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • View Preprint
    A peer-reviewed version of this preprint was published in PeerJ on 6 August 2019. View the peer-reviewed version (peerj.com/articles/7309), which is the preferred citable publication unless you specifically need to cite this preprint. Liu H, Peng Y, Lee H. 2019. miRDRN—miRNA disease regulatory network: a tool for exploring disease and tissue-specific microRNA regulatory networks. PeerJ 7:e7309 https://doi.org/10.7717/peerj.7309 miRDRN – miRNA Disease Regulatory Network: A tool for exploring disease and tissue-specific microRNA regulatory networks Hsueh-Chuan Liu 1 , Yi-Shian Peng 1 , Hoong-Chien Lee Corresp. 1 1 Department of Biomedical Sciences and Engineering, National Central University, Taoyuan City, Taiwan Corresponding Author: Hoong-Chien Lee Email address: [email protected] Background. MiRNA regulates cellular processes through acting on specific target genes. Hundreds of miRNAs and their target genes have been identified, as are many miRNA- disease associations. Cellular processes, including those related to disease, proceed through multiple interactions, are often organized into pathways among genes and gene products. Large databases on protein-protein interactions (PPIs) are available. Here, we have integrated the information mentioned above to build a web service platform, miRNA Disease Regulatory Network, or miRDRN, for users to construct disease and tissue-specific miRNA-protein regulatory networks. Methods. Data on human protein interaction, disease-associated miRNA, tumor-associated gene, miRNA targeted gene, molecular interaction and reaction network or pathway, gene ontology, gene annotation and gene product information, and gene expression were collected from publicly available databases and integrated. A complete set of regulatory sub-pathways (RSPs) having the form (M, T, G1, G2) were built from the integrated data and stored in the database part of miRDRN, where M is a disease-associated miRNA, T is its regulatory target gene, G1 (G2) is a gene/protein interacting with T (G1).
    [Show full text]
  • Transdifferentiation of Human Mesenchymal Stem Cells
    Transdifferentiation of Human Mesenchymal Stem Cells Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Tatjana Schilling aus San Miguel de Tucuman, Argentinien Würzburg, 2007 Eingereicht am: Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Martin J. Müller Gutachter: PD Dr. Norbert Schütze Gutachter: Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbstständig angefertigt und keine anderen als die von mir angegebenen Hilfsmittel und Quellen verwendet habe. Des Weiteren erkläre ich, dass diese Arbeit weder in gleicher noch in ähnlicher Form in einem Prüfungsverfahren vorgelegen hat und ich noch keinen Promotionsversuch unternommen habe. Gerbrunn, 4. Mai 2007 Tatjana Schilling Table of contents i Table of contents 1 Summary ........................................................................................................................ 1 1.1 Summary.................................................................................................................... 1 1.2 Zusammenfassung..................................................................................................... 2 2 Introduction.................................................................................................................... 4 2.1 Osteoporosis and the fatty degeneration of the bone marrow..................................... 4 2.2 Adipose and bone
    [Show full text]
  • Transcriptional Regulation of Neurodevelopmental and Metabolic Pathways by NPAS3
    Molecular Psychiatry (2012) 17, 267–279 & 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12 www.nature.com/mp ORIGINAL ARTICLE Transcriptional regulation of neurodevelopmental and metabolic pathways by NPAS3 L Sha1,7, L MacIntyre2,7, JA Machell1, MP Kelly3, DJ Porteous1, NJ Brandon3, WJ Muir4,{, DH Blackwood4, DG Watson2, SJ Clapcote5 and BS Pickard2,6 1Department of Medical Genetics, Institute for Genetics and Molecular Medicine, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Edinburgh, UK; 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK; 3Pfizer, Pfizer Global Research and Development, Neuroscience Research Unit, Groton, CT, USA; 4Division of Psychiatry, University of Edinburgh, Royal Edinburgh Hospital, Edinburgh, UK; 5Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK and 6CeNsUS—Centre for Neuroscience, University of Strathclyde, Glasgow, UK The basic helix-loop-helix PAS (Per, Arnt, Sim) domain transcription factor gene NPAS3 is a replicated genetic risk factor for psychiatric disorders. A knockout (KO) mouse model exhibits behavioral and adult neurogenesis deficits consistent with human illness. To define the location and mechanism of NPAS3 etiopathology, we combined immunofluorescent, transcriptomic and metabonomic approaches. Intense Npas3 immunoreactivity was observed in the hippocampal subgranular zone—the site of adult neurogenesis—but was restricted to maturing, rather than proliferating, neuronal precursor cells. Microarray analysis of a HEK293 cell line over-expressing NPAS3 showed that transcriptional targets varied according to circadian rhythm context and C-terminal deletion. The most highly up-regulated NPAS3 target gene, VGF, encodes secretory peptides with established roles in neurogenesis, depression and schizophrenia. VGF was just one of many NPAS3 target genes also regulated by the SOX family of transcription factors, suggesting an overlap in neurodevelopmental function.
    [Show full text]
  • Supplemental Data
    Supplementary Table 1. Gene sets from Figure 6. Lists of genes from each individual gene set defined in Figure 6, including the fold-change in expression of each gene in treatment group pair-wise comparisons. ENSEMBL: Ensembl gene identifier; Symbol: official gene symbol; logFC: log fold change; p value: significance of fold-change in a pair-wise comparison, P<0.05 cut-off; FDR: false discovery rate, expected proportion of false positives among the differentially expressed genes in a pair-wise comparison (FDR<0.25 cut-off). Sup. Table 1 SET I CP versus Sal CP versus CP+DCA DCA versus Sal ENSEMBL Symbol logFC PValue FDR logFC PValue FDR logFC PValue FDR Desc ENSMUSG00000020326 Ccng1 2.64 0.00 0.00 -0.06 0.13 0.96 0.40 0.00 0.23 cyclin G1 [Source:MGI Symbol;Acc:MGI:102890] ENSMUSG00000031886 Ces2e 3.97 0.00 0.00 -0.24 0.02 0.28 0.01 1.00 1.00 carboxylesterase 2E [Source:MGI Symbol;Acc:MGI:2443170] ENSMUSG00000041959 S100a10 2.31 0.00 0.00 -0.21 0.02 0.23 -0.11 0.53 1.00 S100 calcium binding protein A10 (calpactin) [Source:MGI Symbol;Acc:MGI:1339468] ENSMUSG00000092341 Malat1 1.09 0.00 0.00 -0.11 0.20 1.00 0.66 0.00 0.00 metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA) [Source:MGI Symbol;Acc:MGI:1919539] ENSMUSG00000072949 Acot1 1.73 0.00 0.00 -0.22 0.01 0.12 -0.44 0.01 1.00 acyl-CoA thioesterase 1 [Source:MGI Symbol;Acc:MGI:1349396] ENSMUSG00000064339 mt-Rnr2 1.09 0.00 0.00 -0.08 0.17 1.00 0.67 0.00 0.07 mitochondrially encoded 16S rRNA [Source:MGI Symbol;Acc:MGI:102492] ENSMUSG00000025934 Gsta3 1.86 0.00 0.00 -0.28
    [Show full text]
  • Table S1. 103 Ferroptosis-Related Genes Retrieved from the Genecards
    Table S1. 103 ferroptosis-related genes retrieved from the GeneCards. Gene Symbol Description Category GPX4 Glutathione Peroxidase 4 Protein Coding AIFM2 Apoptosis Inducing Factor Mitochondria Associated 2 Protein Coding TP53 Tumor Protein P53 Protein Coding ACSL4 Acyl-CoA Synthetase Long Chain Family Member 4 Protein Coding SLC7A11 Solute Carrier Family 7 Member 11 Protein Coding VDAC2 Voltage Dependent Anion Channel 2 Protein Coding VDAC3 Voltage Dependent Anion Channel 3 Protein Coding ATG5 Autophagy Related 5 Protein Coding ATG7 Autophagy Related 7 Protein Coding NCOA4 Nuclear Receptor Coactivator 4 Protein Coding HMOX1 Heme Oxygenase 1 Protein Coding SLC3A2 Solute Carrier Family 3 Member 2 Protein Coding ALOX15 Arachidonate 15-Lipoxygenase Protein Coding BECN1 Beclin 1 Protein Coding PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 Protein Coding SAT1 Spermidine/Spermine N1-Acetyltransferase 1 Protein Coding NF2 Neurofibromin 2 Protein Coding YAP1 Yes1 Associated Transcriptional Regulator Protein Coding FTH1 Ferritin Heavy Chain 1 Protein Coding TF Transferrin Protein Coding TFRC Transferrin Receptor Protein Coding FTL Ferritin Light Chain Protein Coding CYBB Cytochrome B-245 Beta Chain Protein Coding GSS Glutathione Synthetase Protein Coding CP Ceruloplasmin Protein Coding PRNP Prion Protein Protein Coding SLC11A2 Solute Carrier Family 11 Member 2 Protein Coding SLC40A1 Solute Carrier Family 40 Member 1 Protein Coding STEAP3 STEAP3 Metalloreductase Protein Coding ACSL1 Acyl-CoA Synthetase Long Chain Family Member 1 Protein
    [Show full text]
  • The Genomic Variation in the Aosta Cattle Breeds Raised in an Extensive Alpine Farming System
    animals Article The Genomic Variation in the Aosta Cattle Breeds Raised in an Extensive Alpine Farming System Maria Giuseppina Strillacci 1 , Mario Vevey 2, Veruska Blanchet 2, Roberto Mantovani 3 , Cristina Sartori 3 and Alessandro Bagnato 1,* 1 Department of Veterinary Medicine, Università degli Studi di Milano, Via dell’Università 6, 20133 Milano, Italy; [email protected] 2 Associazione Nazionale Bovini di Razza Valdostana, Fraz. Favret, 5, 11020 Gressan, Italy; [email protected] (M.V.); [email protected] (V.B.) 3 Department of Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), Università degli Studi di Padova, Viale dell’Università 16, 35020 Legnaro, Italy; [email protected] (R.M.); [email protected] (C.S.) * Correspondence: [email protected]; Tel.: +39-02-5033-4583 Received: 6 November 2020; Accepted: 10 December 2020; Published: 12 December 2020 Simple Summary: Genetic variability among native cattle breeds can disclose the important features that make a population adapted to harsh environments. The Aosta cattle breeds have been raised and selected for centuries to be farmed in a mountain environment, characterized by a semi-intensive system, i.e., summer pasture with winter recovery on the farms. To disclose the genomic variation and its association with known genes, it is important to genetically characterize these breeds. Abstract: The Aosta Red Pied (Valdostana Pezzata Rossa (VRP)), the Aosta Black Pied (Valdostana Pezzata Nera (VBP)) and the Aosta Chestnut (Valdostana Castana (CAS)) are dual-purpose cattle breeds (meat and milk), very well adapted to the harsh environmental conditions of alpine territories: their farming is in fact characterized by summer pasture at very high altitude.
    [Show full text]
  • Supplemental Figures
    Control #1 Control #2 Control #3 83972 #1 83972 #2 83972 #3 CFT073 #1 CFT073 #2 CFT073 #3 Log-intensity ratios (M-values) ratios Log-intensity Log-intensity averages (A-values) Supplemental Figure 1. MA-plots showing normalized raw data using cross- correlation. The data was found to be of high quality with high replicate correlation (correlation>0.98) and with no systematic bias. A 83972 B CFT073 Supplemental Figure 2. Inversely regulated gene networks generated by IPA. The five inversely regulated genes belonged to one functional network in which HSPA6, FOSB and RN7SK interact with TATA box binding protein (TBP), a general transcription factor required for RNA polymerase II activity. The ABU strain 83972 (A) suppressed FOSB and other genes in this network while the CFT073 strain (B) enhanced their expression. 83972 also suppressed RGS4, a negative regulator of G-protein-coupled receptor-induced signaling and IFIT1, a gene of the type I IFN signaling pathway essential for the antibacterial effector response. A No of affected Network genes Score Top functions genes BAIAP2, BANP, BRIX1, Caspase, CTNNB1, Cyclin A, DACH1, Infection Mechanisms, EMP1, EPHA4, FGFBP1, GADD45G, HES1, HIST2H2BE, IgG, Infectious Disease, IL12, Interferon α, Jnk, MAVS, MDM2, NFkB, NOD1, P38 MAPK, 51 24 Endocrine System PDCD4, PELI2, PEMT, Ras homolog, TFAP2A, TLR5, Development and TNFRSF10D, UBC, Ubiquitin, VANGL2, VARS, Vegf, ZC3HAV1 Function AHCTF1, AKR1C2, ALOX5AP, ARL4A, BAMBI, BAZ2B, Bcl9- Cbp/p300-Ctnnb1-Lef/Tcf, BRF2, CA9, Ca2+, CNN2, CRIP1, Small Molecule
    [Show full text]
  • Integrative Multi-Omic Analysis Identifies New Drivers and Pathways
    www.nature.com/scientificreports OPEN Integrative multi-omic analysis identifes new drivers and pathways in molecularly distinct subtypes of Received: 16 October 2018 Accepted: 4 June 2019 ALS Published: xx xx xxxx Giovanna Morello1, Maria Guarnaccia1, Antonio Gianmaria Spampinato1, Salvatore Salomone2, Velia D’Agata 3, Francesca Luisa Conforti6, Eleonora Aronica4,5 & Sebastiano Cavallaro1 Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease. Increasing the chances of success for future clinical strategies requires more in-depth knowledge of the molecular basis underlying disease heterogeneity. We recently laid the foundation for a molecular taxonomy of ALS by whole-genome expression profling of motor cortex from sporadic ALS (SALS) patients. Here, we analyzed copy number variants (CNVs) occurring in the same patients, by using a customized exon-centered comparative genomic hybridization array (aCGH) covering a large panel of ALS-related genes. A large number of novel and known disease-associated CNVs were detected in SALS samples, including several subgroup-specifc loci, suggestive of a great divergence of two subgroups at the molecular level. Integrative analysis of copy number profles with their associated transcriptomic data revealed subtype-specifc genomic perturbations and candidate driver genes positively correlated with transcriptional signatures, suggesting a strong interaction between genomic and transcriptomic events in ALS pathogenesis. The functional analysis confrmed our previous pathway-based characterization of SALS subtypes and identifed 24 potential candidates for genomic-based patient stratifcation. To our knowledge, this is the frst comprehensive “omics” analysis of molecular events characterizing SALS pathology, providing a road map to facilitate genome-guided personalized diagnosis and treatments for this devastating disease.
    [Show full text]
  • Supplementary Data
    SUPPLEMENTARY DATA Bioinformatic analysis of ChIP-seq data Define the panel of genes associated with energy storage and expenditure We searched the genes list from NCBI Gene, Gene Ontology and UniProt database with the terms “obesity”, “energy homeostasis”, "energy metabolism", “energy storage”, and “energy expenditure” by two independent investigators, and through which we identified 742 genes from all these databases (supplementary Table 8). We then selected the genes associated either with energy storage or expenditure by searching the literatures from PubMed and Google Scholar with the term “gene name" and "obesity" or "energy metabolism" or "energy homeostasis" and "adipose tissue" or "adipocyte”. For a particular gene, if its deficiency protected mice from obesity or overexpression exacerbated obesity, we then categorized it into the panel of energy storage. On the contrary, if its deficiency or overexpression generated opposite phenotype, we classified it into the panel of energy expenditure. We next searched them in the BioGPS database to check their expression in the adipose tissue or adipocyte. If the transcriptional activity index is lower than 50, the gene was considered without expression in adipose tissue. Those genes were also excluded in which they are only expressed in the central nervous system to involve in energy metabolism. The above screenings allowed us to characterize 216 genes associated with energy storage (supplementary Table 9) and 159 genes associated with energy expenditure (supplementary Table 10). Characterization of differential methylated genes. We first compared the signal of chip-seq data in the promoter region to check the DNA methylation state of two panels. Promoter region of those differential methylated genes was defined as 5kb upstream TSS sites.
    [Show full text]
  • Iron Transporters in the Human Mammary Gland and Their
    Iron transporters in the human mammary gland and their relationship to low iron concentrations in human milk By Chenxi Cai A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of Doctor of Philosophy Department of Human Nutritional Sciences University of Manitoba Winnipeg Copyright © 2017 by Chenxi Cai Abstract The capacity of milk to provide adequate iron may be critical to breastfed infants as human milk is often their sole source of iron during early infancy. Extremely low iron content in human milk has been documented, yet whether the iron in breastmilk is adequate for exclusively breastfed infants and the underlying mechanism responsible for low iron in human milk are still unclarified. This study was designed to investigate the adequacy of iron intake in breastfed infants and the iron trafficking pathway in lactating human epithelial cells. First, through the meta-analysis of randomised controlled feeding trials (RCT) it was determined that iron supplementation to healthy exclusively breastfed infants improves their iron status and cognitive development, but may delay their physical growth. There was no evidence to suggest iron supplementation could cause other adverse effects. In order to address why iron concentrations are low in human milk, the expression of iron membrane transporters and iron-binding proteins genes in human breast milk samples was determined. RNA extracted from the breast milk fat globular fraction originates exclusively from epithelial cells when mothers are healthy. Transcript for the transferrin receptor 1 (TFRC), divalent metal transporter 1 (DMT1, SLC11A2), transferrin (TF) and lactoferrin (LTF) were present.
    [Show full text]