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Arabidopsis Thaliana Atypical Aspartic Proteases Involved in Primary Root Development and Lateral Root Formation

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André Filipe Marques Soares RLR1 and RLR2, two novel Arabidopsis thaliana atypical aspartic proteases involved in primary root development and lateral root formation 2016 Thesis submitted to the Institute for Interdisciplinary Research of the University of Coimbra to apply for the degree of Doctor in Philosophy in the area of Experimental Biology and Biomedicine, specialization in Molecular, Cell and Developmental Biology This work was conducted at the Center for Neuroscience and Cell Biology (CNC) of University of Coimbra and at Biocant - Technology Transfer Association, under the scientific supervision of Doctor Isaura Simões and at the Department of Biochemistry of University of Massachusetts, Amherst, under the scientific supervision of Doctor Alice Y. Cheung. Part of this work was also performed at the Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, under the scientific supervision of Doctor Herta Steinkellner and also at the Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, under the schientific supervision of Doctor Pitter F. Huesgen. André Filipe Marques Soares was a student of the Doctoral Programme in Experimental Biology and Biomedicine coordinated by the Center for Neuroscience and Cell Biology (CNC) of the University of Coimbra and a recipient of the fellowship SFRH/BD/51676/2011 from the Portuguese Foundation for Science and Technology (FCT). The execution of this work was supported by a PPP grant of the German Academic Exchange Service with funding from the Federal Ministry of Education and Research (Project-ID 57128819 to PFH) and the Fundação para a Ciência e a Tecnologia (FCT) (grant: Scientific and Technological Bilateral Agreement 2015/2016 to IS) Agradecimentos/Acknowledgments Esta tese e todo o percurso que culminou na sua escrita não teriam sido possíveis sem o apoio, o carinho e a amizade de várias pessoas que ainda estão ou estiveram presentes na minha vida. Sem estas pessoas eu tenho a certeza que nada disto seria possível portanto aqui fica o meu muito obrigado por todo o apoio que me deram. Os próximos parágrafos são a expressão escrita de todo o meu agradecimento mas todas as palavras serão sempre poucas para vos agradecer! Em primeiro lugar, gostaria de agradecer ao Professor Doutor Carlos Faro e ao Professor Doutor Euclides Pires por terem proporcionado todas as condições necessárias para a realização deste trabalho quer na Unidade de Biotecnologia Molecular do Biocant, quer no Laboratório de Biotecnologia Molecular do Centro de Neurociências e Biologia Celular de Coimbra. Se nos agradecimentos da minha tese de Mestrado me faltaram as palavras para poder agradecer devidamente à Doutora Isaura Simões, nesta tese de Doutoramento todas as palavras serão sempre poucas para lhe agradecer tudo o que fez e continua a fazer por mim. Muito obrigado por voltar a apostar em mim Professora e por me ter acompanhado ao longo destes quase 5 anos! Obrigado por ter acreditado em mim quando eu próprio duvidava. Obrigado por toda a sua preocupação e por tudo o que me ensinou ao longo de todos estes anos que trabalhámos juntos. Se sou o cientista que sou neste momento devo-o a si e a tudo o que me ensinou! Tal como escrevi na minha tese de Mestrado, espero sinceramente não a ter desiludido Professora. To Dr. Alice Cheung and Dr. Hen-ming Wu I would like to thank the opportunity that they gave me to work in their lab at the University of Massachusetts, Amherst. Thank you for giving me all the conditions necessary to perform my work in your lab and for all the things that I learn during my stay. Especially I would like to express my deepest gratefulness to Dr. Alice for agreeing to be the co-supervisor of this thesis. Thank you for all the time and effort that you spent guiding me and discussing with me my results and ideas. Thank you very much. To Dr. Pitter Huesgen I would like to thank the opportunity of working with you in your laboratory at Forschungszentrum Jülich. Thank you very much for all the time that you spent and continue spending analyzing my data and for all the great ideas that you gave me. To Dr. Herta Steinkellner I also would like to thank the opportunity of working in your lab at BOKU University, Vienna. Thank you very much for allowing me in your lab and for giving me all the conditions to express both proteins in Nicotiana plants. It was a great opportunity for me and also a great experience! A todas as pessoas que fazem parte da Unidade de Biotecnologia Molecular do Biocant ou que por lá passaram enquanto eu lá trabalhei, o meu muito obrigado por tornarem o laboratório um sítio onde não só se trabalhava à séria mas onde também era possível dar umas boas gargalhadas! Apesar de ter andado a saltitar de um lado para o outro ao longo deste doutoramento, era no nosso laboratório que eu mais me sentia em casa e agora que parto tenho a certeza que vou sentir sempre a vossa falta! Queria agradecer também à Dona Alda, à Dona Adelaide e à Filipa todo o trabalho que tiveram comigo ao longo destes anos, por todas as provetas, frascos e fraquinhos que tiveram de lavar à minha conta! Muito obrigado por todo o apoio que me deram, por todas as gargalhadas partilhadas e pela forma incansável com que não só me apoiavam a mim mas também ao resto das pessoas do Biocant. Aproveito também para agradecer a todos os meus amigos que ainda estão ou já estiveram no Biocant e que ajudaram a fazer do Biocant muito mais do que um simples local de trabalho. Muito obrigado! To the people from the Amherst, Vienna and Jülich Laboratories, I thank you for the very good environment that I experience during my stay in your labs and for all the help that you were always willing to give me! A especial thanks to Yanjao (Amherst lab) for helping me taking my first baby steps in plant/pollen research, to Andreas and Alexandra (Vienna Lab) for all the help and advices, and finally to Stefan (Jülich lab) for all the time that you spent (and continue spending) doing my proteomics experiments and analyzing my data! Thank you very very much for everything! A todos os meus amigos da X edição do BEB, Catarina, Tânia, Sara, Joana, Dominique, Mariline, Sofia, Patrícia, António, Marcelo e Ribas, o meu muito obrigado por serem os amigos que são. O primeiro ano do Programa Doutoral foi sem dúvida o melhor ano que passei em Coimbra e se assim foi, devo-o a todos vós! Deixo um agradecimento especial à Nina e à Tânia pela vossa amizade e por estarem sempre presentes e dispostas a dois dedos de conversa sobre tudo ou mesmo sobre nada! Aos restantes amigos de Coimbra e arredores, o meu muito obrigado por todo o apoio que me têm dado! Deixo aqui também um agradecimento especial à Tábata e à Kaline, duas pessoas que tive a sorte de conhecer em Amherst e que tornaram a minha estadia naquele canto do mundo bastante mais alegre. Meninas, eu se calhar devia escrever este agradecimento em inglês porque eu não sei se vocês vão perceber este meu “Old Portuguese” mas o dicionário existe para essas coisas! Eheh! To the people that I met during my stay in Vienna, thank you very much for making those 3 months that I spent there unforgettable. A special thanks to my great friend Kemo, a.k.a. “The Bosnian Guy”, and to Anja and Heidi! Those last two months that I spent in your company were truly great and I will never forget them! Aos meus Pais, Avós e Irmã agradeço todo o incansável apoio que me deram ao longo de toda a minha vida. Eu posso andar sempre de um lado para o outro, a saltitar de país em país mas sei que terei sempre o vosso apoio e o vosso carinho! Muito obrigado por terem estado sempre presentes na minha vida, mesmo quando era eu que parecia estar mais ausente! O meu último e mais especial agradecimento vai para a Maria. Nós só nos conhecemos a meio desta jornada mas sem ti tudo isto teria sido bem mais difícil. Muito obrigado por toda a força que me deste especialmente quando estávamos separados por todo um oceano. Muito obrigado por acreditares sempre em mim mesmo quando eu próprio duvidava! Acima de tudo, muito obrigado por fazeres parte da minha vida desta forma tão especial. Abstract ................................................................................................... I Resumo .................................................................................................. V Abbreviations ......................................................................................... IX Chapter I - Introduction ........................................................................... 1 1.1 Plant Atypical and Nucellin-like Aspartic Proteases ................................................ 4 Primary structure organization ................................................................................................... 4 Cleavage Specificity and Inhibition ............................................................................................. 7 Distribution and localization ..................................................................................................... 10 1.2 Biological functions of plant atypical and nucellin-like APs ................................... 12 Protein degradation as a nitrogen source ................................................................................. 12 Chloroplast homeostasis and protein turnover ......................................................................... 13 Defense against biotic injury ....................................................................................................
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  • Discovery of Digestive Enzymes in Carnivorous Plants with Focus on Proteases

    Discovery of Digestive Enzymes in Carnivorous Plants with Focus on Proteases

    A peer-reviewed version of this preprint was published in PeerJ on 5 June 2018. View the peer-reviewed version (peerj.com/articles/4914), which is the preferred citable publication unless you specifically need to cite this preprint. Ravee R, Mohd Salleh F‘, Goh H. 2018. Discovery of digestive enzymes in carnivorous plants with focus on proteases. PeerJ 6:e4914 https://doi.org/10.7717/peerj.4914 Discovery of digestive enzymes in carnivorous plants with focus on proteases Rishiesvari Ravee 1 , Faris ‘Imadi Mohd Salleh 1 , Hoe-Han Goh Corresp. 1 1 Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Corresponding Author: Hoe-Han Goh Email address: [email protected] Background. Carnivorous plants have been fascinating researchers with their unique characters and bioinspired applications. These include medicinal trait of some carnivorous plants with potentials for pharmaceutical industry. Methods. This review will cover recent progress based on current studies on digestive enzymes secreted by different genera of carnivorous plants: Drosera (sundews), Dionaea (Venus flytrap), Nepenthes (tropical pitcher plants), Sarracenia (North American pitcher plants), Cephalotus (Australian pitcher plants), Genlisea (corkscrew plants), and Utricularia (bladderworts). Results. Since the discovery of secreted protease nepenthesin in Nepenthes pitcher, digestive enzymes from carnivorous plants have been the focus of many studies. Recent genomics approaches have accelerated digestive enzyme discovery. Furthermore, the advancement in recombinant technology and protein purification helped in the identification and characterisation of enzymes in carnivorous plants. Discussion. These different aspects will be described and discussed in this review with focus on the role of secreted plant proteases and their potential industrial applications.
  • A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of Dimiq, a Potent Indolo[2,3- B]Quinoline Agent, Against Candida Albicans Biofilms

    A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of Dimiq, a Potent Indolo[2,3- B]Quinoline Agent, Against Candida Albicans Biofilms

    A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of DiMIQ, a Potent Indolo[2,3- b]Quinoline Agent, against Candida albicans Biofilms Robert Zarnowski 1,2*, Anna Jaromin 3*, Agnieszka Zagórska 4, Eddie G. Dominguez 1,2, Katarzyna Sidoryk 5, Jerzy Gubernator 3 and David R. Andes 1,2 1 Department of Medicine, School of Medicine & Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA; [email protected] (E.G.D.); [email protected] (D.R.A.) 2 Department of Medical Microbiology, School of Medicine & Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA 3 Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, 50-383 Wroclaw, Poland; [email protected] 4 Department of Medicinal Chemistry, Jagiellonian University Medical College, 30-688 Cracow, Poland; [email protected] 5 Department of Pharmacy, Cosmetic Chemicals and Biotechnology, Team of Chemistry, Łukasiewicz Research Network-Industrial Chemistry Institute, 01-793 Warsaw, Poland; [email protected] * Correspondence: [email protected] (R.Z.); [email protected] (A.J.); Tel.: +1-608-265-8578 (R.Z.); +48-71-3756203 (A.J.) Label-Free Cellular Proteomics of Candida albicans biofilms treated with DiMIQ Identified Proteins Accession # Alternate ID Gene names (ORF ) WT DIMIQ Z SCORE Proteins induced by DiMIQ Arginase (EC 3.5.3.1) A0A1D8PP00 CAR1 CAALFM_C504490CA 0.000 6.648 drug induced Glucan 1,3-beta-glucosidase BGL2 (EC 3.2.1.58) (Exo-1Q5AMT2 BGL2 CAALFM_C402250CA