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A fosforilação reversível de proteínas em resíduos de serina e treonina, catalisada por quinases e fosfatases desempenha papel chave na regulação do crescimento e na diferenciação celular em eucariotos. As serina/treonina proteínas fosfatases (PSTPs) são atualmente divididas em três famílias denominadas PPP (PhosphoProtein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) e FCP/SCP (RNA polymerase II CTD phosphatase), sendo que os membros da família PPP são, frequentemente, holoenzimas compostas de uma subunidade catalítica associada a uma ou mais subunidades reguladoras, as quais definem a função, localização e especificidade ao substrato da fosfatase. Neste trabalho, analisamos, através de RT-qPCR, o perfil de expressão dos genes codificadores de subunidades catalíticas de PPPs de Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c e PP5c) e de 16 potenciais parceiros moleculares de algumas destas subunidades catalíticas, tais como DdI-2 e DdI-3, sabidamente inibidores da PP1c. Em resposta ao estresse térmico de células da fase de crescimento, detectamos o aumento dos níveis de transcritos de PP4c e PP6c e também de DdI-2, DdI-3 e DDB_G0292194, esta última, uma proteína de função desconhecida que interage com a PP1c em ensaios de duplo-híbrido em leveduras. Por outro lado, durante o estresse hiper-osmótico observamos a diminuição dos níveis de transcritos de quase todos os genes analisados com exceção de DdI-2 e DDB_G0292194. O nível de expressão de DdPP1c, DdI-2, DdI-3 e DDB_G0292194 também foi analisado em resposta ao estresse oxidativo e apenas o DDB_G0292194 foi induzido nesta condição. Os genes de PP1c, PP4, PP5c e PP6c são expressos durante todo o ciclo de vida de D. discoideum, mas a expressão de alguns dos genes analisados aumenta em uma fase definida do ciclo de desenvolvimento como é o caso de DDB_G0292194 que tem níveis de transcritos aumentados na fase de agregação. Este gene codifica uma proteína hipotética de 559 aminoácidos, que apresenta um domínio FHA (ForkHead-Associated) em sua região aminoterminal, além de uma sequência similar ao motivo consenso de ligação à PP1c. Ensaios no sistema de duplo-híbrido em leveduras confirmaram que a interação entre DDB_G0292194 e DdPP1c independe do domínio FHA. Verificamos, também, que o mutante nocaute de DDB_G0292194 apresenta uma morfologia alterada em condições padrões de cultivo, tanto na fase de crescimento como durante o desenvolvimento, além de uma maior sensibilidade ao estresse oxidativo causado pelo peróxido de hidrogênio quando comparado à linhagem selvagem. Em conjunto, nossos resultados evidenciam a importância das PPPs na resposta a diferentes tipos de estresse e para o crescimento e desenvolvimento de D. discoideum. / Reversible phosphorylation of proteins on serine and threonine residues, catalyzed by kinases and phosphatases plays a key role in growth and cell differentiation regulation in eukaryotes. Protein serine/threonine phosphatases (PSTPs) are currently divided into three families named PPP (Phosphoprotein Phosphatase), PPM (Phosphoprotein Phosphatase Magnesium-dependent) and FCP/SCP (RNA polymerase II CTD phosphatase). The PPP family members are often holoenzymes composed of a catalytic subunit associated with one or more regulatory subunits, which define function, localization and substrate specificity of the phosphatase. In this work, we have examined, by RT-qPCR, the expression profile of genes encoding PPP catalytic subunits of Dictyostelium discoideum (PP1c, PP2Ac, PP4c, PP4c-like, PP6c and PP5c) and 16 potential molecular partners for some of these catalytic subunits, such as DdI-2 and DdI-3, both known as PP1c inhibitors. In response to heat stress of growth phase cells, we detected increased levels of transcripts of PP4c and PP6c as well as of DdI-2, DdI-3, and DDB_G0292194, the latter a protein of unknown function that interacts with PP1c in yeast two-hybrid assays. Moreover, during the hyperosmotic stress we observed decreased transcript levels of nearly all genes examined except DdI-2 and DDB_G0292194. The expression level of DdPP1c, DdI-2, DdI-3 and DDB_G0292194 was also analyzed in response to oxidative stress and only DDB_G0292194 was induced in this condition. PP1c, PP4c, PP5c and PP6c genes are expressed throughout growth and development of D. discoideum while transcript levels of some the analysed genes were increased at a defined stage of the developmental cycle as in the case of DDB_G0292194, which increased during aggregation. This gene encodes a hypothetical protein of 559 amino acids bearing a FHA (ForkHead-Associated) domain in its aminoterminal region and a sequence matching the PP1c binding consensus motif. Yeast two-hybrid assays confirmed that DDB_G0292194 and DdPP1c interaction does not depend on FHA domain. We also found that DDB_G0292194 knockout mutant exibits an altered morphology on standard growth and developmental conditions and shows an increased sensitivity to oxidative stress induced by hydrogen peroxide in comparison to the wild type strain. Taken together, our results highlight the importance of PPPs in the response to different types of stress and for growth and development of D. discoideum.

A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados, sendo a interação entre NtSCI1 e NtCDKG;2 confirmada por BiFC e localizada no nucléolo. Uma biblioteca de cDNAs de estigmas/estiletes de N. tabacum, no sistema de duplo-híbrido de levedura, foi construída com sucesso. O screening desta biblioteca, utilizando BD-NtSCI1 como \"isca\", permitiu a identificação de vários parceiros de interação com NtSCI1, entre eles: uma helicase de RNA DEAD-BOX, a proteína 14-3-3D2, dois fatores de transcrição (HOMEOBOX-22 e STOREKEEPER), um fator de splicing portador do domínio SWAP, uma quinase de adenosina e uma transposase. As interações entre NtSCI1 e os três primeiros parceiros citados já foram confirmadas por BiFC (observadas no núcleo e nucléolo) e a interação entre NtSCI1 e Nt14-3-3D2 foi confirmada também por co-imunoprecipitação. O envolvimento de NtSCI1 com a regulação do ciclo celular foi corroborado pela interação entre NtSCI1 e a proteína NtCICLINA-L1 (subunidade regulatória de CDKG;2), confirmada por duplo-híbrido e por BiFC, no nucléolo. A interação entre NtSCI1 e NtCICLINA-RELATED também foi confirmada por BiFC. Para entender a dinâmica de NtSCI1 no nucléolo, foi estudada a localização subcelular da proteína de fusão NtSCI1-GFP durante as fases do ciclo celular. NtSCI1-GFP foi observada no nucléolo de células BY-2 em interfase e prófase, desaparecendo na metáfase e anáfase e reaparecendo no nucléolo no final da telófase, mostrando que a presença de NtSCI1 na célula é controlada pelo ciclo celular. A construção de uma primeira versão do interactoma de NtSCI1 mostrou seu envolvimento direto e indireto com proteínas relacionadas ao metabolismo de RNAs, controle da transcrição e regulação do ciclo celular. Estes resultados sugerem que NtSCI1 possa atuar no controle do ciclo celular de forma não canônica, por meio de múltiplos processos paralelos que interconectam aspectos da regulação da transcrição e o processamento de RNAs com o controle do ciclo celular. / The biology of plant reproduction is a field of great interest, since most of the food consumed by humans is composed of reproductive parts of plants (fruits and seeds). The pistil is the female reproductive organ, composed of stigma, style and ovary. Due to the central importance of the pistil in the success of plant reproduction, a better knowledge of the genes and processes that regulate pistil development and function is necessary. Comparative studies of gene expression in vegetative and reproductive organs of Nicotiana tabacum have revealed genes preferentially expressed in the reproductive organs, among them some encoding proteins of unknown function. One of these genes was characterized and denominated SCI1 (Stigma/style Cell-cycle Inhibitor 1), since it has an important role in stigma/style development, acting as a tissue-specific cell-cycle inhibitor (DePaoli et al., 2011). The objective of the present work was to study the molecular mechanisms through which NtSCI1 regulates the cell cycle investigating its interaction partners. In a pull-down assay, using nuclear protein extracts from N. tabacum stigmas/styles, several putative cell cycle regulators were identified. Among them, the interaction between NtSCI1 and NtCDKG;2 was confirmed by BiFC and localized in the nucleolus. A N. tabacum stigma/style cDNA library in the yeast two-hybrid system was successfully constructed. The screening of this library, using BD-NtSCI1 as bait, allowed the identification of several NtSCI1 interaction partners, among them: a DEAD-BOX RNA helicase; the 14-3-3D2 protein; two transcription factors (HOMEOBOX-22 and STOREKEEPER); a splicing factor containing a SWAP domain; an adenosine kinase; and a transposase. The interactions between NtSCI1 and the first three mentioned partners have already been confirmed by BiFC (observed in the nucleus and nucleolus) and the interaction between NtSCI1 and Nt14-3-3D2 was also wconfirmed by co-immunoprecipitation. The NtSCI1 involvement in cell cycle regulation was corroborated by the interaction between NtSCI1 and the NtCYCLIN-L1 (a regulatory subunit of CDKG;2), which was confirmed by two-hybrid and BiFC in the nucleolus. The interaction between NtSCI1 and NtCYCLIN-RELATED was also confirmed by BiFC. To understand the dynamics of NtSCI1 in the nucleolus, the subcellular localization of the fusion protein NtSCI1-GFP was studied during the different cell cycle phases. NtSCI1-GFP was observed in the nucleolus of BY-2 cells at interphase and prophase, disappearing at metaphase and anaphase and reappearing in the nucleolus at the end of telophase, showing that NtSCI1 presence in the cell is controlled by the cell cycle. The construction of the first version of NtSCI1 interactome showed its direct and indirect involvement with proteins related to RNA metabolism, transcription control and cell cycle regulation. These results suggest that NtSCI1 may act in cell cycle control in a non-canonical way, through multiple parallel processes interconnecting aspects of transcription regulation, RNA processing and cell cycle control.

Human immunodeficiency virus (HIV-1) is the pathogenic agent of HIV infection thatprecedes the total breakdown of cellular immunity, a condition known as acquiredimmunodeficiency syndrome (AIDS). The pandemic nature of the disease has promptedintense research into its biology. Already, much is known about HIV-1 infection, lifecycle,and progression to aids. Systems biology enables the combination of complex data fromthese studies into a framework where their effect on the various levels of cellularorganization (i.e. Pathways, cells, tissues, organs and the whole body) could be studied insilico. In this thesis, first, we reviewed our knowledge of the HIV-1 Human InteractionDatabase. We examined its contents and identified processes that HIV-1 was not previouslyknown to interact with. Then, we attempted an in silico dynamic model of HIV-1 interaction. We built a model of HIV-1 interaction with the CD4 T cell activation pathway comprised of137 nodes (16 HIV-1, 121 human) and 336 interactions. The model reproduced expectedpatterns of T cell activation. Using interaction graph properties, we identified 26 host cellfactors, including MAPK1&3, Ikkb-Ikky-Ikka and PKA, which contribute to the net activationor inhibition of viral proteins. By following a logical Boolean formalism, we identified 9 hostcell factors essential to the functions of viral proteins in the activation pathway. This wasthe first attempt to model dynamic viral-host interaction relationships. Then, we organize HIV-1 interacting host genes into modules to represent cellular processesneeded by the virus. We combined HIV-1 interactions with host gene GO annotations toclassify host genes according to these needed cellular processes. We obtained 201 modulesand found the same set of viral proteins do not interact with host genes having similarmodules suggesting intelligence in its co-ordination of host processes. This work is one of agrowing list that explores coordination of HIV-1 interactions. But more importantly, it would bebeneficial to functionally downsize the large dynamic HIV-1 interaction network. Finally, in our discussion, we discuss our results and suggest possible ways in which our workon dynamic models could be improved. This work is opening up a new field of systems virologythat studies the effect of viruses on the host in terms of its temporal and spatial aspects.

L’élucidation des interactions non-covalentes des complexes biologiques revêt d’une importance majeure dans la compréhension du fonctionnement cellulaire. L’objectif de ce travail de thèse est d’approfondir les développements de la spectrométrie de masse (MS) pour l’étude de ces complexes, que ce soit par MALDI-MS (la désorption-ionisation laser assistée par matrice) ou par ESI-MS (l’ionisation électrospray). Ce travail s’est articulé autour de trois axes : i) étude de la stœchiométrie et de la topologie du complexe SAGA HAT (Spt-Ada-Gcn5 Acétyltransferase, module Histone Acétyl Transferase) par pontage chimique couplé à la MS ; ii) suivi de la dimérisation des complexes formés par RAR-RXR (récepteur de l’acide rétinoïque - récepteur X des rétinoïdes) avec différents ADNs ; iii) mesure de la constante de dissociation des complexes RXR-ligand. Les méthodologies développées ont permis de repousser le potentiel de la MS et d’obtenir des informations structurales des complexes biologiques. / Elucidation of non-covalent interactions of biological complexes takes on great importance for the understanding of cellular function. The purpose of this thesis is a further development of mass spectrometry (MS) for the study of these complexes, either by MALDI-MS (matrix-assisted laser desorption-ionization) or by ESI-MS (electrospray ionization). This work was focused on three main lines: i) study of the stoichiometry and the topology of SAGA HAT (Spt-Ada-Gcn5 Acetyltransferase, Histone Acetyl Transferase module) complex by chemical cross-linking coupled to MS; ii) monitoring the dimerization of the complexes formed by RAR-RXR (retinoic acid receptor - retinoid X receptor) with different DNAs; iii) measuring the dissociation constant of RXR-ligand complexes. The developed methodologies made it possible to expand the potential of MS and get insight into structure of biological complexes.

Dans les thérapies anticancéreuses, les traitements anti-angiogéniques agissant sur l’axe VEGF/VEGFR ont une place importante en clinique. Dans ce contexte, nous avons conçu et évalué l’activité de nouveaux inhibiteurs de l’interaction VEGF/VEGFR. Une première approche a été la conception de molécules antagonistes du VEGFR1. Différents analogues hétérocycliques dérivant d’un composé de type (3-carboxy-2-ureido) thiophène ont été synthétisés. Des réactivités chimiques intéressantes ont été mises en évidence, mais l’activité biochimique de ces molécules ne s’est pas révélée concluante. Une seconde approche reposant sur la conception de peptides ciblant le VEGF a alors été initiée. A partir d’un peptide cyclique connu de 19 résidus ayant une affinité submicromolaire pour le VEGF, de nouveaux peptides et peptidomimétiques ont été développés.L’objectif a été de concevoir des composés de structures chimiques potentiellement plus simples et plus stables en milieu biologique, tout en optimisant l’affinité pour le VEGF. L’interaction de ces peptides avec le VEGF a été étudiée in vitro par ELISA et ITC, ainsi que par cristallographie pour le composé le plus affin. En parallèle, nous avons étudié l’effet du cuivre et d’autres métaux divalents sur l’interaction VEGF/VEGFR1. Au travers d’expériences réalisées au laboratoire ainsi qu’en collaboration, nous avons montré que certains métaux étaient capables non seulement d’inhiber l’interaction VEGF/VEGFR1 mais également d’induire une dimérisation non classique du domaine 2du récepteur. Sachant que les métaux, et en particulier le cuivre, sont connus pour jouer un rôle important dans l’angiogenèse, cette découverte apporte de nouveaux éléments de réponse sur leur mécanisme d’action. Ce travail de thèse s’inscrit donc non seulement dans une démarche de développement de nouveaux composés anti-angiogéniques mais également de compréhension du mécanisme de régulation de l’angiogenèse. / Inhibiting angiogenesis is an effective strategy of targeting therapy against cancer. In thiscontext, we develop an antiangiogenic strategy consisting in the design and evaluation of compoundsblocking the VEGF/VEGFR interaction. The first approach was the conception of antagonists of theVEGFR1. Starting from a (3-carboxy-2-ureido) thiophene hit, a variety of heterocyclic analogs wasdeveloped. Interesting chemical observations were made during the synthesis, but no optimization ofthe biochemical activity was achieved. The second approach was the design of peptides that bind tothe receptor-recognition surface of the VEGF. Starting from a cyclic peptide known to bind to theVEGF with a sub-micromolar affinity, new peptides and peptidomimetics were developed. Thestrategy was to design simplified and potentially more stable compounds, and to improve at thesame time the VEGF affinity. The interaction of VEGF with these ligands was studied in vitro by ELISAand ITC experiments, as well as X-ray diffraction for the best compound. Moreover, the investigationof the effects of copper and other divalent metals on the VEGF/VEGFR1 interaction was undertaken.Experiments realized in the laboratory and in collaboration showed that metals were able to displacethe VEGF/VEGFR1 interaction and to induce the dimerisation of the domain 2 of the receptor. Metalsare well known to play an important role in angiogenic phenomena, but their specific targets are stilla matter of debate. In this context, this discovery brings new response elements regarding theirmechanisms of action. Therefore, the objectives of this PhD thesis were the development of newantiangiogenic compounds, as well as the understanding of some aspects of the regulation of angiogenesis.

Proteins are the fundamental macromolecules within a cell that carry out most of the biological functions. The computational study of protein structure and its functions, using machine learning and data analytics, is elemental in advancing the life-science research due to the fast-growing biological data and the extensive complexities involved in their analyses towards discovering meaningful insights. Mapping of protein’s primary sequence is not only limited to its structure, we extend that to its disordered component known as Intrinsically Disordered Proteins or Regions in proteins (IDPs/IDRs), and hence the involved dynamics, which help us explain complex interaction within a cell that is otherwise obscured. The objective of this dissertation is to develop machine learning based effective tools to predict disordered protein, its properties and dynamics, and interaction paradigm by systematically mining and analyzing large-scale biological data. In this dissertation, we propose a robust framework to predict disordered proteins given only sequence information, using an optimized SVM with RBF kernel. Through appropriate reasoning, we highlight the structure-like behavior of IDPs in disease-associated complexes. Further, we develop a fast and effective predictor of Accessible Surface Area (ASA) of protein residues, a useful structural property that defines protein’s exposure to partners, using regularized regression with 3rd-degree polynomial kernel function and genetic algorithm. As a key outcome of this research, we then introduce a novel method to extract position specific energy (PSEE) of protein residues by modeling the pairwise thermodynamic interactions and hydrophobic effect. PSEE is found to be an effective feature in identifying the enthalpy-gain of the folded state of a protein and otherwise the neutral state of the unstructured proteins. Moreover, we study the peptide-protein transient interactions that involve the induced folding of short peptides through disorder-to-order conformational changes to bind to an appropriate partner. A suite of predictors is developed to identify the residue-patterns of Peptide-Recognition Domains from protein sequence that can recognize and bind to the peptide-motifs and phospho-peptides with post-translational-modifications (PTMs) of amino acid, responsible for critical human diseases, using the stacked generalization ensemble technique. The involved biologically relevant case-studies demonstrate possibilities of discovering new knowledge using the developed tools.

PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...

Influenza virus causes severe respiratory infections in birds and mammals and it is responsible for up to half a million deaths of human beings worldwide each year. Two molecular targets in influenza viral life cycle, neuraminidase and M2 proton channel are exploited in treatment. However, the recent emergence of new pandemic type along with increasing resistance against approved drugs has urged the need for a new drug target discovery and potential search of its inhibitor. Recently, an interesting protein-protein interaction between two subunits PA and PB1 of influenza A viral polymerase has been identified by X-ray crystallography as a new promising drug target. The fact that relatively few residues drive the binding and that the binding interface is highly conserved presents an intriguing possibility to identify antiviral lead compounds effective against all subtypes of influenza A virus. In our laboratory, we expressed and purified two fusion tag constructs of the recombinant C-terminal domain of polymerase acidic subunit (CPA) from the pandemic isolate A/California/07/2009 H1N1. First, GST-CPA fusion protein was used for kinetic evaluation of PA-PB1 interaction by surface plasmon resonance. Moreover, this construct was used in the development of high-throughput screening method for search of...

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