Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane.

L’un des obstacles majeurs à l’éradication du VIH-1 est sa capacité à faire évoluer rapidement son matériel génétique. Ceci lui permet de contourner le système immunitaire de l’hôte ainsi que la pharmacologie antirétrovirale due à l’émergence de formes mutantes et résistantes des enzymes ciblées. A l’origine de ses recombinaisons génétiques, se trouvent d'une part les erreurs commises par la RT lors de l’étape de transcription inverse et d'autre part les processus de transfert de brins qui sont facilités par la protéine de nucléocapside (NC). Notre objectif est de mieux comprendre les propriétés chaperon de la NC pendant le premier transfert de brin ; au cours de celui-ci, la NC déstabilise les structures secondaires des acides nucléiques impliqués ARN et ADN afin de les hybrider pour former un duplex ARN/ADN. Nous souhaitons notamment savoir si la NC est capable de reconnaître la polarité des chaines d’acides nucléiques selon leur nature et si cette capacité module ses propriétés déstabilisatrices. Nos travaux sur la dynamique interne de la protéine ont permis de montrer que certains mouvements étaient corrélés avec les modalités de fixation sur l’ARN. Nous avons ainsi pu montrer, en comparant les propriétés des différentes formes maturées de la NC au cours du cycle viral, que les domaines p1 et p6 présents dans les formes immatures (NCp9 et NCp15) modulaient les propriétés d’interaction comparé à la forme mature (NCp7). Ceci nous amène à reconsidérer le rôle des différentes parties de la polyprotéine Gag sur les propriétés du domaine NC au sein de ce précurseur Gag. Ce domaine apparaissant largement responsable de la reconnaissance et de la sélection du génome viral ARN pour l’encapsidation dans les particules néosynthétisées. Pour réaliser cette étude, nous avons étudié différents complexes entre la NC avec différents acides nucléiques (ARN et ADN), ce en utilisant principalement la résonance magnétique nucléaire couplée à d’autres méthodes biophysiques et biochimiques dans le but d’obtenir des informations à l’échelle atomique. Ce travail peut également être utile dans la conception d’une nouvelle classe d’inhibiteurs dirigés contre la NC sachant que celle-ci représente une cible thérapeutique attrayante vu qu’elle est très conservée et très importante pour l’infectiosité. / One of the major difficulty in the eradication of HIV-1 is its ability to evolve rapidly its genome. This permit to the virus to escape the host immune response and the antiretroviral pharmacology due to the emergence of mutant and resistant forms of the target enzymes.The origin of these genetic recombination is, in one hand the errors commited by the RT during the reverse transcription stage and in the other hand the strand transfer processes facilitated by the nucleocapsid protein (NC).Our goal is to understand the chaperonnig properties of NC protein during the first strand transfer ; where NC destabilizes the involved RNA and DNA secondary structures to anneal them and form a hybrid RNA/DNA duplex.In order to determine this mecanism, we seek, if NC protein have the property to recognize the polarity of nucleic acids chains and if this modulates its destabilization properties. Also, our work on the internal dynamic of the protein showed that some motions are correlated to its binding to RNA.We compared the properties of different maturated forms of NC protein during the viral life cycle and we concluded that p1 and p6 domains present within the immature forms (NCp9 and NCp15) adjusted differently the interaction properties to nucleic acids compared to the mature form (NCp7). It leads us to reconsider the role of the different parts of the Gag polyprotein on the properties of the NC domain within this Gag precursor. This domain appears to be largely responsible for the recognition and selection of the viral genomic RNA in order to package it in the newly formed viral particles.To permform this study, we studied different complexes between NC and nucleic acids sequences (RNA and DNA) using mainly NMR spectroscopy and biophysical or biochemical methods to obtain informations at the atomic scale. This work can also be useful in the design of a new class of inhibitors against NC protein which is an attractive therapeutic target due to its conservation and importance in viral infectivity.

Les chaperons moléculaires, une famille de protéines diverses en structure et taille, sont dédiés à accompagner, replier et protéger d’autres protéines afin qu’elles atteignent leur conformation finale et leur emplacement dans la cellule. Dans ce but, les chaperons moléculaires doivent être hautement spécialisés dans l’exécution de tâches spécifiques, telles que le repliement, le transport ou la désagrégation, et polyvalents dans leur motifs de reconnais- sance, afin de pouvoir interagir avec un grand nombre de protéines di érentes. Di érents chaperons moléculaires collaborent au sein de la cellule, formant ainsi un réseau complexe qui assure le contrôle de la qualité du protéome. Les interactions entre les di érents partenaires de ce réseau et entre les chap- erones et leurs substrats sont souvent dynamiques, ce qui rend leur obser- vation structurale particulièrement di cile pour les techniques de biologie structurale. Par conséquent, il y a à ce jour peu d’information sur les struc- tures et mécanismes d’interaction au sein des complexes chaperon-substrate. Dans cette thèse, je présente des études sur la structure, la dynamique et les interactions entre les substrats de deux chaperons moléculaires, en utilisant diverses méthodes biophysiques et in vivo.Dans la première partie, je montre que la chaperone TIM910, située dans l’espace inter-membranaire des mitochondries, lie ses substrats, des protéines membranaires destinées aux deux membranes mitochondriales, d’une manière très dynamique. Non seulement le complexe TIM910 est en échange constant entre les espèces monomèriques et hexameriques, mais aussi le substrat lié échange entre mulitples conformations à une échelle de millisecondes. Sur la base de la résonance magnétique nucléaire (RMN), de small-angle X-ray scat- tering (SAXS), de l’ultracentrifugation analytique (AUC) et des expériences mutationnelles in vivo et des tests fonctionnels d’import dans les mitochon- dries, je propose un modèle structurale de l’interaction entre le chaperon et la protéine membranaire. TIM910 lie ses substrats dans une poche hydrophobe à l’extérieur du chaperon. Cette interaction est modulaire et se fait avec un ou deux hexamères de TIM910, en fonction de la longueur du substrat.Dans la deuxième partie, nous avons étudié le comportement du récepteur N-terminal du unfoldase ClpC1 de M. tuberculosis en présence d’antibiotiques et de ligands di érents. Le domaine N-terminal de ClpC1 est le site de liai- son de divers antibiotiques nouveaux contre M. tuberculosis. L’antibiotique Cyclomarin A supprime complètement la dynamique induite par le ligand arginine-phosphate. Nous proposons que cette suppression de la dynamique soit le principe fondamental du mécanisme d’action de cet antibiotique.Dans les deux cas, les structures X-ray des chaperons dans leur état apo et la structure de ClpC-NTD liée à des antibiotiques étaient disponibles, mais ces structures statiques ne su sent pas pour expliquer le mécanisme d’action. La structure X-ray de TIM910 n’a pas fourni d’ indication sur l’endroit ou la façon dont les substrats sont liés. De même, les structures X-ray du domaine N-terminal de apo et de Cyclomarine A de ClpC1 ne présentent que des di érences de structure mineures. Les deux exemples montrent que les données structurelles statiques souvent ne permettent pas d’expliquer le fonctionnement d’un système moléculaire, donc la combinaison de di érentes techniques et le développement de nouvelles méthodes pour étudier les complexes chaperon-substrat sont primordiaux pour comprendre leur fonction. / The diverse group of molecular chaperones is dedicated to accompany, fold and protect other proteins until they reach their final conformation and loca- tion inside the cell. To this end, molecular chaperones need to be specialized in performing specific tasks, like folding, transport or disaggregation, and versatile in their recognition pattern to engage many di erent client pro- teins. Moreover, molecular chaperones need to be able to interact with each other and with other components of the protein quality control system in a complex network. Interactions between the di erent partners in this network and between the substrate and the chaperone are often dynamic processes, which are especially di cult to study using standard structural biology tech- niques. Consequently, structural data on chaperone/substrate complexes are sparse, and the mechanisms of chaperone action are poorly understood. In this thesis I present investigations of the structure, dynamics and substrate- interactions of two molecular chaperones, using various biophysical and in vivo methods.In the first part I show that the mitochondrial membrane protein chap- erone TIM910 binds its substrates in a highly dynamic manner. Not only is the TIM910 complex in constant exchange between monomeric and hex- americ species, but also the bound substrate samples multiple conformations on a millisecond timescale. Based on nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC) and in vivo mutational experiments I propose a structural model of the chap- erone/membrane protein interaction. TIM910 binds its substrates in a hy- drophobic pocket on the exterior of the chaperone in a modular fashion, where the number of TIM910 complexes bound depends on the length of the substrate.In the second part I studied the behavior of the N-terminal receptor do- main of the ClpC1 unfoldase from M.tuberculosis in the presence of di erent antibiotics and ligands. The N-terminal domain of ClpC1 is the binding site for various new antibiotics against M.tuberculosis. The antibiotic cyclomarin completely abolishes dynamics induced by the ligand arginine-phosphate. We propose that this suppression of dynamics is the underlying principle for the mechanism of action of this antibiotic.In both cases X-ray structures of the apo or antibiotic bound form were available, but not su cient to explain the mechanism of action. The X- ray structure of TIM910 provided no evidence on where or how substrates are bound. Likewise, X-ray structures of the apo and cyclomarin-bound N-terminal domain of ClpC1 show only minor di erences in structure.Both examples show that static structural data is often not enough to explain how a molecular system works, and only the combination of di er- ent techniques, including newly developed methods enable the atomic-level understanding of chaperone/substrate complexes.

My research focuses on understanding the importance of human mitochondrial Hsp70 (Grp75) chaperone machinery for the maintenance of protein quality control inside the mitochondrial matrix. The investigations carried out during this study have been addressed towards gaining better insights into the working of Grp75 chaperone folding machinery in association with its diverse set of co-chaperones residing in human mitochondria. Additionally, the research also focuses on explaining the various modes of Grp75 participation leading to multiple disease conditions. The thesis has been divided into the following sections as follows: Chapter I: An introduction to the mitochondrial import machinery and role of mitochondrial Hsp70 chaperone folding machinery for the maintenance of protein quality control: Mitochondrion is an essential organelle present in the eukaryotic cell and requires more than 1500 proteins for its proper functioning. Although, mitochondria harbour their own genome, it encodes for only 13 proteins in humans. The rest of the entire proteome is encoded by the nuclear genome and requires proper targeting of proteins to different compartments of mitochondria. Remarkably, mitochondrial matrix alone requires more than 60% of the proteome for its suitable functioning. Briefly, the mitochondrial matrix destined polypeptide passes through the outer membrane translocon; the ‘TOM’ complex and then enters the TIM23 translocon present in the inner membrane of mitochondria. The complete translocation of the polypeptide into the mitochondrial matrix side requires the assistance of mtHsp70 based motor system present on the matrix side which pulls the polypeptide into the matrix in an ATP-dependent manner and with the assistance of various co-chaperones. Subsequently, the unfolded polypeptide is to be folded back to its native state, which is ensured again by the mtHsp70 based chaperone folding machinery. Importantly, while 20% of mtHsp70 is involved in protein import, 80% of mtHsp70 is dedicated for protein folding. In addition to mtHsp70, the chaperone folding machinery consists of various soluble co-chaperones such as the J-proteins which stimulate the ATP hydrolysis rate of Hsp70. Furthermore, another co-chaperone termed as a nucleotide exchange factor ensures binding of fresh ATP molecule onto Hsp70 ensuring multiple rounds of folding cycles. To understand the relevance of mitochondrial Hsp70 chaperone folding machine in the maintenance of protein quality control, Chapter I of the thesis has been divided into multiple sections as follows: Briefly, the initial portion of Chapter I provide a glimpse of the translocon components present in mitochondria for targeting of proteins to outer membrane, inner membrane and inter-membrane space. Owing to the vast proteome size of the mitochondrial matrix, the following section describes the detailed mechanism and translocation process of the mitochondrial matrix targeted proteins. Additionally, subsequent sections of Chapter I provide a comprehensive description of each of the mtHsp70 chaperone folding components, which maintain the protein quality control in the matrix. The players that constitute the chaperone folding machines are mitochondrial Hsp70, J-proteins, nucleotide exchange factors and the newly discovered human escort protein. Essentially, the section provides information about the cellular distribution, structure and function of each of these players constituting the mtHsp70 chaperone folding machine. Loss of regulation between these players leads to defects in protein folding. Imbalance in protein homeostasis is one of the primary causes for mitochondrial dysfunction leading to various diseases. Importantly, recent literature has highlighted the involvement of mtHsp70 chaperone folding players in Parkinson’s disease (PD), Myelodysplastic syndrome (MDS) and cancer. In accordance, the last section of the Chapter I has been dedicated to describe the basic cell biology and proposed mechanisms for the above diseased states. Interestingly, in comparison to yeast and bacteria, the composition of mtHsp70 chaperone folding machinery in humans is unique and distinctly different. Owing to a lack of information about the functioning of human mitochondrial Hsp70 chaperone folding machinery and with an emphasis on understanding its role in various disease manifestations, the objectives that were laid for my PhD thesis are as follows: 1) Functional in vitro reconstitution of the human Grp75 chaperone folding machinery by purifying all the Grp75 chaperone folding machinery players namely; Grp75 (human mtHsp70), hTid-1L and hTid-1S (J-proteins), GrpEL1 (nucleotide exchange factor) and Human escort protein (Hep). 2) Dissection of the intrinsic biochemical defects associated with the variants of Grp75 reported in Parkinson’s disease (PD). 3) To understand the correlation between elevated levels of Grp75 and its contribution to malignancy. In conclusion, the current study has highlighted some of the key features of human Grp75 chaperone folding machinery and its regulation in the maintenance of human mitochondrial matrix protein quality control, failure of which leads to pathological conditions. Chapter II: Reconstitution of the human Grp75 chaperone folding machinery to understand the functional interplay between the multiple protein components: The mitochondrial Heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 towards mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5-peptide as compared to the other Hsp70’s. We observed that the two human mitochondrial matrix J-protein splice-variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hep possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C-terminus of mtHsp70 in a full-length context, and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C-terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thereby promoting a similar conformational change that leads to ATPase stimulation. Moreover, we have also dissected out the inter-domain defective nature associated with the point mutant of Grp75 implicated in Myelodysplastic syndrome thus providing an explanation for the loss of function of Grp75 eventually leading to loss of protein quality control in the diseased state. Chapter III: Enhanced J-protein interaction and compromised protein stability of Grp75 variants leads to mitochondrial dysfunction in Parkinson’s disease: Parkinson’s disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multi-factorial in nature, a recent proteomic screen involving PD-patients revealed two mitochondrial Hsp70 variants (P509S and R126W) that are implicated in PD-pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD-variants are centrally involved in PD-progression is totally elusive. In this report, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD-variants. Biochemically, R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, P509S variant exhibits significantly enhanced interaction with J-protein co-chaperones involved in folding and import machinery, thus altering the overall regulation of chaperone mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD-mutations at the cellular level, we have developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD-mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’ such as mitochondrial DNA (mtDNA) loss and increased susceptibility to oxidative stress recapitulating the cellular features of dopaminergic neurons similar to those reported in other PD-models. Together, our observations for both the variants strongly indicate a definite involvement of mtHsp70 as a susceptibility factor in Parkinson’s disease. Chapter IV: To understand the correlation between elevated levels of Grp75 and its contribution to malignancy: Multiple studies carried out by various groups have reported the presence of elevated levels of Grp75 in cancer cells. Furthermore, proteomic screens show a positive correlation with the higher levels of Grp75 and the aggressive or metastatic nature of cancer. Importantly, cancer cells also exhibit altered mitochondrial metabolism and are found to be under constant oxidative stress pressure. Moreover, Grp75 actively participates in maintenance of mitochondrial function and as well is reported to interact with many putative oncoproteins. However, there is little information available on the possible role of Grp75 in modulating the cellular niche which might favor towards increased malignant transformation of cells. To identify pathways for explaining the correlation between Grp75 and cancer, our initial attempts have focused on monitoring the multiple cellular changes influenced by elevated levels of Grp75 in a cell line based system. To our surprise, transient transfection of cells with Grp75 led to a tremendous increase in the reactive oxygen species levels. Furthermore, a strong positive correlation between the extent of increased levels of Grp75 and the amount of ROS generated in these cells was established. As expected, increased ROS levels observed in Grp75 overexpressing cells also resulted in reduced cell viability. Notably, mitochondrial superoxide generation was found to be the major source for the observed increment in ROS levels in Grp75 expressing cells. In addition, the localization profile of the exogenously expressed Grp75 protein highlighted the fact that the protein was found to be predominantly targeted to mitochondria. Strikingly, the elevated Grp75 levels led to an increase in mitochondrial mass and also displayed a higher proportion of circular and fragmented mitochondria in these cells. Together, the above preliminary observations hint towards a strong correlation between the levels of Grp75 and its influence on the redox biology of cells providing an additional and a possible explanation of the mode of participation of Grp75 in generation and progression of malignancy.

Master of Science / Department of Human Nutrition / Denis M. Medeiros / Copper deficiency has been linked with many health problems, among them cardiac hypertrophy. Because of its potential for causing oxidative damage, copper within the cell must be bound to chaperone proteins. In this thesis, we examined the role of dietary copper deficiency in the regulation of select copper chaperone proteins in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper deficient diet (< 1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Rats were sacrificed and a small blood sample was removed to determine hematocrit. Also, heart and liver tissues were removed for subsequent analysis. Rats fed the copper deficient diet had lower body weights but greater heart weights and heart:body weight. Hematocrit levels and liver copper concentrations were markedly decreased in copper deficient rats. These variables indicated that the copper deficient diet did in fact induce a copper deficiency in these animals. Non-myofibrillar proteins from the hearts were removed and separated by SDS-PAGE. Western Blotting was used to determine the concentrations of CTR1, CCS, Cox17, SCO1, Cox1 and Cox4. No changes were observed in the concentrations of CTR1 and Cox17. CCS and SCO1 were up-regulated as a result of copper deficiency, while Cox1 and Cox4 were both down-regulated. However, use of another antibody against Cox subunits suggested that only the nuclear encoded subunits including subunit IV were decreased, but not subunits I and II. These data provide new insight into the cardiac hypertrophy observed in copper deficiency, which suggests that select chaperone proteins may be up-regulated by a dietary copper deficiency.

Le cancer de la prostate représente la deuxième cause de décès liée au cancer. Des stratégies thérapeutiques ciblant des mécanismes moléculaires conduisant à la résistance doivent donc être développées. Une stratégie visant à améliorer les traitements du cancer de la prostate consiste à cibler les gènes qui sont activés lors de la disparition des androgènes, soit pour retarder ou empêcher l'émergence du phénotype de résistance à la castration. Le but de cette thèse est d'identifier et de développer des petites molécules inhibitrices ciblant des interactions protéine-protéine impliquées dans le cancer de la prostate. Cette thèse porte sur l'étude de deux protéines cruciales liées au cancer de la prostate, à savoir, la protéine de choc thermique de bas poids moléculaire (Hsp27) et la protéine TCTP. Nous avons validé deux composés ciblant TCTP en utilisant une chimiothèque dédiée à l'inhibition d'interaction protéine-protéine. Des tests fonctionnels sont actuellement mis au point pour évaluer la capacité de ces molécules à être proposées comme composés potentiels contre le cancer de la prostate. / Prostate Cancer (PCa) is one of most common malignancies, being the second leading cause among cancer-related death. Additional therapeutic strategies targeting molecular mechanisms mediating resistance must be developed because of the defects of docetaxel-based treatments. One strategy to improve therapies in advanced PCa involves targeting genes that are activated by androgen withdrawal, either to delay or prevent the emergence of the CR phenotype. The purpose of my thesis is to identify & develop small molecules inhibitors targeting PPIs involved in prostate cancer. we focuses on 2 crucial prostate cancer related proteins, namely, the small molecular weight Heat shock protein 27 (Hsp27) and the Translationally Controlled Tumor Protein (TCTP). We have validated 2 compounds targeting TCTP by using a "PPI Inhibitor-like" dedicated chemical library. Functional tests are now being developed to evaluate the capacity of such molecules to be proposed as potential compounds against prostate cancer.

L'adaptation des plantes face au stress thermique est cruciale pour leur survie et implique des réponses spécifiques telles que l’induction de protéines chaperonnes et la production d'espèces réactives de l'oxygène (ROS). Les glutaredoxines (GRX), une famille de protéines thiol anti- oxydantes, jouent un rôle important dans la régulation redox et la réponse au stress oxydatif. Mes études se concentrent sur GRXS17, une protéine multi-domaine à cœur fer-soufre. Malgré un phénotype de développement sévère du mutant grxs17 à des températures normales et plus élevées, peu est connu sur les fonctions biochimiques et les rôles intracellulaires précis de GRXS17. J’ai montré au cours de ma thèse que GRXS17 fonctionne comme une chaperonnedépendante de l’oxydation de la cellule. Elle présente à la fois une activité de type foldase mais également holdase. L'exposition aux stress oxydatif et thermique provoque le passage d'une forme dimérique à des complexes à poids moléculaires élevés ce qui est consistant avec une activité holdase. J’ai également montré que GRXS17 est requis pour la tolérance à des hausses de température de manière dépendante de ses cystéines catalytiques. Des approches de transcriptomique, métabolomique et protéomique montrent une réponse au stress thermique retardée dans le mutant grxs17, des défauts dans l’accumulation de certains métabolites clés, et ont permis d'identifier de potentiels nouveaux interactants de GRXS17 dans des conditions de stress thermique. Ces éléments soutiennent la fonction chaperonne de GRXS17 dans desconditions normales et de stress thermiques. / Adaption of plants to heat stress is crucial for their survival and involves dedicated response such as chaperones proteins induction and production of reactive oxygen species (ROS). Glutaredoxins (GRX), a family of thiol redox proteins, play an important role in redox regulation and response to oxidative stress. The focus of our studies is on GRXS17 which is a multi-subunit iron-sulfur glutaredoxin. Despite the severe developmental phenotype of the grxs17 mutant at normal and elevated temperatures, relatively little is known about the biochemical functions and precise intracellular roles of GRXS17. I show during my thesis that GRXS17 function as a foldase and holdase redox-dependent chaperone. Oxidative and heat stress exposure cause a shift from a dimeric form to high MW complexes which is concordant with a holdase activity. I show that GRXS17 is required for the tolerance to moderated heatstress in a Cys-dependent manner. Transcriptomic, metabolomic and proteomic approaches show heat stress response delayed in grxs17, key-metabolites defects and allowed to identifynew potential GRXS17-interactor under heat stress conditions, supporting a potential protecting function of GRXS17 against stress damage.

C. trachomatis est une bactérie Gram-négative intracellulaire obligatoire et un pathogène humain. Première cause de maladie sexuellement transmissible d'origine bactérienne, elle est également responsable, dans les pays en développement, d'infections oculaires pouvant conduire à la cécité (trachome). Son cycle de développement bi-phasique a lieu au sein d'un compartiment appelé inclusion. Grâce à un système de sécrétion de type 3 (SST3), Chlamydia sécrète des protéines dans le cytosol de la cellule afin de promouvoir sa survie et sa multiplication. Ces protéines sont désignées sous le terme d'effecteurs. / C. trachomatis is an obligate intracellular Gram-negative bacteria and a human pathogen. It is the most prevalent cause of sexually transmitted diseases of bacterial origin and a leading cause of preventable blindness in the developing world. During their biphasic developmental cycle the bacteria remains in a membrane-bounded cellular compartment called an inclusion. Using a type 3 secretion system (T3SS) they translocate effector proteins inside the cytosol of the cell to promote its survival and multiplication.The aim of the PhD was to study the function of CT622, a hypothetic protein from C. trachomatis. We showed that CT622 is an effector protein from the T3SS and that it is secreted early during the infection. We identified a bacterial protein that binds to CT622, and we showed that it acts as a chaperone, stabilizing CT622 and enhancing its secretion. We obtained bacteria lacking CT622 expression, thus demonstrating that CT622 is not essential for bacterial growth in vitro. However, preliminary studies indicate that in the absence of CT622 bacterial development is delayed and T3SS is defective.We identified several molecules interacting with CT622: geranylgeranyl diphosphate, Rab39 and Atg16L1 proteins. Future work will aim at understanding how these identified interactions, or other bacterial or cellular partners still to be discovered, contribute to the establishment of a niche favorable to bacterial development.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2018-04-19T17:09:39Z No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-23T11:47:10Z (GMT) No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-23T11:47:10Z (GMT). No. of bitstreams: 2 Dissertação - Camila Oliveira Barbosa de Morais - 2018.pdf: 2456994 bytes, checksum: d3e60d3fd27acd984ae3a91b457086b5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-13 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Paracoccidioidomycosis (PCM) is an important mycosis of Latin America, caused by thermodymorphic fungi of the genus Paracoccidioides. Homeostasis of metals such as copper, zinc and iron is important for the survival of fungi in the host environment. In this context, copper is an important cofactor for several enzymes, such as as superoxide dismutases and cytochrome c oxidase. The excess free copper in the cell promotes accumulation of reactive oxygen species, causing damage to nucleic acids, lipids and proteins. Thus, organisms shall to maintain cytoplasmic levels of that micronutrient at non-toxic levels, just sufficient for cell growth and vital metabolic processes. The metabolism of that metal is strictly controlled by high and low affinity uptake systems. The objective of this work is to perform heterologous expression of the Atx1 protein, described in the literature as a copper chaperone, and to analyze its cellular location, as well as the protein behavior upon copper deprivation in Paracoccidioides spp. Heterologous expression was performed on electrocompetent cells of Escherichia coli strain BL21. Identity of the recombinant protein was confirmed by LC-MS / MS. BALB/c mice were immunized to obtain anti-Atx1 polyclonal antibodies. After performing immunoblotting the produced antibodies were used in immunofluorescence assays. Analysis by qRT-PCR allowed us to evaluate the levels of the transcript encoding the Atx1 protein during copper deprivation in Paracoccidioides spp. / A paracoccidioidomicose (PCM) é uma importante micose da América Latina, causada por fungos termodimórficos do gênero Paracoccidioides. A homeostase de metais como cobre, zinco e ferro é importante para a sobrevivência dos fungos no ambiente do hospedeiro. Nesse contexto, o cobre é um importante cofator para várias enzimas, como as superóxido dismutases e a citocromo c oxidase. O excesso de cobre livre na célula promove o acúmulo de espécies reativas de oxigênio, causando danos a ácidos nucléicos, lipídeos e proteínas. Assim, os organismos devem manter concentrações citoplasmáticas desses micronutrientes em níveis não tóxicos, apenas suficiente para o crescimento celular e processos metabólicos vitais. O metabolismo desse metal é rigorosamente controlado por sistemas de captação de alta e baixa afinidade. O objetivo do trabalho é realizar a expressão heteróloga da proteína Atx1, descrita na literatura como uma chaperona citosólica de cobre e analisar a sua localização celular, bem como o comportamento diante da privação de cobre em Paracoccidioides spp. A expressão heteróloga foi realizada em células eletrocompetentes de Escherichia coli, cepa BL21. A identidade da proteína recombinante foi confirmada por LC-MS/MS. Camundongos BALB/c foram imunizados para obtenção de anticorpos policlonais anti-Atx1. Após realização de imunoblotting, os anticorpos produzidos foram utilizados para ensaios de imunofluorescência em células leveduriformes. Análise por qRT-PCR nos permitiram avaliar os níveis do transcrito codificante da proteína Atx1 durante a privação de cobre em Paracoccidioides spp.

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