Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder characterized by proliferation of cells expressing BCR-ABL fusion protein. In the BCR-ABL+ leukemia murine model, 12B1, we explored the therapeutic applicability of chaperone-rich cell lysate (CRCL) in the context of syngeneic hematopoietic cell transplantation (HCT) to treat pre-existing leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL immunized donors compared to animals receiving HCT from non-immunized donors. CRCL immunization post-immune HCT further hindered tumor growth when compared to immune HCT without post-transplant vaccination. The magnitude of the immune response was consistent with the anti-tumor effects observed in vivo. We also demonstrated that cured mice had developed long-term tumor specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and NK cells contributed to the anti-tumor effect of CRCL vaccination as depletion of either subset hampered tumor growth delay. Thus, our results suggest that CRCL represents a promising vaccine capable of generating specific immune responses. This anti-tumor immunity can be effectively transferred to a host via HCT and further enhanced post-HCT with additional tumor CRCL immunizations.CD4+CD25+ regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4+CD25+ FoxP3+ regulatory T cells may modulate immunity against human tuberculosis (TB). Ourresults indicate that the number of CD4+CD25+FoxP3+ Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4+CD25+FoxP3+ Treg in pleural fluid inversely correlates with local MTB-specific immunity(p<0.002). These CD4+CD25+FoxP3+ T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-γ and IL-10 production in TB patients. Therefore, CD4+CD25+FoxP3+ Treg expanded in TB patients suppress Mycobacterium tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

Signal termination processes of GPCRs are well established, unlike processes that regulate the assembly and intracellular trafficking of these signaling complexes. Bimolecular fluorescence complementation was used to study GPCR dimer formation in two projects. Firstly, the importance of Rab GTPases on the cell surface expression and signaling of two chemokine receptors expressed on prostate cancer cells was examined. Rab GTPases necessary for CXCR4 and CCR2 cell surface expression and signaling were different from those necessary for the CXCR4/CCR2 heterodimer. Therefore, this project emphasizes the importance of studying heterodimers as unique entities from their constituent receptors. Secondly, interactions between molecular chaperones and two coreceptors necessary for HIV infection – CCR5, a chemokine GPCR, and the main HIV receptor, CD4, a glycoprotein – were investigated. Further emphasizing the unique characteristics of GPCR dimers, this project found that molecular chaperones interact differently with CCR5 homodimers, when compared to CCR5/CD4 heterodimers.

The family of Deg/HtrA proteases is present in a wide range of organisms from bacteria, archaea to eukaryota. These ATP-independent serine endopeptidases play key roles in the cellular protein quality control. The cyanobacterium Synechocystis sp. PCC 6803, a model organism for studies on photosynthesis, metabolism and renewable energy, contains three Deg proteases known as HhoA, HhoB and HtrA. The three proteases are important for survival in stress conditions, such as high light or temperature. In my work the biochemical characteristics of each protease were revealed in vitro and in vivo. In vitro studies performed using recombinant Synechocystis Deg proteases allowed conclusions about their oligomerization states, proteolytic activities and tertiary structure. The in vivo studies addressed their sub-cellular localization, expression and physiological importance by comparing wild-type Synechocystis cells with the three single mutants lacking one of the Deg proteases. HhoA seems to be involved in the cytoplasmic protein quality control. This protease is regulated post-transcriptional and post-translational: oligomerization, pH and/or cation-binding are some of the important factors to stimulate its proteolytic activity. Instead HhoB acts on periplasmic proteins and seems to be important for the transportation/secretion of proteins. While it has low proteolytic capacity, it may act as a chaperone. The stress-induced HtrA functions in the cellular tolerance against photosynthetic stress; additionally it might act as a protease partner of HhoB, generating a protease/chaperone complex. The results presented in this thesis lay the foundation for a better understanding of the dynamic protein quality control in cyanobacteria, which is undoubtedly important for various cellular metabolic pathways.

Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.

La variante d’histone H2AZ joue un rôle important dans l’activation de la transcription, la prolifération cellulaire, le développement et la différentiation. H2AZ orne les promoteurs de la majorité des gènes, mais les mécanismes de bases de cette localisation sont inconnus. La compréhension de l’assemblage et du désassemblage du nucléosome passe par la caractérisation de la dynamique du nucléosome et des chaperonnes d’histones. L’objectif de ma thèse était d’identifier des chaperonnes spécifiques impliqués dans la dynamique de H2AZ en utilisant une approche de protéomique. Pour élucider les mécanismes de déposition/éviction de H2AZ, j’ai purifié le complexe prénucléosomale de H2AZ et j’ai caractérisé toutes les protéines associées. J’ai trouvé que Anp32e fait partie du complexe p400/TIP60 qui est présumée pour être responsable de l’échange d’H2AZ sur la chromatine. Anp32e présente une spécificité pour le dimère H2AZ-H2B, car il n’interagit pas avec le dimère H2A-H2B. L’interaction est accomplie au niveau d’une petite région dans le domaine d’ancrage sur H2AZ et au niveau d’un nouveau domaine ZID sur Anp32e. Finalement, j’ai montré que la suppression d’Anp32e entraine : un défaut dans la dé-répression des gènes dont l’expression est contrôlée par une hormone et une accumulation sur les promoteurs de ces derniers. Dans l’ensemble ces résultats identifient Anp32e comme une nouvelle chaperonne de la variante d’histoneH2AZ impliquée dans l’éviction de H2AZ chez les mammifères. / The histone variant H2AZ has emerged as a key regulator of chromatin function and plays an essential role in transcriptional activation, cell proliferation, development, and differentiation. H2AZ marks nucleosomes flanking the promoters of most genes, but the mechanistic basis for this localization is unknown. A mechanistic understanding of nucleosome assembly/disassembly requiresa detailed knowledge of nucleosome thermodynamics and histone chaperones. The aim of my thesis was to identify specific chaperone involved in H2AZ dynamic by using biochemical and proteomic strategies. To elucidate the mechanism of H2AZ deposition/eviction, I purified the prenucelosomal H2AZ complex and characterized in details the interacting protein partners. I found that Anp32e is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. Bacterially expressed Anp32e binds only to the H2AZ/H2B dimers but not to the H2A/H2B. Anp32e interacts with a short region of the docking domain of H2A.Z. The binding occurred through a novel Anp32e motif, termed ZID. Finally, I show that down regulation of Anp32e interferes with both the de-repression of hormone dependent genes and H2A.Z removal from their promoter. Our data identified Anp32e as a novel mammalian H2AZ chaperone invoved in H2AZ eviction.

La thérapie chaperon représente une approche thérapeutique stratégique et innovante, en particulier dans le traitement des maladies lysosomales. Ces maladies génétiques rares ont une gravité variable, qui peut aller de la létalité avant la naissance jusqu’à la nécessité d‟une prise en charge permanente ; elles apparaissent à tous les stades de la vie. Des mimes du substrat appelé iminosucres, vont agir en allant au coeur du site actif de l’enzyme, stabiliser l’enzyme mutée qui est instable mais non inactive. Paradoxalement, la plupart des chaperons pharmacologiques sont des inhibiteurs de l’enzyme visée mais leur administration à faible concentration leur permet de réaliser leur mission de sauvetage de l’enzyme mutée. Dans cette optique, des recherches effectuées au sein de notre laboratoire ont fait état de la synthèse d’iminosucres, tels que les α-1-C-alkyl iminoxylitols qui sont de très bons inhibiteurs de la β-glucocérébrosidase, l’enzyme défaillante dans la maladie de Gaucher, mais aussi qui doublent l’activité enzymatique résiduelle. Une nouvelle voie de synthèse plus efficace a été réalisée afin d’obtenir plus efficacement ce type d’iminosucres et d’autres dérivés. Ces travaux ont également été l’occasion de développer des iminoxylitols structurellement simplifiés qui agissent comme chaperons pharmacologiques toujours pour le traitement de la maladie de Gaucher. Une partie de ces travaux a aussi été consacrée à la recherche d‟inhibiteurs de la β-galactocérébrosidase, l’enzyme impliquée dans la maladie de Krabbé, et qui pourront agir comme chaperons pharmacologiques. Différentes évaluations pharmacologiques ont été réalisées, notamment des tests d’inhibition et la détermination des effets chaperons. / Chaperone Mediated Therapy represents an innovative and strategic approach to treat lysosomal storage disorders which a class of rare genetic diseases. Competitive inhibitors for some of these lysosomal enzymes can, at sub inhibitory concentrations, act as chaperones and rescue the mutant proteins. In fact, enzymes carrying some mutations are still catalytically active. α-1-C-alkyl iminoxylitols represent a class of iminosugars which mimic the “gluco” configuration of the substrate and give powerful inhibitors of β-glucocerebrosidase, the enzyme involved in Gaucher disease. Moreover, this class of iminosugars, synthesized by our group, act as pharmacological chaperones and are able to double the residual activity of the N370S mutant. In order to synthesize more efficiently these iminosugars, the synthetic strategy was improved and optimized. Moreover, we focused our investigations on structural variations on our lead compound (α-1-C9 iminoxylitol) and draw important conclusions on structure-activity relationship. Then, we extended our expertise on iminosugars as pharmacological chaperones to another lysosomal glycosidase. In paricular, we targeted β-galactocerebrosidase, the enzyme responsible for Krabbe disease, and synthesized a series of iminosugars which mimic the “galacto” configuration. Biological assays were performed on our compounds to determine their activity as inhibitors and for some of them, their chaperone effects.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of IRE1, a conserved transmembrane ER stress receptor. Despite significant research, the mechanism of coupling ER stress to IRE1 oligomerisation and activation has remained contested. There are two proposed mechanisms by which IRE1 may sense accumulating unfolded proteins. In the direct binding mechanism, unfolded proteins are able to bind directly to IRE1 to drive its oligomerisation. In the chaperone inhibition mechanism, unfolded proteins compete for the repressive BiP bound to IRE1 leaving IRE1 free to oligomerise. Currently, these two mechanisms respectively lack compelling in vivo and in vitro evidence required to assess their validity. The work presented here first describes in vivo experiments that identify a role of the ER co-chaperone ERdj4 as an IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). This is then built on by a series of in vitro experiments showing that ERdj4 catalyses formation of a repressive BiP-IRE1LD complex and that this complex can be disrupted by the presence of competing unfolded protein substrates to restore IRE1LD to its default, dimeric, and active state. The identification of ERdj4 and the in vitro reconstitution of chaperone inhibition establish BiP and its J-domain co-chaperones as key regulators of the UPR. This thesis also utilises the power of Cas9-CRISPR technology to introduce specific mutations into the endogenous IRE1α locus and to screen for derepressing IRE1α mutations. Via this methodology, two predicted unstructured regions of IRE1 are found to be important for IRE1 repression. Finally, this thesis challenges recent in vitro findings concerning the direct binding mechanism.

Aim of this thesis was to shed light on the function(s) of Iba57 and Ssc1 proteins in both life cycle stages of T. brucei using RNA interference. Depletion of Ssc1 resulted in severe grow phenotype, decrease in activities of iron-sulphur cluster-containing enzyme aconitase but no increase in oxidative stress sensitivity or accumulation of ROS in mitochondrion. Down regulation of Iba57, specialized maturation factor of aconitase and homoaconitase, lead to depletion of aconitase, destabilization of Isa1 and increased sensitivity to oxidative stress and accumulation of ROS in both stages.

A proteína prion celular (PrPC) consiste em uma glicoproteína de membrana que atua como receptora para diversas moléculas, desencadeando sinais intracelulares. Ao interagir com a co-chaperona STIP1, PrPC promove a autorrenovação e proliferação de células-tronco/progenitoras neurais (NSPCs) durante a fase embrionária. De fato, PrPC tem se destacado por sua participação na neurogênese embrionária e adulta, porém o papel de sua interação com a proteína STIP1 na neurogênese adulta permanece obscuro. Deste modo, o presente trabalho adotou abordagens in vitro para avaliação do complexo PrPC-STIP1 em processos celulares que culminam na neurogênese adulta. Para isso, culturas primárias de NSPCs de camundongos deficientes (Prnp-/-) e tipo-selvagens (Prnp+/+) para PrPC foram realizadas, e a cultura foi devidamente padronizada e caracterizada. Através de ensaios de autorrenovação, proliferação e migração celular sugere-se que PrPC promove estes eventos celulares independentemente de STIP1, e que possivelmente a proteína laminina seja um alvo crítico para migração via PrPC. / Cellular prion protein (PrPC) consists in a membrane glycoprotein that acts as a receptor to several molecules, triggering intracellular signals. By interacting with co-chaperone STIP1, PrPC promotes self-renewal and proliferation of neural stem/progenitor cells (NSPCs) during embryonic stage. Indeed, PrPC has excelled for its participation in embryonic and adult neurogenesis, but the role of its interaction with STIP1 protein in adult neurogenesis remains unclear. Thus, herein it was adopted in vitro approaches in order to evaluate the PrPC-STIP1 complex on cellular processes that culminate in adult neurogenesis. In order to assess that, NSPC primary cultures of PrPC deficient (Prnp-/-) and wild-type (Prnp+/+) mice were performed, and the culture was properly standardized and characterized. Through self-renewal, proliferation and cell migration assays, it was suggested that PrPC promotes these cellular events regardless of STIP1, and possibly the laminin protein is a critical target for migration via PrPC.

As chaperonas moleculares da família Hsp70 desempenham funções cruciais nas células de todos os organismos vivos, de procariotos a eucariotos. Nestes, estão presentes em todos os compartimentos celulares e nas mitocôndrias é expressa uma isoforma própria (mtHsp70), que participa dos processos enovelamento e maturação de proteínas bem como de sua importação para a matriz mitocondrial. Diante da crescente demanda de pesquisa sobre doenças tropicais negligenciadas, foi tomado como objeto de estudo neste trabalho uma Hsp70 mitocondrial de Leishmania braziliensis (LbmtHsp70) com o intuito de caracterizá-la estrutural e funcionalmente em comparação à ortóloga humana com maior identidade: a mtHsp70 também chamada de mortalina, GRP75, HspA9 ou PBP74. A LbmtHsp70 foi purificada em sua forma enovelada, em sistema monodisperso apresentando dados hidrodinâmicos condizentes com a forma monomérica, foi testada sua estabilidade quanto à influência de nucleotídeos de adenosina (ATP e ADP) à sua estrutura e, por fim, foram feitos ensaios para avaliar sua atividade ATPásica e de energia de interação com nucleotídeos. De forma geral, a LbmtHsp70 é bastante similar à mortalina, como pode ser evidenciado pelos resultados obtidos com algumas particularidades. / The molecular chaperones from the Hsp70 family perform critical cell roles in all organisms, from prokaryotes to eukaryotes. In the last ones, they are found in all cell compartments and a particular isoform is expressed in the mitochondria, where it carries out folding and maturation processes as well as the import of proteins to the mitochondrial matrix. In face of the growing demand for research about neglected tropical diseases, in this study a Hsp70 from Leishmania braziliensis\'s mitochondria was taken as object of study for further structural and functional characterization in comparison to the human orthologous which presents the highest identity to LbmtHsp70: the mtHsp70 also known as mortalin, GRP75, HspA9 or PB74. LbmtHsp70 was obtained in folded state in monodisperse system with hydrodynamic data consistent to monomeric conformer, stability and adenosine nucleotides influence to its structure were analyzed, and were performed assays for ATPase activity and nucleotide interaction energy. In a general way LbmtHsp70 is very similar to mortalin as can be shown through the results, but with some peculiarities.