CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O linfoma difuso de grandes cÃlulas B (LDGCB) corresponde a 50 % dos casos de linfoma nÃo-Hodgkin (LNH). Seu tratamento de escolha à a quimioterapia de associaÃÃo, em especial o esquema CHOP (ciclofosfamida, doxorrubicina, vincristina e prednisona), considerado o tratamento inicial padrÃo dos LDGCB. VariaÃÃes deste esquema, como o protocolo CHOP-Bleo (ciclofosfamida, doxorrubicina, vincristina, prednisona e bleomicina) tem sido utilizadas com a intenÃÃo de se obter maiores taxas de remissÃo completa pelos pacientes. No Brasil, pouco se conhece a respeito da incidÃncia, do comportamento clÃnico, da resposta Ãs terapÃuticas utilizadas e da sobrevida de pacientes com LDGCB. Este estudo teve como objetivos traÃar o perfil epidemiolÃgico dos pacientes portadores de linfoma difuso de grandes cÃlulas B, atendidos no Hospital UniversitÃrio Walter CantÃdio (HUWC), com data de primeiro atendimento de janeiro de 1989 a dezembro de 2003, e que fizeram uso dos esquemas quimioterÃpicos CHOP e/ou CHOP-Bleo; avaliar a eficÃcia e seguranÃa terapÃutica dos esquemas propostos atravÃs da anÃlise do tipo de resposta terapÃutica, achados clÃnicos e laboratoriais destes pacientes. A coleta dos dados foi realizada a partir dos prontuÃrios mÃdicos dos 31 pacientes analisados. Destes, 21 (67,74%) eram do sexo masculino e 10 (32,26%) do feminino, com idade mÃdia de 45,81  16,3 anos. A ocupaÃÃo trabalhador agrÃcola representou 25,82% (8/31). O estÃdio III da classificaÃÃo de Ann Arbor foi o mais freqÃente (32,26%), mas apenas 45% dos pacientes apresentaram sintomas B. A lactato desidrogenase (LDH) sÃrica de 49% dos pacientes encontrava-se elevada à Ãpoca do diagnÃstico, sendo que outros 16% dos pacientes nÃo apresentavam resultado desta enzima em seus prontuÃrios. Quanto ao IPI, 71% foram classificados como de risco baixo e intermediÃrio, 13% de alto risco intermediÃrio, nenhum dos pacientes do estudo apresentou IPI compatÃvel com de alto risco e em 16% dos pacientes nÃo foi possÃvel estabelecer a classificaÃÃo devido à ausÃncia de dados nos prontuÃrios. Quanto à utilizaÃÃo dos protocolos quimioterÃpicos, 58% (18/31) dos pacientes fizeram uso do esquema CHOP, 36% (11/31) utilizaram CHOP-Bleo e 6% (2/31) utilizaram os dois esquemas quimioterÃpicos. Entre os pacientes que utilizaram o esquema CHOP, 78% atingiram a remissÃo completa (RC), 17% apresentaram recidiva da doenÃa e apenas 5 % foram a Ãbito. No grupo que utilizou o esquema CHOP-Bleo, 63% atingiram a RC, 18% apresentaram recidiva da doenÃa e 19% foram a Ãbito. Os 2 pacientes que utilizaram os dois esquemas como tratamento apresentaram recidiva da doenÃa. Os valores de LDH dos pacientes apÃs a quimioterapia apresentam-se reduzidos tanto em pacientes que atingiram a remissÃo completa como naqueles que tiveram recidiva. Verificamos que a sobrevida global (SG) e a sobrevida livre de doenÃa (SLD) nÃo foram influenciadas pelo estÃdio clÃnico e LDH inicial dos pacientes. A regressÃo logÃstica nÃo mostrou significÃncia estatÃstica quando analisou a remissÃo completa dos pacientes a partir dos resultados das variÃveis em estudo pÃs QT, com exceÃÃo da proporÃÃo de reduÃÃo da LDH e a resposta ao tratamento. Os resultados mostraram a eficÃcia e seguranÃa dos esquemas terapÃuticos CHOP e CHOP-Bleo em nossa populaÃÃo de estudo. Os resultados demonstram ainda que se faz necessÃrio o estudo epidemiolÃgico de diferentes populaÃÃes com LDGCB para que haja seguranÃa na escolha de esquemas quimioterÃpicos, bem como a uniformidade em descrever e classificar os linfomas e os seus fatores prognÃstico por parte dos patologistas e oncologistas. / Diffuse Large B-Cell Lymphomas (DLBCL) corresponds to 50% of non-Hodgkinâs lymphomas (LNH). Their treatment of choice is the association chemotherapy, in special the CHOP therapy (cyclophosphamide, doxorubicin, vincristine and prednisone) considered the standard treatment initial of the DLBCL. Variations of this therapy, with the CHOP-Bleo protocol (cyclophosphamide, doxorubicin, vincristine, prednisone and bleomycin) have been used with the intention of obtaining complete response rates for the patients. In Brazil, little is known about the incidence, clinical behavior, response to therapy and survival of the patients with DLBCL. This study aimed to set out the epidemiological profile of patients with diffuse large B-Cell lymphomas, who received medical care at Hospital UniversitÃrio Walter CantÃdio (HUWC), outline in Cearà state, with the first attendment from January 1989 to December 2003, who the used the CHOP and/or CHOP-Bleo therapy; Evaluating the security and efficiency of the protocols proposed by analysis of the kind of therapeutical response, clinical and laboratorial outcomes of these patients. The data collection was performed from medical recording of the 31 patients analyzed. These, 21 (67,74%) were the men and 10 (32,26%) women. The average age was 45,81  16,3 anos. Agriculturists represented 25,82% (8/31) of all patients. The stage III the Ann Arbor classification were the most frequent (32,26%), but only 45% of the patients had B symptoms. The values of lactate dehydrogenises (LDH) enzyme were elevated in 49% of the patients at diagnosis, but in 16% of the patients these values at diagnosis were unknown. As much as the IPI, 71% were classified as an IPI low and intermediate risk, 13% as an IPI intermediate-high risk, none of the study patients showed as an IPI high risk and 16% there is not possible the classification to establish due to the data is unknown. As much as the chemotherapy protocols used, 58% (18/31) of the patients was received CHOP chemotherapy, 36% (11/31) CHOP-Bleo chemotherapy and 6% (2/31) received CHOP associated with CHOP-Bleo chemotherapy. Among the patients who used CHOP chemotherapy, 78% was achieving complete response (CR), 17% was achieving relapse of the disease and only 5% were the death. In the group who used CHOP-Bleo chemotherapy, 63% was achieving RC, 18% was achieving relapse of the disease and 19% died. The 2 patients who used CHOP and CHOP-Bleo chemotherapy were achieving relapse of the disease. The values of the LDH after chemotherapy showed decreased in patients with RC as much as the relapsed patients. We verified that the overall survival (OS) and disease-free survival (DFS) were not influenced by the clinic stage and initial values of the LDH patients. The logistic regression did not show statistical differences when the complete response was analyzed comparing to outcomes the studied variables after QT, except for the proportion of reduction of LDH levels and response to the treatment. The results stress the security and efficiency of the protocols CHOP e CHOP-Bleo in our study population. The data obtained also the need epidemiological studies in different DLBCL populations for the security in the choice chemotherapy, well as standardized the classification and description of the DLBCL and prognoses factures by pathologists and oncologists.

Les lymphomes T périphériques (PTCL) sont des néoplasmes rares et agressifs représentant environ 12% des lymphomes chez l’Homme. Nos travaux récents dans des souris p53-/- ont révélé une nouvelle entité de PTCL, émergeant de cellules Natural-Killer T-cell (NKT), un type particulier de lymphocyte T reconnaissant des antigènes lipidiques. Nous avons montré que ces lymphomes NKT (PTCL-NKT) présentent des caractéristiques de NKT stimulés chroniquement, et que la lymphomagenèse est initiée via l’activation chronique du TCR. Chez l’Homme, de nombreux PTCL sont suspectés pour être associés à des stimulations antigéniques chroniques, mais les mécanismes de transformation impliqués sont encore mal connus. Borrelia burgdorferi (Bb), l’agent responsable de la maladie de Lyme, provoque des infections chroniques dont l’implication dans certains lymphomes T cutanés (CTCL) a été suggérée. Cependant, cette observation manque de preuves cliniques et expérimentales. De manière intéressante, Bb est connue pour exprimer des glycolipides activateurs des NKT. Nous avons donc infecté des souris p53-/- avec des Bb vivantes, et montré que l’infection augmente significativement la fréquence des PTCL-NKT. Par traitement antibiotique précoce de souris infectées et par injections de Bb inactivées, nous avons également démontré que la chronicité de l’infection est nécessaire au développement de ces lymphomes. L’analyse phénotypique de ces PTCL-NKT a confirmé nos observations précédentes, montrant des caractéristiques de cellules NKT activées chroniquement, telles que l’expression de marqueurs d’activation et d’exhaustion (perte de NK1.1, surexpression de PD-1). Ces résultats suggèrent une implication de Borrelia dans la lymphomagenèse T. En se basant sur l’analyse de différents marqueurs phénotypiques et de leur production cytokinique, nous avons également montré que ces lymphomes présentent un profil dérégulé se rapprochant du sous-type NKT2. Une étude génomique par séquençage whole-exome sur 6 PTCL-NKT a révélé de larges pertes récurrentes du chromosome 13. Au sein de la zone minimale de délétion, nous avons identifié Jarid2, codant un facteur épigénétique impliqué dans le développement NKT par une activité histone-methytransférase. Ce gène est retrouvé altéré dans 20% des CTCL. De manière intéressante, les souris Jarid2-/- présentent une expansion périphérique de NKT au profil immature/NKT2, partageant donc des caractéristiques avec les PTCL-NKT. La perte de Jarid2 a été détectée dans presque tous les PTCL-NKT. Nous avons confirmé la perte de Jarid2 au niveau ARN et protéique. Nos résultats préliminaires montrent une hypométhylation de la lysine 9 de l’histone H3 (H3K9), la cible de Jarid2, soutenant un effet fonctionnel dans la physiopathologie des PTCL-NKT. Par conséquent, nous pensons que la perte de Jarid2 pourrait être un événement important de la lymphomagenèse NKT, puisque de plus en plus d’altérations de facteurs épigénétiques sont retrouvées dans les PTCL humains. Pour réponse à cette question, nous sommes notamment en train de générer des souris p53-/- x Jarid2-/-. En conclusion, nos données viennent renforcer le concept selon lequel certaines infections peuvent initier la transformation des cellules T par l’activation chronique du TCR. Nous avons également identifié un nouveau facteur épigénétique potentiellement impliqué dans la lymphomagenèse NKT / Peripheral T-cell lymphomas (PTCL) are aggressive and heterogeneous neoplasms that represent around 12% of Human lymphomas. Our recent work in p53-/- mice revealed a new PTCL entity, arising from Natural-Killer T-cell (NKT), a particular type of T cell recognizing lipidic antigens. We found that NKT lymphomas (NKTL) present features of chronically stimulated NKT-cells and that lymphomagenesis is driven through chronic TCR activation by microbial glycolipids. In human, many PTCL are suspected to be associated with chronic antigenic stimulation, but this transformation mechanism is still poorly understood.Borrelia burgdorferi (Bb), the causative agent of Lyme disease, induces chronic infection and has recently been suggested to be involved in cutaneous T-cell lymphomas (CTCL). However, this observation lacks clinical and experimental proofs. Interestingly, Bb is known to express NKT-activating glycolipids. We therefore infected p53-/- mice by live intradermal Bb injection and showed that Bb infection significantly increased NKTL rate. Phenotypic characterization of these NKTL confirmed our previously described features of chronically stimulated NKT-cells, with expression of activation and exhaustion markers (loss of NK1.1, upregulation of PD-1). Based on surface markers, transcription factors and cytokine production analysis, we also found that our lymphomas mostly present a NKT2 subtype profile, sometimes surprisingly mixed with NKT17 or NKT1. Genomic study by whole-exome sequencing on few of these lymphomas revealed recurrent large losses in the chromosome 13. Within the minimal deletion region, we identified Jarid2, a gene involved in NKT development by epigenetic regulation and which is found altered in 20% of CTCL. Jarid2 loss was detected in almost all NKTL. Interestingly, Jarid2-/- mice show increased NKT number in the periphery with an immature/NKT2 phenotype, sharing features with our NKTL.Thus, we believe that Jarid2 loss may be an important event in NKT lymphomagenesis, as more and more epigenetic factors are found mutated in several human PTCL. To answer this question we are currently breeding p53-/- x Jarid2-/- mice. In conclusion, our data reinforced the concept that chronic bacterial activation of T-cells through their TCR can effectively drive T-cell transformation. We also identified a new potential epigenetic factor that may be involved in lymphomagenesis

Les lymphomes B non-Hodgkiniens sont caractérisés par des altérations génétiques à l’origine du syndrôme néoplasique. Dans ce travail, nous démontrons que l’association dans les microdomaines rafts de la membrane de Lyn, une kinase de la famille Src, avec l’adaptateur PAG, participe également au processus néoplasique. Dans les cellules B non-néoplasiques, Lyn et PAG sont des molécules impliquées dans la signalisation du récepteur des cellules B en réponse à une stimulation antigénique. Dans ce contexte, l’adaptateur PAG est décrit comme un inhibiteur de l’activité kinase de Lyn. Au contraire, dans les rafts des lymphomes B non-Hodgkiniens, nous avons mis en évidence que PAG retient la kinase Lyn sous une conformation active. Au sein des rafts, la kinase Lyn semble responsable de l’activation oncogénique de la voie de signalisation PI3K/Akt et de la voie de signalisation STAT3. L’inhibition pharmacologique et génétique de la kinase Lyn induit l’apoptose et l’inhibition de la prolifération de lignées cellulaires dérivées de lymphomes B non-Hodgkiniens. Ce résultat est révélateur d’une dépendance des lignées cellulaires à l’association oncogénique de Lyn avec PAG. Le ciblage thérapeutique du complexe Lyn/PAG constitue donc une opportunité pour le traitement des lymphomes B non-Hodgkiniens. / Non Hodgkin B lymphomas are caracterized by genetic alerations that condition their neoplastic development. In this work, we demonstrate that an association between the Src family kinase Lyn and the PAG adaptor in rafts is also involved in the neoplastic phenotype. In normal B cells, Lyn and PAG contribute to BCR signalling, where PAG functions as a Lyn kinase inihibitor. In contrast, we demonstrate that Lyn remains active when associated with PAG in B non Hodgkin lymphomas rafts. In those rafts, the Lyn kinase participates in the oncogenic activation of the PI3K/Akt and STAT3 signaling pathways. Pharmacologic and genetic inhibition of the Lyn kinase leads to apoptosis induction and proliferation inhibition of B non Hodgkin lymphoma cell lines. Our results reveal the dependence (or addiction) of these cell lines to the Lyn and PAG complex in rafts. The Lyn/PAG complex therefore constitutes an appropriate therapeutic target in certain non Hodgkin B lymphoma treatment.

Les lymphomes B sont des cancers du système lymphatique se développant à partir des cellules B. Il devient évident que le développement des cellules B malignes dépend d’interactions avec les cellules immunes dans leur microenvironnement. Nous avons étudié la caractérisation des lymphocytes T intra tumoraux afin de comprendre leur contribution dans la lymphomagenèse et leur potentiel thérapeutique dans les lymphomes B comme le lymphome diffus à grandes cellules B (DLBCL), le lymphome folliculaire (FL) et le lymphome Hodgkinien classique (cHL)Nous avons mis en évidence une différence importante, quantitative et qualitative, entre la composition immunitaire de différents lymphomes B, notamment au niveau des lymphocytes T intra tumoraux. Le FL se caractérise par une accumulation des lymphocytes T régulateurs (Tregs) exprimant ICOS, pouvant supprimer les cellules B lymphomateuses. La génération des Tregs ICOS+ est favorisée par le contact avec les cellules B lymphomateuses exprimant ICOSL. Quant à lui, le DLBCL a beaucoup de lymphocytes TCD8 coexprimant PD1 et TIM3 possédant un état de dysfonctionnement dit « épuisement », lymphocytes dont la proportion est corrélée à leur niveau de dysfonctionnement et à leur capacité de réponse au blocage des récepteurs inhibiteurs. Enfin, dans certains lymphomes B, en particulier le cHL, nous avons découvert une sous population de TCD8, dite « TFH-like » pour leur similarité phénotypique et fonctionnelle avec les lymphocytes T auxiliaires folliculaires (TFH). Ces données indiquent l’hétérogénéité des composants immunitaires entre différents lymphomes B et sont une piste pour une future thérapie ciblée dans le traitement du lymphome. / B-cell lymphomas represent a heterogeneous group of cancers that affect B cells in the lymphatic system. It has become evidence that malignant B cells depend on various interactions with microenvironmental immune cells for their development. Our study focuses on characterization of intra-tumoral T cells in order to understand their contribution in pathogenesis and their therapeutic potentials in the most frequent B cell-lymphoma such as Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma (FL) and classical Hodgkin lymphoma (cHL).During this work, we have demonstrated a significant quantitative and qualitative difference between different B-cell lymphoma immune composition, especially between their intra-tumoral T cells. FL is characterized by the accumulation of regulatory T cells (Tregs) expressing ICOS, with ability to suppress lymphoma B cells. Generation of Tregs ICOS+ is prompted by cell contact with the lymphoma B cells expressing ICOSL. On the other hand, DLBCL have high level of TCD8 coexpressing PD1 and TIM3 displaying an exhaustion state, which proportion is correlated with their dysfunction level and with their responsiveness to inhibitor receptors blockade. Finally, in some B-cell lymphoma cases, especially cHL, we found the existence of a TCD8 subset, called TFH-like due to their phenotypic and functional similarity with follicular helper T cells (TFH).These data show heterogeneity of immune components between the different B lymphomas, and give opportunity for targeted therapy in lymphoma treatment

Oncogenic microRNAs (miRNAs) are small RNA molecules that inhibit post-translational regulatory mechanisms at the epigenetic level. miRNAs are often deregulated in malignancies and due to their stability are detectable in non-cellular fractions of peripheral blood. In our laboratory, we have performed several studies that have investigated and utilized miRNAs as biomarkers for various hematological tumors (e.g., chronic lymphocytic leukemia, Hodgkin`s lymphoma) and solid tumors (e.g., breast cancer). The aim of these studies was to find the association of miRNAs with pathophysiological and clinical aspects of each disease. Here, we confirmed the importance of particular miRNA or its complex during disease monitoring. Combining clinical, molecular biological and statistical analyses, we were able to find miRNA sets that fulfilled not only a diagnostic role but also a prognostic role beyond expectations. The main focus of this thesis is on the investigation of microRNAs in the diagnosis of a hematological malignancy - primary cutaneous T-cell lymphoma (CTCL). Tumor specificity of some miRNAs has been demonstrated. Their aberrant expression in tissue samples of CTCL patients obtained from skin biopsies, correctly distinguished malignant disease from control samples of benign skin lesions. Here, we...

L'infection par le virus de l'immunodéficience humaine (VIH) est associée à la survenue de lymphomes B chez des patients infectés et l’incidence de certains lymphomes reste élevée même chez les individus infectés dont la fonction immunitaire est reconstituée sous traitement antirétroviral combiné. La contribution du VIH-1 à l'oncogenèse des cellules B reste donc énigmatique. Le VIH-1 induit un stress oxydant et des dommages à l'ADN (DA) dans les cellules infectées via de multiples mécanismes. Cependant il n'infecte pas les lymphocytes B. En revanche, la protéine virale Tat qui circule dans le sang des individus infectés est capable de pénétrer spontanément dans des cellules non infectables par VIH. Nous avons détecté des niveaux élevés d’espèces réactives de l’oxygène (ROS), principalement mitochondriales, et des DA dans les cellules B d'individus infectés par le VIH. Nous avons ainsi émis l'hypothèse que Tat pourrait induire des DA oxydants dans les cellules B et favoriser ainsi l'instabilité génétique et la transformation maligne de ces cellules.Dans des cellules B isolées à partir du sang périphérique de donneurs sains et incubées en présence de protéine Tat recombinante, un stress oxydant a été induit, la capacité antioxydante a diminué avec la diminution de taux du glutathion, le facteur de transcription NF-κB a été activé, et sont apparus des DA accompagnés d'aberrations chromosomiques. En outre, tous les effets induits par Tat dépendaient de son activité transcriptionnelle. Dans le but de mieux comprendre le(s) mécanisme(s) d’action de Tat chez les patients séropositifs, des extraits bruts de plantes endémiques du Liban ont été utilisés pour leur potentiel antioxydant. L’effet pro-oxydant de Tat a été contrecarré, le stress oxydant inhibé ainsi que les DA induits par la protéine virale. En conclusion, nous proposons que les dommages oxydants causés à l’ADN et les aberrations chromosomiques induites par Tat correspondent à de nouveaux facteurs oncogéniques favorisant le développement de lymphomes B chez les individus infectés par le VIH-1. / An infection with the Human immunodeficiency virus (HIV) is associated with Bcell lymphomas in infected patients. The incidence of some lymphomas remains elevated in HIVinfected individuals whose immune function has been reconstituted under combined antiretroviral therapy. Its contribution to B-cell oncogenesis cells remains enigmatic. HIV-1 is known to induce an oxidative stress and DNA damage (DD) in infected cells via multiple mechanisms. However, it does not infect B lymphocytes. This contrasts with the viral transactivator protein Tat which circulates in the blood of infected individuals and spontaneously penetrates even non infectable cells. We have detected high levels of reactive oxygen species (ROS), mainly from mitochondria, and DDs in Bcells of HIV-infected individuals. We have thus hypothesized that Tat could induce oxidative DD in B-cells thereby promoting genetic instability and malignant transformation in these cells.In B-cells isolated from peripheral blood of healthy donors and incubated in the presence of purified recombinant protein Tat, an oxidative stress has been induced, the antioxidant capacity was decreased due to diminished glutathione levels, the transcription factor NF-κB was activated, and DD and chromosomal aberrations induced. All the effects induced by Tat were shown to depend on its transcriptional activity. To better understand the mechanism(s) of action of this viral protein, crude extracts from endemic plants of Lebanon were tested for their antioxidant potential. The prooxidative effect of Tat was inhibited, as well as the DD and chromosoml aberrations induced by the viral protein. In conclusion, we propose that the oxidative DNA damage and chromosomal aberrations induced by the Tat protein correspond to novel oncogenic factors that favor the development of B-cell lymphomas in HIV-1 infected individuals.

Animal models have greatly contributed to the understanding of different aspects of human biology, as well as a variety of human-related pathogens and diseases. In order to study them, humanized mice susceptible to pathogens that replicate in human immune cells have been developed (e.g., humanized Rag2-/-γc-/- mice). These animals are engrafted with human hematopoietic stem cells (HSCs), resulting in the de novo development and maturation of the major functional components of the human adaptive immune system and the production of a variety of human cell types. Primary and secondary lymphoid organs in the mouse are populated with human cells, and animals have long term engraftment. These features make humanized mice an excellent in vivo model to study pathogenesis of human-specific viruses in the context of a human antiviral immune response. In addition, humanized mice have been shown to be useful preclinical models for the development and validation of antiviral therapeutics. In the present study, we aimed to successfully re-establish the humanized Rag2-/-γc-/- mouse model using cord blood-derived HSCs in our laboratory. We have shown that these mice sustain long term engraftment and systemic expansion of human cells, including the major targets of Kaposi's sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus type 1 (HIV-1), in peripheral blood and different lymphoid organs. Further, we have begun to evaluate the susceptibility of the humanized Rag2-/-γc-/- mouse model to infection with KSHV. We demonstrate that human lymphocytes differentiated in reconstituted Rag2-/-γc-/- mice are permissive to KSHV infection ex vivo. This finding was corroborated by detection of KSHV mRNA expression in the spleen of a humanized mouse at 6 months post infection. In a different study, we tested the in vivo antiviral efficacy of a novel HIV-1 fusion inhibitor (PIE-12-trimer) in humanized Rag2-/-γc-/- mice. We have determined the half life of PIE-12-trimer in mouse plasma. Furthermore, the administration of PIE-12-trimer to HIV-1 infected humanized Rag2-/-γc-/- mice prevents depletion of CD4+ T cells in blood, thus it may be useful to prevent AIDS in human patients.

<p>The proteasome is a multi-catalytic, multiprotein enzymatic machinery that is responsible for most of the protein degradation in the cell. Cellular protein homeostasis through the proteasome is regulated through the ubiquitin-independent or ubiquitin-dependent degradation pathway, which both utilize different isoforms of the enzymatic machinery. Over the past twenty years, the proteasome has been a well-validated therapeutic target by inhibition of its catalytic particle function, and more recently, through targeted protein degradation with the use of proteolysis targeting chimeras (PROTACs). Inhibition of the proteasome’s catalytic function has been previously shown to be therapeutically advantageous due to the need for high proteasomal activity for the survival of hematological cancer cells, which produce an overabundance of misfolded and unwanted proteins. Despite this success, off-target toxicities and drug-resistant mechanisms remain as dose-limiting factors for proteasome catalytic inhibition. Herein, we describe a variety of peptidomimetic (or “peptide-like”) approaches that target the proteasome beyond standard catalytic inhibition to serve as alternative therapies for hematological cancer. We investigate <em>(1)</em> the preferential structural properties of peptide-conjugated unnatural substrates for different proteasome isoforms’ substrate channels, <em>(2)</em> the effectiveness of an immunoproteasome-targeting peptide-conjugated prodrug strategy, and <em>(3)</em> the unknown binding site of a peptoid probe on the proteasome’s non-catalytic ubiquitin receptor, Rpn-13. This work not only showcases novel strategies to target the proteasome system but also describes methods that could be applied to other challenging enzymes or non-catalytic protein targets.</p>

Thesis (M.S.)--University of Missouri-Columbia, 2009. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Thesis advisor: Yang Gong. "May 2009" Includes bibliographical references.

Le virus d’Epstein-Barr (EBV) est un herpèsvirus humain qui infecte plus de 90% de la population mondiale. Il est décrit comme associé à plusieurs pathologies cancéreuses humaines comme les carcinomes nasopharyngés et gastriques et divers lymphomes, comme le lymphome de Burkitt, les lymphomes NK/T et certains lymphomes de Hodgkin. Le lymphome T angio-immunoblastique (LTAI), un cancer des cellules T folliculaires helper TFH, contient souvent des cellules B porteuses de l’EBV. Mais jusqu’à présent le rôle de l’EBV dans la pathogenèse de cette maladie reste inconnu. Dans ce contexte, notre travail avait pour objectif de déterminer si l’EBV associé au LTAI présentait une particularité laissant envisager son rôle dans cette pathologie. Pour ce faire, nous avons étudié la séquence complète de l’EBV au sein d’échantillons de LTAI et comparé les résultats à ceux obtenus pour d’autres lymphomes (B, NK/T) ainsi qu’aux séquences publiées. Le séquençage a tout d’abord été réalisé sur 7 lignées cellulaires positives pour l’EBV, afin de valider la technique, et a ensuite été appliqué aux échantillons d’adénopathies de 40 patients atteints de syndrome lymphoprolifératif, parmi lesquels 20 souffraient de LTAI. L’enrichissement en génome viral a été réalisé par capture à l’aide de sondes spécifiques du génome de l’EBV. Ensuite les librairies ont été synthétisées et séquencées sur les plateformes Illumina MiSeq et NextSeq. Dans un deuxième temps, nous avons réalisé l’assemblage de novo des reads et déterminé la séquence complète du virus majoritaire dans chaque échantillon. Les données obtenues ont été analysées bioinformatiquement. D’une manière intéressante, le virus a été trouvé clonal ou quasi-clonal dans les LTAI alors que les lymphocytes B étaient dans la plupart des cas polyclonaux. En outre, le profil de mutations trouvé présentait des similitudes avec ce qui était trouvé pour les autres lymphomes associés à l’EBV, notamment au niveau des épitopes cibles des cellules de l’immunité suggérant un processus de sélection de la souche virale identique à celui d’une tumeur clonale associée à l’EBV. Ceci pourrait jouer un rôle important dans l’échappement au système immunitaire du virus dans ce contexte multicellulaire complexe. La présence de cellules B polyclonales avec un EBV clonal dans un compartiment T tumoral clonal pourrait relever d’une double sélection tumorale, endogène T et exogène EBV clonal, et pourrait suggérer l’existence de cross-talk entre les cellules B-T. / More than 90% of the world's population is infected by Epstein-Barr virus (EBV), a human herpesvirus. EBV is thought to be implicated in the pathogenesis of several human malignancies including epithelial tumors such as nasopharyngeal and gastric carcinomas as well as lymphoproliferative diseases such as Burkitt's lymphoma, NK/T lymphomas and some Hodgkin lymphomas. In angioimmunoblastic T-cell lymphoma (AITL), a peripheral neoplasm of follicular helper T (TFH) cells, a recurrent finding is the presence of EBV-positive B lymphocytes at the beginning of the disease. However, whether this EBV infection of B cells plays a role in AITL pathogenesis remains unclear. In this context, our work aimed to determine if the EBV associated with the AITL presented an oncogenic profile allowing us to consider its role in this pathology. To do this, we sequenced the whole EBV genomes in AIL samples and compared the results to those obtained for other lymphomas (B, NK / T) as well as to previously published sequences. Sequencing was first performed on 7 EBV-positive cell lines to validate the technique, and then was applied to lymphadenopathy specimens from 40 patients with lymphoproliferative disease, of whom 20 had AITL. Enrichment of the viral genome was performed by capture using specific EBV genome probes. The libraries were synthesized and sequenced on Illumina MiSeq and NextSeq platforms. In a second step, we performed de novo assembly and determined the sequence of the virus in each sample. The data obtained were analyzed bioinformatically. Interestingly, the virus was found to be clonal or quasi-clonal in AITL, while B cells were in some cases polyclonal. In addition, the mutational pattern was similar to other EBV-associated lymphomas, especially at the level of the target epitopes of immune cells suggesting a process of selection of the viral strain identical to that of a clone tumor associated with EBV. This could play an important role in the virus escape from the immune system in this context. The presence of polyclonal B cells with clonal EBV in a clonal tumor T cell compartment could be a dual tumor selection; or that is endogenous T and exogenous clonal EBV, and could therefore suggest the existence of a cross-talk between B-T cells.