Introdução: O Câncer de Próstata (CaP) é o tumor mais comum do homem e a segunda causa de óbito por câncer no Brasil. MicroRNA (miRNA) é uma classe de pequenos RNA regulatórios não codificantes de proteínas que tem papel fundamental no controle da expressão dos genes. São responsáveis pelo controle de processos fundamentais na célula e estão envolvidos na tumorigênese em humanos. Previamente demonstramos alterações no perfil de expressão dos miRNA 100, let7c e 218 comparando carcinomas localizados e metastáticos. A caracterização de perfis de expressão de suas proteínas alvo no CaP é crucial para a compreensão dos processos envolvidos na carcinogênese, dando-nos a oportunidade do descobrimento de novos marcadores diagnósticos, prognósticos e mais importante identificação de alvos para o desenvolvimento de terapias inovadoras. Objetivo: Analisar a expressão das proteínas controladas pelo miR-let7c (Ras, c-Myc e Bub1), miR-100 (Smarca5 e Retinoblastoma) e miR-218 (Laminina 5 3) e a atividade proliferativa (Ki-67) no câncer de próstata com a técnica de imuno-histoquímica utilizando microarranjos teciduais representativos de CaP localizado e suas metástases linfonodais e ósseas. Correlacionar os níveis de expressão dos miRNA com suas proteínas alvo. Analisar a expressão dos miRNA, proteínas e atividade proliferativa com os fatores prognósticos do câncer de próstata e com a evolução da doença. Material e Métodos: A imunoexpressão de Smarca5, Retinoblastoma, Laminina, Ras, c- Myc, Bub1 e Ki-67 foi avaliada através de IH pela técnica de microarranjo tecidual caracterizando três estágios do CaP, sendo 112 casos de CaP localizado, 19 metástases linfonodais e 28 metástases ósseas. As imagens obtidas foram submetidas a um software de análise de imagem digital MacBiophotonics ImageJ do National Institutes of Health, EUA, onde a intensidade de luminescência foi quantificada densitometricamente. O perfil de expressão dos miR-let7c, 100 e 218 foi analisado utilizando o bloco de parafina de 61 pacientes dos 112 pacientes com carcinoma localizado, que foram submetidos a analise protéica por IH. O processamento dos miRNA envolveu três etapas: extração do miRNA com kit específico, geração do DNA complementar e amplificação do miRNA por PCR quantitativo em tempo real (qRT-PCR) cujo controle endógeno foi RNU-43 (Applied Biosystems). Os resultados foram analisados usando o método 2-CT. Como controle, utilizamos amostras de tecido com hiperplasia prostática benigna (HPB). Avaliamos a relação entre a expressão dos miRNA e suas proteínas alvo, com o escore de Gleason, estadiamento patológico e evolução da doença considerando recidiva bioquímica, níveis de PSA>0,4 ng/mL, em uma média de seguimento de 77,5 meses. A análise estatística foi realizada através do software SPSS 19.0, utilizamos o test T de Student, Mann-Whitney, Kruskal-Wallis e qui-quadrado. O valor de p foi considerado estatisticamente significante quando inferior na 0,05 em todos os cálculos. Resultados: Observamos uma diminuição de expressão de Ras (p=0,017) e Laminina (p<0,0001) conforme a progressão tumoral do CaP localizado a metástase linfonodal e óssea. Houve um aumento de expressão de Rb (p=0,0361) e aumento da atividade proliferativa avaliada pelo Ki- 67 (p<0,0001). Encontramos ainda uma tendência a relação entre a positividade de expressão de c-Myc com estadiamento patológico pT3 (p=0,070). Todos os miRNA se mostraram superexpressos no CaP localizado. Laminina apresentou uma média de intensidade de expressão maior quanto maior a expressão de miR-218 (p=0,038). Porém os demais miRNA não apresentaram relação de expressão com suas proteínas alvo. Também não houve relação entre a expressão de miRNA e expressão das proteínas por IH com a recidiva bioquímica. Conclusões: Apesar de confirmarmos os nossos achados de superexpressão dos miRNA 100, let7c e 218 no CaP localizado, não houve correlação entre esses e a imunoexpressão de suas proteínas alvo. Demonstramos que houve alteração de imunoexpressão de Ras, Laminina 5 3, Retinoblastoma e Ki-67 de acordo com a progressão tumoral no CaP. E uma maior expressão de c-Myc por IH mostrou uma significância tendência a relacionar-se com tumores não confinados estadiados pT3 / Introduction: Prostate cancer (PCa) is the most common tumor in men and the second leading cause of cancer death in men in Brazil. MicroRNA (miRNA) is a class of small non-coding RNA that plays a key role in the control of gene expression. They are responsible for the control of key processes in the cell and are involved in tumorigenesis in humans. Previously, we demonstrated alterations in the expression profile of miRNA 100, 218 and let7c comparing localized and metastatic carcinomas. The characterization of expression profiles of their target proteins in PCa is crucial to understanding the processes involved in carcinogenesis, giving us the opportunity to discover new diagnostic or prognostic markers, and most importantly to find new targets for the development of innovative therapies. Objective: To analyze the expression of proteins controlled by miR-let7c (Ras, c- Myc and Bub1), miR-100 (Smarca5 and Retinoblastoma) and miR- 218 (Laminin 5 3) and proliferative activity (Ki-67) in prostate cancer with immunohistochemistry using tissue microarrays representing localized PCa, lymph node and bone metastases. To correlate the expression levels of miRNAs with their target proteins. To analyze the expression of miRNAs, proteins and proliferative activity with prognostic factors of prostate cancer and disease progression. Methods: The immunoexpression of Smarca5, Retinoblastoma, Laminin, Ras, c-Myc, Bub1 and Ki-67 was evaluated by IHC by tissue microarray technique featuring three stages of PCa, with 112 cases of localized PCa, 19 lymph node metastases and 28 bone metastases. The images obtained from IHC were submitted to analysis using the digital image software MacBiophotonics ImageJ from the National Institutes of Health, USA, where the intensity of luminescence was quantified densitometrically. We studied the expression profile of the miRNAs in the paraffin blocks of 61 patients out of the 112 patients with localized carcinoma, who underwent protein analysis by IHC. The processing of miRNA involved three steps: extraction of miRNA, generation of complementary DNA and amplification of the miRNA by quantitative real time PCR (qRT-PCR). To analyze the data we used a control endogenous RNU-43. The results were analyzed using the 2-CT formula. As control, we used the tissue from five patients with benign prostate hyperplasia (BPH) submitted to surgery. The relationship between the expression of miRNAs and their target proteins were analyzed as well as their expression with Gleason score, pathological stage and disease progression considered as PSA>0.4 ng/mL in a mean follow-up of 77.5 months. The statistical analysis was performed using SPSS 19.0 software, we used the Student t test, Mann-Whitney test, Kruskal- Wallis and chi-square. The value was considered statistically significant when p0.05. Results: There was a decrease in the expression of Ras (p=0.017) and Laminin (p<0.0001) according to PCa progression from localized to lymph node and bone metastases. There was an increase in the expression of Retinoblastoma (p=0.0361) and an increase in proliferative activity assessed by Ki-67 (p<0.0001). We also found a relationship between the positivity of c-Myc expression with pT3 staged tumors (p=0.070). All miRNAs showed overexpression in PCa samples. Laminin showed a higher expression together with higher expression of miR-218 (p=0.038). The other miRNAs did not show a relationship with protein expression by IHC. There was no correlation between the expression of miRNAs and protein expression by IHC with biochemical recurrence. Conclusions: Although our findings confirm the overexpression of miR-100, 218 and let7c in localized PCa, there was no correlation between their expression and the protein of their target using immunohistochemistry. We demonstrated that there was a change in immunostatining of Ras, Laminin 5 3, Retinoblastoma and Ki- 67 according to tumor progression. The increased expression of c- Myc per IHC showed a significant tendency to relate to tumor unconfined staged pT3

INTRODUÇÃO: O linfoma difuso de grandes células B, sem outras especificaçoes (LDGCB, SOE) é uma neoplasia agressiva caracterizada pela heterogeneidade morfológica, imunofenotípica e molecular, porém o atual tratamento padrão utilizando imunoquimioterapia (R-CHOP) não considera tal diversidade. Há percentual significativo de pacientes que são refratários à terapia de primeira linha e alguns que apresentam recidiva precoce ou tardia, os quais representam as vítimas desta doença. O estudo imuno-histoquímico (IHQ), que é um método simples e universalmente disponível, vem sendo utilizado para reconhecer a diversidade biológica do LDGCB, SOE, identificando biomarcadores e subgrupos distintos da doença, que poderiam predizer a resposta terapêutica ao tratamento padrão e apontar possíveis candidatos a novas estratégias terapêuticas. OBJETIVOS: Este estudo avalia o valor prognóstico de cinco algoritmos para classificação do LDGCB segundo a célula de origem (COO) e da expressão de três biomarcadores (BCL2, CD30 e MYC) tendo como endpoint a sobrevida global. MÉTODOS: Foi realizado estudo retrospectivo com setenta e nove pacientes com LDGCB,SOE de novo tratados com imunoquimioterapia padrão, estadiados e acompanhados protocolarmente. Os casos foram classificados como subgrupo célula B centrogerminativa símile (GCB) ou como subgrupo célula B não-centrogerminativa símile (NGCB), de acordo com três algoritmos IHQ (Hans, Choi, e Visco-Young) pareados com estudo do perfil de expressão gênica (PEG) e dois algoritmos IHQ não-PEG pareados (Muris e Nyman). Foi estimado o valor prognóstico destes algoritmos e também avaliado a concordância entre eles. O valor prognóstico da expressão do BCL2, CD30 e MYC utilizando IHQ também foi analisado. RESULTADOS: Os algoritmos IHQ PEG pareados revelaram maior concordância entre si, porém nenhum deles revelou força prognóstica. A expressão do CD30 mostrou tendência a melhor prognóstico, porém a expressão de BCL2 e MYC avaliados isoladamente não revelaram impacto prognóstico. Contudo, a coexpressão do BCL2 e MYC, denominado como fenótipo linfoma duplo-expressor (LDE), revelou-se importante marcador prognóstico desfavorável. Foram identificados três subgrupos de risco baseado no fenótipo LDE e o Índice Prognóstico Internacional (IPI). CONCLUSÃO: Em pacientes com LDGCB, SOE de novo tratados com esquema terapêutico padrão, a pesquisa da expressão do fenótipo LDE é mais relevante do ponto vista prognóstico que a classificação em subgrupo GCB ou NGCB. Além disso, a expressão do CD30 pode ser relevante tanto para identificar subgrupo com tendência a melhor prognóstico como para identificar possíveis candidatos a nova terapia alvo / BACKGROUND: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is an aggressive neoplasm characterized by morphological, phenotypic and molecular heterogeneity, but the current standard therapy using immunochemotherapy (R-CHOP) does not consider such diversity. There is a significant percentage of patients who are refractory to first-line therapy and those with early or late recurrence, whose represent the victims of this disease. Immunohistochemistry (IHC), a simple and universally available method, has been used to recognize the biological diversity of DLBCL, NOS, to identify biomarkers and distinct subgroups of the disease, which would predict the therapeutic response to standard treatment and point possible candidates for novel therapeutic strategies. OBJECTIVES: The current study was conducted to evaluate the prognostic value from five algorithms for classification of DLBCL based on cell of origin (COO) and the expression of three biomarkers (BCL2, CD30 and MYC) with overall survival (OS) as an endpoint. METHODS: We retrospectively evaluated seventy nine patients with de novo DLBCL, NOS treated with R-CHOP-like immunochemotherapy. The cases were assigned as germinal center B-cell like (GCB) or non-GCB subgroup (NGCB) according to five different IHC algorithms, including three algorithms based on gene expressing profile study (GEP), proposed by Hans, Choi, and Visco-Young, and two non-GEP based algoritms proposed by Muris, and Nyman. We evaluated their prognostic relevance and the concordance between these algorithms. The prognostic power of BCL2, CD30 and MYC expression were also assessed by IHC. RESULTS: None of the profiles assessed by IHC algorithms was able to predict overall survival (OS). The positive expression of CD30 showed a trend toward a better outcome. Neither the positive expression of BCL2 nor the positive expression of MYC were associated with outcome. However, the double-expressor lymphoma phenotype (DEL), represented by the concurrent expression of MYC and BCL2, exhibited a negative prognostic impact. Three different risk subgroups were identified based on the DEL phenotype and the International Prognostic Index (IPI) score. CONCLUSIONS: These data suggest that the DEL, rather than the cell of origin classification based on IHC, is a better predictor of OS in patients with DLBCL treated with R-CHOP-like immunochemotherapy. Besides, the CD30 expression may be a useful prognostic marker and a possible therapeutic target

Les facteurs de transcription STAT3 sont activés dans de nombreuses tumeurs et leurs effets sur la prolifération et la survie suggéraient fortement que ces protéines puissent être impliquées à la fois dans la transformation cellulaire et dans l'échappement aux traitements classiques de chimiothérapie.<br />Nous nous sommes donc intéressés aux différents aspects impliquant STAT3 dans la réponse de lignées cellulaires aux agents génotoxiques de chimiothérapie, et plus particulièrement aux inhibiteurs de topoisomérase. Durant ces traitements, STAT3 interagit avec le répresseur transcriptionnel Rb et l'inhibiteur du cycle cellulaire p21. Ces deux protéines inhibent son activité transcriptionnelle, notamment sur les gènes c-myc et cdc25A, et permettent la mise en place de la sénescence induite par les dommages de l'ADN et la catastrophe mitotique. Cependant, en présence d'une activité constitutive de STAT3 induite par l'oncogène v-src, STAT3 empêche l'activation de Rb et de p21, favorise la résistance des cellules aux inhibiteurs de topoisomérase II, et génère de l'instabilité génomique. Finalement,<br />deux inhibiteurs de STAT3, le cetuximab, un anticorps monoclonal dirigé contre l'EGFR, et un inhibiteur de la tyrosine kinase c-src, sensibilisent des cellules colorectales aux inhibiteurs de topoisomérase I. L'inhibition de STAT3 empêche l'activation du gène Eme1 qui induit l'expression d'une protéine de réparation de l'ADN.<br />STAT3 est donc un facteur de résistance aux inhibiteurs de topoisomérase. Sa détection pourrait ainsi permettre de mieux prédire la réponse des patients à ces inhibiteurs, et son inhibition, dans les tumeurs où il est actif, pourrait permettre de les sensibiliser aux inhibiteurs de topoisomérase.

<p>An important step in tumorgenesis is the acquisition of cellular immortality. Tumor cells accomplish this by activating the enzyme telomerase, and thereby avoiding replicative senescence. The aim of this thesis was to study the activity and regulation of telomerase in a panel of malignant cell types.</p><p>We found that TGF-β1 (transforming growth factor-β1) mediated differential effects on telomerase activity in five ATC (anaplastic thyroid carcinoma) cell lines. Cells that harbored a <i>p53</i> mutation responded by up-regulation of telomerase activity after TGF-β1 treatment, whereas cell lines displaying wt <i>p53 </i>responded by down-regulation of telomerase activity. Thus, these results indicate a possible connection between <i>p53</i> genotype and telomerase response to TGF-β1 treatment. Furthermore, the decreased telomerase activity appeared to be due to transcriptional repression of the <i>hTERT</i> promoter and the increased activity possibly involved hTERT activation via phosphorylation. </p><p>We have previously shown that IFNs (interferons) sensitize MM (multiple myeloma) cells to Fas-mediated apoptosis. In the present investigation both IFN-α and IFN-γ down regulated telomerase activity in the MM cell line U-266-1970. The mechanism underlying the reduction of telomerase activity by IFN was shown to be transcriptional repression of the <i>hTERT </i>gene. We suggest that one potential mechanism whereby IFN sensitize MM cells to Fas-mediated apoptosis is by repressing <i>hTERT</i> activity at the transcriptional level. </p><p>In the next study we demonstrated that basal telomerase activity is not a key determinant of sensitivity to cytotoxic drugs in ESCC (esophageal squamous cell carcinoma) cell lines. Furthermore, we observed no correlation between <i>c-Myc</i> amplification, <i>p53</i> mutations and high telomerase activity levels in these cell lines. </p><p>Finally, neuroblastoma cell lines were shown to up-regulate telomerase activity in response to hypoxic exposure and the main regulatory mechanism was not mediated by increased hTERT mRNA expression. This finding might constitute an adaptive stress response of tumor cells exposed to hypoxia. </p>

An important step in tumorgenesis is the acquisition of cellular immortality. Tumor cells accomplish this by activating the enzyme telomerase, and thereby avoiding replicative senescence. The aim of this thesis was to study the activity and regulation of telomerase in a panel of malignant cell types. We found that TGF-β1 (transforming growth factor-β1) mediated differential effects on telomerase activity in five ATC (anaplastic thyroid carcinoma) cell lines. Cells that harbored a p53 mutation responded by up-regulation of telomerase activity after TGF-β1 treatment, whereas cell lines displaying wt p53 responded by down-regulation of telomerase activity. Thus, these results indicate a possible connection between p53 genotype and telomerase response to TGF-β1 treatment. Furthermore, the decreased telomerase activity appeared to be due to transcriptional repression of the hTERT promoter and the increased activity possibly involved hTERT activation via phosphorylation. We have previously shown that IFNs (interferons) sensitize MM (multiple myeloma) cells to Fas-mediated apoptosis. In the present investigation both IFN-α and IFN-γ down regulated telomerase activity in the MM cell line U-266-1970. The mechanism underlying the reduction of telomerase activity by IFN was shown to be transcriptional repression of the hTERT gene. We suggest that one potential mechanism whereby IFN sensitize MM cells to Fas-mediated apoptosis is by repressing hTERT activity at the transcriptional level. In the next study we demonstrated that basal telomerase activity is not a key determinant of sensitivity to cytotoxic drugs in ESCC (esophageal squamous cell carcinoma) cell lines. Furthermore, we observed no correlation between c-Myc amplification, p53 mutations and high telomerase activity levels in these cell lines. Finally, neuroblastoma cell lines were shown to up-regulate telomerase activity in response to hypoxic exposure and the main regulatory mechanism was not mediated by increased hTERT mRNA expression. This finding might constitute an adaptive stress response of tumor cells exposed to hypoxia.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus that has been associated with all epidemiological forms of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV undergoes two phases of life cycle (latent and lytic replication). During latency, the viral genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed, namely LANA (latency- associated nuclear antigen), Kaposin, vFLIP (viral FLICE inhibitory protein), vCyclin, and vIRF3/LANA2 (viral interferon regulatory factor 3). These viral genes are responsible for regulation of host cell proliferation, prevention of apoptosis, facilitation of immune evasion, and maintenance of the extrachromosomal viral genome during cell divisions. vIRF3 is a multifunctional nuclear protein that is constitutively expressed in KSHV positive PEL cells and Castleman's disease tumors, which expression causes dramatic changes of critical host pathways that are involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. In our study, we have demonstrated and elucidated predicted mechanism, by which vIRF3 enhances transcription activity of c-Myc. Moreover, we have clarified the previously unappreciated...

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.

Le carcinome hépatocellulaire (CHC) est la première néoplasie létale du foie, représentant 80 à 90% des cas. Actuellement, la majeure partie des patients bénéficient de solutions thérapeutiques avec des efficacités très modestes. La haute variété étiologique, l’hétérogénéité des tumeurs ainsi que l’absence de médiateurs oncogéniques clés connus dans le développement de cette pathologie sont responsables du manque d’options thérapeutiques. À partir d’un crible génétique du kinome humain, nous avons identifié la tyrosine kinase Yes comme un acteur majeur de la prolifération des cellules de CHC. Yes appartient à la famille des kinases Src qui contrôlent de nombreux processus cellulaires notamment la prolifération, la motilité et la survie. La sur-expression ou activation anormale de Yes est retrouvée dans de nombreux cancers et est souvent associée à un mauvais pronostic. Nous avons démontré par des expériences in vitro et in vivo l’activité pro-proliférative de Yes ainsi que son potentiel oncogénique. Notamment, dans un modèle murin de carcinogenèse hépatique induit par le diéthylnitrosamine, la déplétion génétique de Yes abolit totalement la formation de tumeurs. Grâce aux profils transcriptionnels obtenus dans plusieurs modèles cellulaires de CHC, nous avons découvert que l’activité de Yes est associée à une augmentation des signatures géniques des régulateurs transcriptionnels YAP et TAZ ainsi que du facteur transcriptionnel c-Myc. Ces observations ont abouti à identifier YAP, TAZ et c-Myc comme des nouveaux substrats de la tyrosine kinase Yes. Nous avons montré que la phosphorylation par Yes de YAP et TAZ médie leur recrutement dans le noyau ce qui conduit à une hausse de leur activité transcriptionnelle. Nous avons d’ailleurs confirmé l’importance de YAP et TAZ dans les propriétés prolifératives de Yes, notamment dans différents modèles murins de carcinogenèse hépatique. De manière intéressante, nous avons observé que près de 50% de CHCs humains présentent une activation anormale des kinases Src qui corrèle avec la phosphorylation et activation de YAP. Enfin, nous avons observé in vitro et in vivo que Yes stabilise c-Myc. En effet, l’expression transgénique de Yes constitutivement actif dans des hépatocytes entraine la stabilisation de c-Myc à des stades précoces du développement tumoral et une induction de plusieurs de ses gènes cibles à des stades plus tardifs. En plus de leur synergie d’action, cette étude propose que la tyrosine Yes intervient dans les propriétés oncogéniques de c-Myc. Finalement, nous avons découvert que la kinase Yes joue un rôle dans la progression de la stéatose hépatique. En effet, la progression de la pathologie est abolie à la suite de la déplétion de Yes ou suivant l’inhibition pharmacologique des kinases Src. De plus, la survie des cellules tumorales face à leur élimination par le système immunitaire semble être favorisé par la signalisation Yes qui induit l’expression des points de contrôle immuns PD-L1/2. En conclusion nous avons découvert et caractérisé trois nouveaux effecteurs clés de la signalisation oncogénique de la tyrosine kinase Yes dans le CHC. D’ailleurs, la signature génique induite par Yes permet de prédire la survie des patients atteints de CHC. Ces données fournissent de robustes évidences qui placent la tyrosine kinase Yes comme une cible thérapeutique de choix pour la maladie du CHC. / Hepatocellular carcinoma (HCC) is the first lethal neoplasia of the liver, representing 80 to 90% of cases. Currently, for most patients the therapeutic option only provides modest efficiencies. The high etiological variety and heterogeneity of the tumors as well as the absence of known key oncogenic mediator in the development of this pathology is mainly responsible for the lack of therapeutic option. Based on a genetic screen of the human kinome, we identified the tyrosine kinase Yes as a major player in the proliferation of HCC cells. Yes belongs to the family of Src kinases which control many cellular processes including proliferation, motility and survival. The over-expression or abnormal activation of Yes is detected in many cancers and is often associated with poor prognosis. We have demonstrated in vitro and in vivo the pro-proliferative activity of Yes as well as its oncogenic potential. In particular, in a mouse model of hepatic carcinogenesis induced by diethylnitrosamine, the genetic depletion of Yes completely abolishes the formation of tumors. Thanks to the transcriptional profiles obtained in several cellular models of CHC, we discovered that the activity of Yes is associated with an increase in the gene signatures of the transcriptional regulators YAP and TAZ as well as of the transcriptional factor c-Myc. These observations led to the identification of YAP, TAZ and c-Myc as new substrates for the tyrosine kinase Yes. We have shown that the phosphorylation of YAP and TAZ by Yes mediates their recruitment into the nucleus associated with an increase in their transcriptional activity. We have also confirmed the importance of YAP and TAZ in the proliferative properties of Yes in various mouse models of hepatocarcinogenesis. Interestingly, we observed that nearly 50% of human CHCs exhibit an abnormal activation of Src kinases that correlates with phosphorylation and activation of YAP. Moreover, in vitro and in vivo experiments revealed that Yes stabilizes c-Myc. Indeed, the transgenic expression of constitutively active Yes into hepatocytes leads to the accumulation of c-Myc protein at early stages of tumor development and to the induction of several of its target genes at later stages. In addition to their synergistic action, this study suggests that Yes is involved in the oncogenic properties of c-Myc. Finally, we discovered that Yes kinase plays a role in the progression of fatty liver diseases. Indeed, the progression of the pathology is abolished following the depletion of Yes or the pharmacological inhibition of Src kinases. In addition, the survival of Yes-active tumor cells is associated with the induction of PD-L1/2 immune checkpoints that protect cells from immune elimination. In conclusion, we have discovered and characterized three new key effectors of the oncogenic tyrosine kinase Yes in HCC. Interestingly, the gene signature induced by Yes can predict the survival of patients with HCC. These data provide strong evidence for targeting the tyrosine kinase Yes in HCC.

No description available.

This dissertation is a composite of some of the research that I have conducted during the course of my PhD study. The larger goal of this dissertation is to renew the interests among the scientific community for an otherwise under-appreciated technique called Isothermal Titration Calorimetry. The resurgence of calorimetry in the biophysical community and the shift to investigations of more complex biological systems signal a real need for more sophisticated analysis techniques. This dissertation expounds on new ITC analysis methods that we have developed as well as results from the study of thermodynamic properties of higher order DNA structures. In 1978, Peter Privalov described the first use of microcalorimetry to obtain the thermodynamic properties for removing calcium from parvalbumin III protein. Fast forward 36 years: modern day electronics, highly efficient thermally conductive and chemically inert materials, in conjunction with sensitive thermal detectors, has transformed the original calorimeter into a device capable of measuring heat changes as small as 0.05 nanowatts, which is equivalent to capturing heat from an incandescent light bulb a kilometer away. However, analytical methods have not kept pace with this technology. Commercial ITC instruments are typically supplied with software that only includes a number of simple interaction models. As a result, the lack of analysis tools for more complex models has become a limiting factor for many researchers. We have recently developed new ITC fitting algorithms that we have incorporated into a userriendly program (CHASM©) for the analysis of complex ITC equilibria. In a little over a year, CHASM© has been downloaded by over 370 unique users. Several chapters in this dissertation demonstrate this software’s power and versatility in the thermodynamic investigations of two model systems in both aqueous and non-aqueous media. In chapter VI, we assembled a model NHE-III1 : a novel structure of Gquadruplex in a double stranded form and studied its structural complexity and binding interactions with a classical G-quaduplex interactive ligand known as TMPyP4. In chapter VII, we reported the thermodynamic properties of a novel PAH system in which weak dispersion forces are solely responsible for formation of the supramolecular complexes.