O câncer de esôfago é a sexta neoplasia maligna mais comum no mundo. No Rio Grande do Sul, Brasil, o carcinoma epidermóide de esôfago apresenta coeficientes de mortalidade elevados e com tendência ascendente com, pelo menos, o dobro dos coeficientes padronizados de mortalidade encontrados em outros estados brasileiros ou em países do cone sul da América latina. O diagnóstico tardio parece ser o principal responsável pelo mau prognóstico. Nos últimos anos, diversos estudos têm demonstrado a possibilidade de identificação das lesões precursoras do câncer esofágico, mas sem repercussão no prognóstico, até o momento. Considera-se, atualmente, que a carcinogênese esofágica está relacionada a uma interação entre fatores ambientais e anormalidades genéticas Recentemente, estudos em biologia molecular têm demonstrado a influência dos fatores reguladores do ciclo celular no prognóstico de diversas moléstias, inclusive o câncer. O Rb é um gene supressor tumoral envolvido no mecanismo de controle do ciclo celular, cuja expressão tem sido demonstrada no câncer do esôfago. O objetivo deste estudo foi determinar a prevalência da perda da expressão da proteína pRb na mucosa esofágica de indivíduos sob risco para o carcinoma epidermóide de esôfago, bem como relacionar esta expressão com o consumo de tabaco, álcool e chimarrão, achados histopatológicos e cromoscopia com lugol. Foram estudados 170 casos e 20 controles através de reação imunohistoquímica utilizando anticorpo monoclonal anti-pRb em amostras teciduais fixadas em formalina e armazenadas em parafina. Um total de 33 casos demonstrou perda da expressão imunohistoquímica da proteína pRb, determinando uma prevalência de 19,4% na amostra estudada. Não houve associação estatisticamente significativa entre a perda da expressão da proteína pRb e as variáveis idade, raça, exposição ao fumo, álcool e chimarrão, bem como a cromoendoscopia com lugol. Foi demonstrada uma associação significativa entre a perda da expressão da pRb com a história de câncer na família. Da mesma forma, foi demonstrada uma associação linear significativa entre a perda da pRb e o grau histopatológico das lesões. Estes resultados demonstraram uma influência da proteína pRb na evolução da carcinogênese esofágica e permitem sugerir que os indivíduos expostos aos fatores de risco estudados sejam candidatos a uma maior vigilância.

O câncer de esôfago é a sexta neoplasia maligna mais comum no mundo. No Rio Grande do Sul, Brasil, o carcinoma epidermóide de esôfago apresenta coeficientes de mortalidade elevados e com tendência ascendente com, pelo menos, o dobro dos coeficientes padronizados de mortalidade encontrados em outros estados brasileiros ou em países do cone sul da América latina. O diagnóstico tardio parece ser o principal responsável pelo mau prognóstico. Nos últimos anos, diversos estudos têm demonstrado a possibilidade de identificação das lesões precursoras do câncer esofágico, mas sem repercussão no prognóstico, até o momento. Considera-se, atualmente, que a carcinogênese esofágica está relacionada a uma interação entre fatores ambientais e anormalidades genéticas Recentemente, estudos em biologia molecular têm demonstrado a influência dos fatores reguladores do ciclo celular no prognóstico de diversas moléstias, inclusive o câncer. O Rb é um gene supressor tumoral envolvido no mecanismo de controle do ciclo celular, cuja expressão tem sido demonstrada no câncer do esôfago. O objetivo deste estudo foi determinar a prevalência da perda da expressão da proteína pRb na mucosa esofágica de indivíduos sob risco para o carcinoma epidermóide de esôfago, bem como relacionar esta expressão com o consumo de tabaco, álcool e chimarrão, achados histopatológicos e cromoscopia com lugol. Foram estudados 170 casos e 20 controles através de reação imunohistoquímica utilizando anticorpo monoclonal anti-pRb em amostras teciduais fixadas em formalina e armazenadas em parafina. Um total de 33 casos demonstrou perda da expressão imunohistoquímica da proteína pRb, determinando uma prevalência de 19,4% na amostra estudada. Não houve associação estatisticamente significativa entre a perda da expressão da proteína pRb e as variáveis idade, raça, exposição ao fumo, álcool e chimarrão, bem como a cromoendoscopia com lugol. Foi demonstrada uma associação significativa entre a perda da expressão da pRb com a história de câncer na família. Da mesma forma, foi demonstrada uma associação linear significativa entre a perda da pRb e o grau histopatológico das lesões. Estes resultados demonstraram uma influência da proteína pRb na evolução da carcinogênese esofágica e permitem sugerir que os indivíduos expostos aos fatores de risco estudados sejam candidatos a uma maior vigilância.

Development, tissue renewal and longevity of multi-cellular organisms require the ability to switch between a proliferative state and quiescence, a reversible arrest from the cell cycle. The balance of quiescence and proliferation underlies the fundamental feature of generating and maintaining the appropriate number of cells, which is essential for tissue architecture, regeneration, and function. Disruption of quiescence and proliferation balance leads to hypo- or hyper-proliferative diseases. To date, the regulatory mechanism of proliferation has been well established, while cellular quiescence has remained a phenotypic description without a clearly defined molecular control mechanism. Simply, quiescence has long been considered a passive counterpart to proliferation. However, recent findings have revealed that quiescence is an actively maintained state exhibiting a unique gene expression pattern. While quiescence has been traditionally considered as a state (namely G0) outside of the cell cycle, it is in fact a collection of heterogeneous states. In studies conducted in the 70's and 80's using fibroblasts and lymphocytes, it has been observed that the longer the cells were kept under quiescence inducing conditions such as contact inhibition, the deeper the cells moved into quiescence. Deep quiescent cells are still able to reenter the cell cycle upon growth stimulation but they exhibit a longer pre-DNA synthesis phase [1-4]. Shallow quiescent state has also been recently reported in muscle and neural stem cells termed GAlert and "prime" quiescent state, respectively. Heterogeneous quiescent depth entails that cells vary in their sensitivity to growth signals, representing an important yet underappreciated layer of complexity in cell growth control. The cellular mechanisms that control the depth of quiescence remains elusive. In my thesis work, I first investigate the strengths of serum stimulation required for cells to exit deep and shallow quiescence as a determinant of quiescence depth. Through model simulations and experimental measurements, I further demonstrate that various components of the Rb-E2F pathway control quiescence depth with varying efficacy. The Rb-E2F pathway interacts with diverse cellular pathways that respond to environmental signals to jointly modulate quiescence depth. Given that certain circadian clock genes (e.g., Cry) affect key components in the Rb-E2F pathway, I tested the effect of Cry activity on quiescence depth. I found that increased Cry activity resulted in deeper quiescence, contrary to our anticipation based on the literature. Next, we constructed a library of mathematical models that represent possible interactions between Cry and the Rb-E2F pathway. We computationally searched this model library for links that could explain the experimental observations. The modeling search suggested that Cry upregulation may lead to increased expression of cyclin dependent kinase inhibitor (e.g., p21), which in turn drives cells into deeper quiescence. This model prediction was confirmed by my follow-up experiments. Collectively, my thesis work establishes an integrated modeling and experimental framework that will help us to further investigate diverse cellular mechanisms controlling the heterogeneous quiescence depth.

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa RING finger-containing protein that plays a role in 3'-end poly-adenylation of mRNA transcripts as well as acting as an E3 ubiquitin ligase against a number of proteins involved in tumourigenesis, including p53. Since the human protein is too large and poorly structured for heterologous expression in bacteria, it would be advantageous to identify smaller fragments suitable for expression in bacteria. Many E3 ubiquitin ligases form homo-dimers and dimerisation is important for their activity; structural studies of the isolated RING finger of RBBP6 showed that it forms a weak homo-dimer. This poses the question of whether the complete RBBP6 protein forms homo-dimers in vivo, and, if so, whether a fragment of RBBP6 containing the RING finger could be identified which would be suitable for structural as well as functional studies. Such a construct would allow detailed investigation of the homo-dimeric state of the fragment, the relationship between dimerisation and ubiquitination activity, and the role of domains such as the DWNN domain and zinc finger in ubiquitination. A fragment consisting of the first 335 residues of RBBP6, dubbed R3 because it contained the first three domains of the protein, was expressed, along with three variants expressing mutations known to disrupt the dimerisation of the isolated RING finger. Size exclusion chromatography showed that R3 forms a strong homo-dimer that was not disrupted by the mutations, suggesting that additional parts of R3 outside of the isolated RING finger form part of the interface. To identify whether this included the DWNN domain or the zinc finger, a shorter fragment dubbed R2, excluding the N-terminal DWNN domain, was cloned and expressed. This was also found to form a strong homo-dimer, suggesting that the DWNN domain may not form an essential part of the dimer interface. Availability of the RING finger samples and monomerising mutations allowed investigation of whether the RING finger from RBBP6 was able to auto-ubiquitinate itself. Using a fully in vitro ubiquitination assay supplemented with intact proteasomes purified from human cell lysates, we found that wild type RING auto-ubiquitinates itself very efficiently, catalysing its own destruction in the proteasome. This provides an answer to the question of why RBBP6 is so difficult to detect in mammalian cells. Surprisingly, monomeric mutant RING fingers were also able to auto-ubiquitinate and catalyse their own destruction, although perhaps not as efficiently as wild type. This result would appear to rule out the hypothesis that dimerisation of RBBP6 is required for ubiquitination activity. Finally, samples of the RING finger from human MDM2 were expressed in bacteria and used to investigate whether the RING fingers of RBBP6 and MDM2 interact directly with each other. If so, this may provide a mechanism whereby RBBP6 and MDM2 cooperate in ubiquitination of p53. The results of a GST pull down assay using GST-MDM2-RING as ''bait'' and RBBP6-RING as ''prey'' provides evidence that such an interaction between the RING does exist. This work lays the foundation for future structural studies of the RING-RING hetero-dimer using protein Nuclear Magnetic Resonance Spectroscopy.

La photothérapie dynamique (PDT) est un nouveau traitement n’induisant potentiellement pas de mutation et utilisable pour lutter contre le rétinoblastome. Les dérivés de porphyrine utilisés comme Ps dans la Thérapie PhotoDynamique (PDT) sont largement étudiés depuis la naissance du premier Ps de synthèse (l’HpD). Une limitation importante de la PDT provient de la faible profondeur (confinée près de la surface) de pénétration de la lumière (λ <700 nm) employée pour l'excitation du Ps. Afin de fournir une énergie d’activation suffisante pour produire l’oxygène singulet dans la fenêtre thérapeutique entre 700-1300, le processus d'absorption à deux photons a été proposé. Les Ps excitables par l’absorption simultanée de deux photons conduisent au concept de PDT à deux photons (PDT-ADP). Ce processus ayant une faible probabilité, son application demande le développement de nouveaux Ps avec une section efficace importante. Une autre limitation de la PDT est la faible sélectivité et spécificité des Ps actuels pour les cellules tumorales. Un ciblage actif de récepteurs membranaires spécifiques des cellules tumorales représente une solution possible. Il a été rapporté que des récepteurs de type lectine reconnaissant certains sucres sont surexprimés par des cellules malignes. Nous présenterons la synthèse et les résultats photocytotoxiques de dimères dissymétriques de porphyrine P-Y-P', inspirée d'études précédentes du laboratoire, privilégiant l’introduction de trois chaînes para-phénoxy-diéthylène glycol mannose sur trois des positions méso de porphyrines optimisés pour l’absorption à deux photons et vectorisés vers des lectines membranaires.Pour contourner le problème de solubilité des porphyrines dimères en milieu aqueux et améliorer l’internalisation de Ps dans les cellules tumorales, nous avons analysé le comportement interfacial des porphyrines dimères à l'interface air-tampon, étudié leur incorporation dans les liposomes à l’aide de la technique de la fluorescence, et évalué l’interaction entre porphyrin dimères et la Concanavaline A (une lectine extraite de Canavalia ensiformis qui reconnaît de manière spécifique l'alpha-D-mannose). / Photodynamic therapy (PDT) is a new potential treatment against retinoblastoma, which doesn’t induce mutations. The porphyrin derivatives used as Ps in PDT are widely studied since the birth of the first synthesis Ps (HpD). An important limitation of PDT comes from low penetration of light (λ<700 nm) used for excitation of the Ps. In order to provide enough energy to enable production of singlet oxygen in the phototherapeutic window between 700-1300, the absorption of two relatively low-energy photons simultaneously has been proposed. Ps excited by simultaneous absorption of two photons leads to the concept of two-photon PDT (TPE-PDT). This process has very low probability; its application in the PDT needs develop new Ps with intensive cross section. Another limitation is the low selectivity and specificity of current Ps for tumor cells. Active targeting to appropriate receptors expressed at the tumor cells give a possible solution. It has been reported that the lectin-like receptors recognizing certain sugars are overexpressed in malignant cells. We will present the synthesis and in vitro photocytotoxical results of asymmetric porphyrin dimers P-Y-P', inspired by previous studies of our laboratory, introducing three para-phenoxy-diethylene glycol mannose chains at the meso positions of porphyrin core which optimized for two-photon absorption and targeted to membrane lectins. To circumvent the solubility problem of porphyrin dimers in aqueous medium and improve internalization of Ps into tumor cells, we analyzed the interfacial behavior of the porphyrin dimers in the air-buffer interface, studied the incorporation of porphyrin dimers in liposomes using the technique of fluorescence, and evaluated the interaction between porphyrin dimers and Concanavalin A (Canavalia ensiformis lectin derived from that specifically recognizes the alpha-D-mannose).

Le rétinoblastome est la tumeur intraoculaire primitive la plus fréquente de l’enfant. Les traitements actuels du rétinoblastome sont associés à de nombreux effets secondaires. De nouvelles approches thérapeutiques (telles que la photothérapie dynamique [PDT] ou les injections intra-vitréennes [IVT] de chimiothérapies) doivent donc être évaluées sur des modèles animaux, en vue d’une éventuelle application clinique.Dans cette thèse nous avons tout d’abord caractérisé un modèle murin obtenu par xénogreffe orthotopique de cellules issues de rétinoblastomes humains. Nous avons montré que la croissance tumorale intraoculaire est possible dans des lignées de souris immunodéficientes (Swiss-nude et SCID [severe combined immunodeficiency]) et dans une lignée immunocompétente (B6Albino). En raison du taux de prise tumorale insuffisant (entre 28.4% et 68.8% selon les lignées de souris utilisées) et des complications oculaires liées à l’injection orthotopique de cellules tumorales (cataracte, décollement de rétine chronique), les tests thérapeutiques (PDT et IVT de chimiothérapies) ont ensuite été réalisés sur un modèle murin transgénique de rétinoblastome (LHBetaTag).En vue du traitement par PDT, une étude de biodistribution par IRM (imagerie par résonance magnétique) du photosensibilisateur (PS, DEG-mannose) couplé au manganèse et une étude par dosage du PS ont été réalisées. Elles ont toutes les deux montré que l’illumination de la tumeur doit être réalisée 24 à 48h après l’administration intra-péritonéale du PS (ce qui correspond au « drug-to-light interval » du traitement par PDT). En utilisant ces paramètres, le traitement par PDT a été efficace sur les tumeurs rétiniennes des souris LHBetaTag. Au niveau de la zone traitée par PDT, il y a ainsi eu 91.7% de cicatrices choriorétiniennes en OCT (optical coherence tomography) pour un « drug-to-light interval » de 24h et 100% de cicatrices choriorétiniennes pour un « drug-to-light interval » de 48h. La rétine non tumorale située en dehors de la zone traitée par PDT avait un aspect normal en histologie, ce qui est en faveur d’une absence de toxicité rétinienne de la PDT sur les tissus sains. Le traitement par laser seul n’a pas eu d’effet anti-tumoral.Des traitements par IVT de chimiothérapies ont aussi été évalués sur les tumeurs rétiniennes des souris LHBetaTag. Les molécules utilisées ont été le melphalan, le carboplatine et le topotecan, administrées en mono ou en bithérapie. Nous avons montré que 4 IVT hebdomadaires de carboplatine à la dose de 1.5 µg ont la meilleure efficacité anti-tumorale (83.3% d’yeux sans masse tumorale en histologie) pour une toxicité rétinienne faible (21.4% d’yeux où il y a eu une diminution de l’épaisseur de la rétine non tumorale en OCT au cours du suivi in vivo). Le carboplatine semble donc être une alternative intéressante au melphalan, qui est actuellement la molécule la plus utilisée en clinique pour les IVT dans le rétinoblastome mais qui est associé à une toxicité rétinienne importante.En conclusion, ces études précliniques réalisées sur un modèle murin de rétinoblastome (LHBetaTag) montrent que la PDT est envisageable pour le traitement des tumeurs rétiniennes dans le rétinoblastome humain. Les IVT de carboplatine sont une perspective pour le traitement des flocons intra-vitréens dans cette maladie. Des évaluations fonctionnelles (électrorétinogramme, étude du réflexe optocinétique) devront cependant être réalisées chez la souris avant un éventuel passage en clinique afin de mieux caractériser une éventuelle toxicité rétinienne de ces traitements. / Retinoblastoma is the most common primary intraocular malignancy in children. Current retinoblastoma treatments have many adverse effects. New therapeutic approaches (like photodynamic therapy [PDT] or intravitreal injections [IVT] of chemotherapy) must therefore be evaluated on animal models, before a clinical application.In this thesis we first characterized an orthotopic xenograft murine model obtained with human retinoblastoma cells. We showed that intraocular tumor growth can be achieved in immunodeficient mouse strains (Swiss-nude and SCID [severe combined immunodeficiency]) and in an immunocompetent strain (B6Albino). Due to insufficient tumor engraftment rates (between 28.4 and 68.8% depending on the mouse strains) and to ocular complications after the injection of tumor cells (cataract, chronic retinal detachment) the treatments (PDT and IVT of chemotherapy) were performed on a transgenic retinoblastoma mouse model (LHBetaTag).In order to perform PDT, an MRI study (magnetic resonance imaging) of the photosensitizer (PS, DEG-mannose) coupled with manganese and a biodistribution study based on the dosage of the PS were performed. Both studies showed that the illumination of the tumor should be performed between 24 and 48h after the intraperitoneal injection of the PS (which corresponds to the “drug-to-light interval” of PDT). Using these parameters, PDT was effective on the retinal tumors of LHBetaTag mice. In the area treated with PDT we found 91.7% chorioretinal scars on OCT (optical coherence tomography) with a “drug-to-light interval” of 24h, and 100% chorioretinal scars with a “drug-to-light interval” of 48h. The retina outside the treated area had a normal aspect on histology, showing that PDT is not toxic on healthy tissues. Laser treatment alone had no anti-tumor effect.IVT of chemotherapy were also performed in LHBetaTag mice. We used melphalan, carboplatin and topotecan, alone or in association. We showed that 4 weekly IVT of carboplatin at the dose of 1.5 µg had the best anti-tumor effect (83.3% of eyes had no tumor mass on histology) and little retinal toxicity (21.4% of eyes had diminished retinal thickness on OCT). Carboplatin seems an interesting alternative to melphalan which is currently the most commonly used chemotherapy for IVT (but has a retinal toxicity).In conclusion, these preclinical studies on a retinoblastoma mouse model (LHBetaTag) show that PDT could be used to treat retinal tumors in human retinoblastoma. IVT of carboplatin could be used to treat vitreous seeds in this disease. Functional tests (electroretinogram, optokinetic reflex) should be performed in mice in order to evaluate more precisely the retinal toxicity of these treatments.

The discovery of mammalian protein prenylation was originally motivated by an effort to identify a nonsterol isoprenoid which indirect evidence suggested was a coregulator of isoprenoid biosynthesis and played a critical role in cellular proliferation. The first prenylated proteins to be identified were the nuclear lamin proteins-B lamins and prelamin A-which were subsequently shown to be farnesylated at a carboxyl-terminal CAAX motif. In both types of lamin, the farnesylation and carboxymethylation play a role in targeting these proteins to the nuclear envelope. The nucleus can be demonstrated to be a CAAX processing compartment for the lamins. In the case of prelamin A, there is removal of a carboxyl-terminal polypeptide which is specifically catalyzed by the enzyme Zmpste24. This processing event is necessary for assembly of lamin A into the lamina and may play a role in cell cycle control. Because the nucleus contains only one target membrane, lamin farnesylation and carboxymethylation may be sufficient to allow association with this membrane. This stands in contrast to farnesylated proteins expressed in the cytoplasm.

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