Le suppresseur de tumeur p53 permet à la cellule de se défendre contre différentes formes de stress. Il joue un rôle de barrière s'opposant à la tumorigenèse: en effet la perte de p53 chez la souris prédispose grandement ces animaux à développer des tumeurs; de même le locus p53 est inactivé dans près de 50% des tumeurs humaines. p53 constitue un facteur de transcription qui se lie à des séquences particulières de l'ADN et active l'expression des gènes adjacents. L'expression orchestrée de ces gènes conduit, directement ou indirectement et suivant le contexte cellulaire, soit à la mort de la cellule soit à l'inhibition de la division cellulaire. Les mécanismes moléculaires médiant ces deux activités biologiques essentielles de p53, de même que les mécanismes influençant le choix de la réponse cellulaire, sont encore mal compris. L'importance de p53 dans ce choix reste également à démontrer. Afin de contribuer à la compréhension de ces mécanismes, le modèle murin déficient pour Mdm4, un régulateur négatif de l'activité de p53, a été choisi. L'inactivation de Mdm4 chez la souris conduit en effet à l'activation ectopique de p53 in vivo et l'induction de deux types de réponse: apoptose dans le neuroépithélium et arrêt de la prolifération cellulaire dans les tissus non neuronaux. Le profil d'expression des gènes dans les tissus neuronaux et non neuronaux a donc été comparé entre embryons de souris sauvage et mdm4-/- par la technique d'hybridation de biopuces à ADN. Les résultats obtenus suggèrent que le type de réponse dépend du type cellulaire et non de p53 lui-même. En effet les profils d'expression des gènes dans les tissus neuronaux (conditions d'apoptose) et non neuronaux (conditions d'arrêt de la prolifération cellulaire) chez l'embryon de souris mdm4-/- sont comparables. Nous nous sommes ensuite particulièrement intéressés à deux nouveaux gènes dont l'expression est augmentée dans les embryons mdm4-/-. Dans un premier temps, leur induction transcriptionnelle chez l'embryon de souris mdm4-/- a été confirmée par différentes techniques et il a été vérifié qu'ils constituaient tous deux des cibles directes de p53 induites suite à un stress génotoxique. Le premier gène code Dapk1, une protéine suppresseur de tumeur pro-apoptotique présentant une activité de type sérine/thréonine kinase. Ce travail a permis d'établir que Dapk1 participait à une boucle de rétroaction du contrôle de l'activité de p53. Le deuxième gène identifié code la protéine Ptprv, un récepteur transmembranaire présentant une activité de type tyrosine phosphatase. En vue d'étudier la signification physiologique de l'induction transcriptionnelle de ptprv suite à l'activation de p53, des expériences effectuées à partir de matériel biologique issu de souris déficientes pour Ptprv ont été réalisées. Ces expériences confirment le rôle essentiel de Ptprv comme médiateur de l'arrêt du cycle cellulaire en phase G1 induit par p53 suite à un stress génotoxique, à la fois in vitro et in vivo. Par contre, Ptprv ne semble pas influencer l'apoptose induite suite à l'activation de p53. Ce travail a également permis d'établir le rôle essentiel de Ptprv dans la suppression de tumeurs induites chez la souris par activation constitutive de l'oncogène Ras.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-06-03T19:14:49Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AnaliseMolecularGenes.pdf: 1713438 bytes, checksum: c87a3877b195a2f8284332c3ab05f12f (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-06-04T14:55:45Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AnaliseMolecularGenes.pdf: 1713438 bytes, checksum: c87a3877b195a2f8284332c3ab05f12f (MD5) / Made available in DSpace on 2013-06-04T14:55:45Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_AnaliseMolecularGenes.pdf: 1713438 bytes, checksum: c87a3877b195a2f8284332c3ab05f12f (MD5) Previous issue date: 2011 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os rotavírus são os principais agentes virais causadores de gastrenterite aguda e responsáveis por 36% dos casos hospitalizações entre crianças menores de cinco anos, resultando em 453.000 óbitos anualmente, principalmente em países em desenvolvimento. Pertencem à família Reoviridae, gênero Rotavirus, possui RNA de dupla fita (dsRNA) com 11 segmentos codificando 12 proteínas. O genótipo G1 se apresenta geralmente com maior frequência nas investigações epidemiológicas, circulando em várias partes do mundo sob diferentes prevalências. Este estudo teve como objetivo analisar a variabilidade genética dos genes VP4, VP7 e NSP4 dos rotavírus G1 circulantes nos municípios de Belém e Marituba, Pará, Brasil, no período de 1982 a 2008. Foram selecionadas 83 amostras previamente caracterizadas como G1 e submetidas a RT-PCR. Os espécimes foram provenientes de sete estudos realizados no IEC. Foi possível a amplificação para os três genes em estudo de 63 (75,9%) espécimes. Foram detectadas as linhagens 1 (8/63, 12,7 %), 2 (29/63, 46,0%), 3 (18/63, 28,6%) e 9 (8/63, 12,7%) para o gene VP7. Co-predominaram as sublinhagens 2E e 3A concorrendo com um total de 57,1% (36/63) das amostras. Foram observadas três substituições de aminoácidos (97 [D→E], 147 [S→N] e 218 [I→V]) no gene VP7 nas regiões antigênicas (A, B e C) nas amostras das linhagens 1, 2 e 9. Todas as amostras apresentaram a especificidade P[8] para o gene VP4 e as linhagens 2 (21/63, 33,3%) e 3 (42/63, 66,7%) foram detectadas. No gene da VP4 ocorreram duas alterações (35 [I→V] e 38 [S→G]) na região antigênica em todas as amostras analisadas. Para o gene NSP4, todas as amostras pertenceram ao tipo E1. Houve mudanças de nucleotídeos nas posições 47 (C→T) e 101 (T→C), resultando em alteração aminoacídica nos resíduos 16 (S→P) e 34 (L→P) em todas as amostras analisadas e nove espécimes demonstraram alteração no sítio de toxicidade da NSP4 (aa 131). Tal análise permitiu ampliar o conhecimento da diversidade genética e da circulação de variantes de rotavírus G1, representando o primeiro estudo da epidemiologia molecular deste genótipo no Brasil e confirmar a alta heterogeneidade que este tipo apresenta. / Rotaviruses are major viral agents of acute gastroenteritis and responsible for 36% of hospitalization for diarrhea among children less than five years of age, resulting in 453.000 deaths annually, mostly in developing countries. Rotavirus is a member of Reoviridae family, and its genome consists of 11 double-stranded RNA (dsRNA) which encode 12 proteins. G1 rotavirus is commonly detected in epidemiological investigations, occurring under different prevalence rates. The aim of this study was to analyze the VP4, VP7 and NSP4 diversity genetic of G1 rotavirus circulating in Belém and Marituba, Pará, Brazil, from 1982 to 2008. We selected 83 samples previously characterized as G1 type and submitted to RT-PCR. The samples were from seven studies conducted in IEC. It was possible amplification for 63 (75.9%) specimens. Lineages 1 (8/63, 12.7%), 2 (29/63, 46.0%), 3 (18/63, 28.6%) and 9 (8/63, 12.7%) of VP7 gene were detected. The sublineages 2E and 3A were co-predominant detected in 57.1% (36/63) of samples. Three amino acid substitutions (97 [D→E], 147 [S→N] and 218 [I→V]) were observed in VP7 antigenic regions (A, B and C) in samples of 1, 2 and 9 lineages. All samples showed P[8] specificity for VP4 gene and lineages 2 (21/63, 33.3%) and 3 (42/63, 66.7%) were detected. Two substitutions (35 [I→V] and 38 [S→G]) occurred in antigenic region of VP4 of samples analyzed. For NSP4 gene, all samples belonged to E1 type. Phylogenetic analysis of NSP4 gene revealed that occurred changes in nucleotide positions 47 (C→T) and 101 (T→C), resulting in amino acid substitutions at positions 16 (S→P) and 34 (L → P) in all samples and 9 specimens displayed amino acid substitution in NSP4 toxicity residue (aa 131). This study allowed us to broaden our understanding about genetic diversity and circulation of G1 variants and represents the first molecular epidemiology analyze of this genotype in Brazil corroborating the high heterogeneity of this genotype.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 144-170). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Exposure to external stimuli promotes a variety of cellular responses including changes in morphology, gene expression and cell division status. These responses are promoted by signaling pathways composed of modules that are conserved from lower to higher eukaryotes. In Saccharomyces cerevisiae response to the external stimuli provided by mating pheromone is governed by the pheromone response pathway. This pathway is composed of a G protein coupled receptor/heterotrimeric G protein (Gαβγ) module and a MAP kinase cascade. Activation of this pathway allows the heterotrimeric G protein βγ dimer (Gβγ) to recruit polarity proteins to promote changes in cell morphology and to activate signaling through the MAP kinase cascade. Here we investigate the regulation of these pheromone-induced responses. We first examine how an asymmetric polarization response is generated. Normally, a gradient of pheromone serves as a spatial cue for formation of a polarized mating projection, but cells can still polarize when pheromone is present uniformly. Here we show that an intact receptor/Gαβγ module is required for polarization in response to both a gradient and uniform concentration of pheromone. Further investigation into regulation of Gβγ by Gα revealed that the two interaction interfaces between Gα and Gβ have qualitatively different roles. Our results suggest that one interface controls signaling whereas the other governs coupling to the receptor. Overall our results indicate that communication between the receptor and Gαβγ is required for proper polarization. We then examine how G1 CDKs regulate MAP kinase signaling. Response to pheromone is restricted to the G1 stage of the cell cycle. Once cells commit to a round of division they become refractory to mating pheromone until that round of division is complete. One contributor to this specificity involves inhibition of signaling through the MAP kinase cascade by G1 CDKs, but it was not known how this occurs. Here, we show that the MAP kinase cascade scaffold Ste5 is the target of this inhibition. Cln/CDKs inhibit signaling by phosphorylating sites surrounding a small membrane-binding domain in Ste5, thereby disrupting the membrane localization of Ste5. Furthermore, we found that disrupting this regulation allows cells to arrest at an aberrant non-G1 position. Our findings define a mechanism and a physiological benefit for restricting pheromone-induced signaling to G1. This thesis describes findings related to generation of an asymmetric polarization response, heterotrimeric G protein function, and coordination of differentiation signaling with cell division status. Lessons learned here might be applicable to the regulation of polarization and differentiation responses in other systems as the signaling modules are conserved.

Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.

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