Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 15, 2009). Includes bibliographical references.

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 96-124.

Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006. / Typescript. Includes bibliographical references (leaves 69-83). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--Ohio University, March, 2007. / Title from PDF t.p. Includes bibliographical references.

Le gène suppresseur de tumeur LKB1 code une protéine sérine/thréonine kinase qui régule le métabolisme et la polarité cellulaires. LKB1 exerce une partie de ses fonctions biologiques en phosphorylant et en activant les 14 kinases appartenant à la famille des protéines kinases activées par l'AMP (AMPK). Le membre éponyme de cette famille, AMPK, agit comme un senseur nutritionnel essentiel dans la cellule. La recherche que j'ai conduite au cours de ma thèse a porté sur le mode de régulation de LKB1. L'holoenzyme LKB1, un hétérotrimère comprenant deux autres protéines appelées STRAD et MO25, est dotée d'une activité catalytique constitutive. Mon travail a permis de montrer que la lysine 48 de LKB1 est acétylée par l´acétyltransférase GCN5. Par des approches biochimiques et des techniques d'imagerie, j'ai montré que l'acétylation de LKB1 par GCN5 favorise sa localisation nucléaire, la fraction non-acétylée étant localisée à la fois dans le cytoplasme et le noyau. GCN5 promeut également l´export cytoplasmique de LKB1 de manière HAT-indépendente et régule son niveau d´expression. Afin de préciser la contribution de cette acétylation à la fonction in vivo de LKB1, j'ai utilisé le modèle expérimental de la crête neurale (CN) chez le poulet. En effet, j'ai été impliquée au cours de ma thèse dans une étude issue du laboratoire, qui a établi que l'activité de LKB1 est requise pour la délamination, la migration polarisée et la survie des cellules de la CN céphalique. Ces dernières contribuent à la formation de la majorité du squelette cranio-facial des vertébrés. Le signal LKB1 dans ces cellules est relayé par l'AMPK et la kinase ROCK et converge sur le moteur moléculaire dépendant de l'actine, la Myosine II. A l'aide du même modèle expérimental, j'ai montré que GCN5 est exprimé dans les cellules de la CN au cours de l'embryogenèse et que l'interaction fonctionnelle entre LKB1 et GCN5 est nécessaire à l'activité de LKB1 au cours de l'ontogénie des cellules de la CN céphalique et donc de la formation de la tête. / The tumor suppressor gene LKB1 encodes a serine/threonine kinase which regulates the cellular metabolism and polarity. Its biological activity is partly exerted through the phosphorylation and activation of 14 kinases which belong to the AMP-activated protein kinases (AMPK). The eponym member of this family acts as an essential nutritional sensor in the cell. The research that I conducted during my PhD focused on the regulation of LKB1. The LKB1 holoenzyme is a constitutively active heterotrimer comprising two other proteins called STRAD and MO25. My PhD project shows that LKB1 is acetylated on the lysine 48 residue by the acetyltransferase GCN5. Using biochemical approaches and cell imaging, I have shown that the acetylation of LKB1 by GCN5 favors its nuclear localization, while the non-acetylated fraction is localized in both the nucleus and the cytoplasm. GCN5 also promotes the cytoplasmic export of LKB1 in an HAT-independent manner and regulates its expression levels. In order to investigate the contribution of this acetylation to the functions of LKB1 in vivo, I have used the experimental model of the neural crest (NC) in chick embryos. Indeed, during my PhD, I have contributed to a study, initiated by my host laboratory, in which we show that LKB1 is required for the delamination, polarized migration and survival of neural crest cells (NCCs) which contribute to the formation of most craniofacial structures in vertebrates. LKB1 signaling is mediated by AMPK and the ROCK kinase and converges towards the actin-dependent molecular motor, Myosin II. Using the same experimental model, I have shown that GCN5 is expressed in NCCs during embryogenesis and that the functional interaction between GCN5 and LKB1 is essential for the activity of LKB1 in the cephalic NCCs ontogenesis and head formation.

Orientadores: Sophie Fraçoise Mauricette Derchain, Luís Otávio Zanatta Sarian / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T00:11:32Z (GMT). No. of bitstreams: 1 Serra_KatiaPiton_D.pdf: 3794654 bytes, checksum: ce5565714883e2e12d99e6f9ed0ca141 (MD5) Previous issue date: 2014 / Resumo: Introdução: Na última década, doferentes subtipos moleculares de cancer de mama foram propostos. A classificação clinic-patológicas dos subtipos vem comprovando ser estratégica para predizer sobrevida e resposta ao tratamento. Modificação recente da classificação considera a avaliação semiquantitativa da expressão dos RP no curso clínico e resposta ao tratamento. Embora exista associação apreciável com o prognóstico e indicação de terapia citotóxica e endócrina, os subtipos parecem falhar em explicar completamente o comçortamento da doença e a resposta ao tratamento. Moléculas como as da família das cicloxigenases (COX), composta por três entidades (COX 1, 2 e 3) vem demonstrando associação com a carcinogênese mamária, e a análise da expressão da p53 nos tumores de mama pode também oferecer informações adicionais para determinação do prognóstico. Objetivos: Foi avaliada a associação entre os subtipos clinic-patológicos do cancer de mama com o prognóstico e fatores preditivos em uma relativamente grande casuística de pacientes Brasileiras com câncer de mama, que foram acompanhadas por cerca de quatro anos. Foram discutidas as vantagens e possíveis ressalvas relacionadas à nova classificação. Também foi mensurada a expressão da COX2 e da p53 em relação aos subtipos clínico-patológicos e avaliada se a expressão destas molécular poderia explicar a variabilidade no prognóstico ainda encontrada entre os subtipos clínico-patológicos do câncer de mama. Metodologia: Um total de 183 amostras de cancer de mama foram obtidas de mulheres tratadas no Hospital da Mulher da Universidade Estadual de Campinas, Campinas, Brasil, entre Junho de 2008 e Janeiro de 2011. Tissue microarrays (TMA) foram construídos dos blocos originais de parafina para realização de imunoistoquímica (IQ) e hibridização fluorescente in situ (FISH). IQ foi realizada para detecção da expressão de RE, RP, ki67, COX2 e p53; o status do HER2 foi avaliado por FISH nas 183 amostras. Os tumores foram classificados em cinco categorias de acordo com a definição correspondente clinic-patológica dos dos subtipos intrínsecos do câncer de mama, definida durante a 13th St Gallen International Breast Cancer Conference (2013). As características clínicas e patológicas das pacientes e seus tumors e a sobrevida foi avaliada em relação aos subtipos clínico-patológicos, a COX2 e a p53. O tempo médio de seguimento foi 2,94 anos (90% faixa central = 0,93 a 4,1 anos). Resultados: Aproximadamente 75% dos tumors foram classificados como luminais-like. OS HER2 positivos (não luminais) somaram 9,3% dos casos e os Triplos-negativos 13,1%. Os Luminais B-like e HER2 positivos (não luminais) foram associados a alto grau histológico quando comparados aos Luminais A-like (p<0,01). Os Luminais A-like associaram-se significativamente com melhor sobrevida global e livre de doença quando comparados aos HER2 positivos (não luminais) e Triplos-negativos. Não houve tendência à expressão de COX2 relacionada aos subtipos de Luminal A-like a Triplo-negativo. Em contraste, a p53 se expressou em cerca de 67% dos tumores Luminais A-like, 50% dos Luminais B-like HER2 positivos, 60,9% dos Luminais B-like HER2 negativos, 82% dos HER2 positivos (não luminais) e 87% dos Triplos-negativos (p para tendências = 0.06). Houve uma significativa expressão de COX2 nos tumors (66,9%) quando a p53 eram também positive, comparada àqueles tumors que não expressavam p53 (em cujo caso apenas 18,0% dos tumores foram positivos para COX2; p<0,001). Nem a COX2, nem a p53 se relacionaram à sobrevida das pacientes. Conclusões: O critério mais estrito para definer os tumors Luminais A-like aumentou a acurácia da classificação para selecionar tumors que partilhem um bom prognóstico e respondam a terapia endócrina. Parece haver uma associação positive entre a expressão da COX2 e da p53. Por outro lado, nem a expressão da COX2 nem a da p53 se associaram aos subtipos clínico-patológicos, características clínicas e do tumor e ao prognóstico. Parece ser muito cedo para eleger a detecção de COX2 usando IQ como ferramenta de prognóstico ou preditiva, mas evidências incipientes apontam para um possível papel para o marcador / Abstract: Background: In the last decade, different molecular subtypes of breast cancer have been proposed. The clinico-pathological surrogate subtypes of breast cancer classification has been proven as straightforward strategy to predict patient survival and response to treatment. Recent modifications to the classification considered the semi quantitative evaluation of the expression of PR in the clinical course and response to treatment. Although displaying appreciable association with disease prognosis and the prognostic value of cytotoxic and endocrine therapeutic modalities, the subtypes seem to fail at completely explaining disease behavior and response to treatment. Molecules such as those of the cyclocooxigenase (COX) family, currently composed of three entities (COX 1, 2 and 3) have been shown to be associated with breast carcinogenesis, and the analysis of p53 expression in breast tumors may also offer some additional prognostic clues. Objectives: We tested the association of the current clinico-pathological surrogate subtypes of breast cancer with the main prognostic and predictive factors in a relatively large dataset of breast cancer Brazilian patients, which were followed up for almost four years. We discuss the advantages and possible caveats related to this new classification. Our study also assessed COX2 and p53 expression in these clinico-pathological subtypes, and evaluated whether the expression of these molecules could help further explain the variability in prognosis still found within the surrogate molecular groups of breast cancer. Methods: A total of 183 breast cancer samples were obtained from women treated at the Women's Hospital of Campinas State University, Campinas, Brazil, between June 2008 and January 2011. Tissue microarrays (TMA) were constructed from the original paraffin blocks for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses. Immunohistochemistry was performed to detect the expression of ER, PR, ki67, COX2, and p53; the HER2 status of the 183 specimens was assessed using FISH. Tumors were subtyped into five distinct categories according to the Clinico-Pathological surrogate definitions of intrinsic subtypes of breast cancer defined during the 13th St Gallen International Breast Cancer Conference (2013). Clinical and pathological features of patients and their tumors, and patients¿ survival were assessed in relation to the surrogate subtypes, COX2 and p53. Mean follow-up time was 2.94 years (90% central range = 0.93 to 4.1 years). Results: Approximately 75% of the tumors were classified as luminal-type-like. HER2 positive (non-luminal) tumors accounted for 9.3% of the cases and Triple-negative tumors for the remainder 13.1%. Luminal B-like and HER2 positive (non-luminal) tumors were associated with higher histological grades when compared to Luminal A-like tumors (p<0.01). Luminal A-like tumors were significantly associated with better disease free and overall survival when compared to HER2 positive (non-luminal) and Triple-negative tumors. There was no trend in COX2 overexpression from Luminal A to Triple-negative subtypes. By contrast, p53 was expressed in roughly 67% of the Luminal A-like tumors, 50% of the Luminal B-like HER2 positive tumors, 60.9% of the Luminal B-like HER2 negative, approximately 82% of the HER2 positive (non-luminal) and 87% of the Triple-negative tumors (p for trends = 0.06). There was a significantly higher proportion of COX2 positive tumors (66.9%) when p53 was also positive compared to when the tumor was negative for p53 (in which case only 18.0% of the tumors were positive for COX2; p<0.001). Neither COX2 nor p53 were found to be associated with patients¿ survival. Conclusions: The more strict criteria to define Luminal A-like tumors increased the accuracy of the classification by selecting tumors that share a good prognosis and response to endocrine therapy.There seems to be a positive association between the expressions of COX2 and p53. On the other hand, neither the expression of COX nor that of p53 was associated with clinic-pathological subtypes, tumor features and prognosis. It seems to be too early to elect the detection of COX2 using IHC as prognostic or predictive tool, but incipient evidence points towards a possible role for the marker / Doutorado / Oncologia Ginecológica e Mamária / Doutora em Ciências da Saúde

Abstract Neurofibromatosis type 1 syndrome (NF1) is caused by mutations of the NF1 gene. The NF1 protein (neurofibromin) contains a domain which is related to the GTPase-activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The NF1 protein has been referred to as a tumor suppressor, since the cells of malignant schwannomas of NF1 patients may display a loss of heterozygosity of the NF1 gene. Psoriasis is characterized by hyperproliferation of the epidermis and by down-regulated levels of NF1 mRNA and protein. Ca2+ is an universal signal transduction element modulating cell growth and differentiation. Many cell types coordinate their activities by transmitting waves of elevated intracellular calcium levels from cell to cell. The propagation of calcium waves had not been studied previously in human keratinocytes. Thus, the aim of the present study was to find out which pathways may play a role in Ca2+ signaling at different extracellular calcium concentrations in NF1 and and psoriatic keratinocytes versus normal control keratinocytes. The results demonstrated that NF1 and psoriatic keratinocytes have a tendency to form cultures characterized by altered Ca2+-mediated cell signaling compared to normal keratinocytes. Specifically, the main route of calcium-mediated signaling was gap-junctional in normal keratinocytes. In contrast, ATP-mediated calcium signaling predominated and capacitative calcium influx was defective in NF1 and psoriatic keratinocytes. The results of the present study suggest that mutations of the NF1 tumor suppressor gene or lowered levels of NF1 protein/mRNA may eventually lead to altered intercellular communication.

Notre laboratoire a caractérisé TP53INP1 comme un gène clé de réponse au stress cellulaire. Cible de p53, TP53INP1 est ubiquitairement exprimé et fortement induit lors de différents stress in vivo et in vitro. Sa surexpression ectopique induit un arrêt du cycle cellulaire et la mort cellulaire. Nous avons montré que l’expression de TP53INP1 est perdue dans plusieurs cancers chez l’homme et que sa réexpression dans les cellules cancéreuses inhibe la croissance tumorale. De plus, les souris TP53INP1-déficientes sont très susceptibles au développement de tumeurs induites et présentent un stress oxydatif permanent caractérisé par une augmentation du taux de ROS. L’ensemble de ces résultats indique que TP53INP1 possède une fonction suppressive de tumeur probablement en lien avec son implication dans la régulation du statut redox cellulaire. Le but de mon travail de thèse est de décrypter cette fonction an niveau cellulaire et moléculaire en exploitant le modèle des souris déficientes. Mes travaux montrent que l’absence de TP53INP1 affecte tous les processus biologiques dérégulés lors de la tumorigenèse. En effet, la perte de TP53INP1 se caractérise par une augmentation de la prolifération cellulaire, de la migration cellulaire, de l’angiogenèse et de l’instabilité génétique. De façon paradoxale, les cellules déficientes pour TP53INP1 présentent une sensibilité accrue à l’apoptose induite par un stress indépendant ou non de p53. Cette sensibilité se base probablement sur un fort défaut de l’autophagie dans ces cellules. Nous observons également une diminution du taux des petites molécules antioxydantes liée au stress oxydatif constitutif observé dans ces souris. Les défauts observés en absence de TP53INP1 sont corrigés par un traitement avec un antioxydant, le N-Acetylcystéine, ce qui démontre le lien entre le rôle suppresseur de tumeur de TP53INP1 et son implication dans le contrôle du stress oxydatif. Ce travail a permis de mieux comprendre le rôle antioxydant TP53INP1 en lien avec sa fonction de suppresseur de tumeur. / Our laboratory has characterized TP53INP1 as a key gene in cell stress response. Target of p53, TP53INP1 is ubiquitously expressed and strongly induced during various stress-inducing treatments in vivo and in vitro. Its ectopic overexpression induces cell cycle arrest and cell death. We have shown that the expression of TP53INP1 is lost in many human cancers and that its re-expression in cancer cells inhibits tumor growth. In addition, TP53INP1-deficient mice are highly susceptible to induced tumors and show a permanent oxidative stress characterized by increased ROS levels. Taken together, these results indicate that TP53INP1 has a tumor suppressor function likely related to its involvement in the regulation of cellular redox status. The purpose of my PhD is to decipher the cellular and molecular function of TP53INP1 by exploiting the model of deficient mice. My work shows that the absence of TP53INP1 affects all biological processes deregulated in tumorigenesis. Indeed, the loss of TP53INP1 is characterized by an increase in cell proliferation, cell migration, angiogenesis and genetic instability. Paradoxically, TP53INP1-deficient cells have increased susceptibility to stress-induced apoptosis, independently or not of p53. This sensitivity is probably based on a strong impairment of autophagy in these cells. We also observed a decrease in the rate of small antioxidant molecules related to constitutive oxidative stress in these mice. The defects observed in the absence of TP53INP1 are corrected by treatment with an antioxidant, N-acetylcysteine, demonstrating the link between the role of TP53INP1 in tumor suppression and its involvement in redox control. This work has led to a better understanding of the antioxidant role of TP53INP1 linked to its function as a tumor suppressor.

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