Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 171 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 124-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

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;

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

<p>The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms.</p><p>The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme <i>Cyp2a5/CYP2A6</i>. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific <i>cis</i> binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis. </p><p>The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine <i>Cyp2a5</i> gene are linked <i>via</i> hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a <i>trans </i>factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, <i>CYP2A6</i>, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, <i>via</i> changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR. </p>

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 131 pages. Includes vita. Includes bibliographical references.

The family of proteins known as heterogeneous nuclear ribonucleoproteins (hnRNPs) is large and diverse. Often, one and the same hnRNP will perform multiple cellular functions, leading to their description as “multifunctional proteins”. The two hnRNPs known as hnRNP A1 and hnRNP C1/C2 are multifunctional proteins found to affect the transcription, splicing, stability, and translation of specific genes’ mRNA. They are implicated in carcinogenesis, apoptosis, and DNA damage response mechanisms. The aims of this thesis were to study the hnRNP A1 and hnRNP C1/C2 dependent regulation of two highly stress responsive genes, the tumor suppressor p53 and the cytochrome P450 enzyme Cyp2a5/CYP2A6. We identified hnRNP C1/C2 as a DNA damage induced binding protein towards the coding region of p53 mRNA, and found that while a specific cis binding site appears to have a positive function in p53 expression, interaction of hnRNP C1/C2 with this site represses the expression. The data suggest that two distinct molecular mechanisms exist for the down-regulation of p53 by hnRNP C1/C2. One mechanism, active during transcriptional stress, is dependent upon the aforementioned site, and the other, independent. We discuss how hnRNP C1/C2 dependent repression of p53 may play a role in apoptosis. The data presented here further suggest that the transcriptional and post-transcriptional processes controlling the expression of the murine Cyp2a5 gene are linked via hnRNP A1, by performing functions in the nucleus as a transcription factor, or in the cytoplasmic compartment as a trans factor bound to the 3’UTR of the mRNA as needed. Our studies of the human ortholog of this gene, CYP2A6, suggest that this gene is regulated post-transcriptionally in a manner similar to that of its murine counterpart, via changes in mRNA stability and interaction of hnRNP A1 with its 3’ UTR.

The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation. / In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein. / Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.

The p53 tumor suppressor protein is upregulated in response to DNA damage and other stress signals. The upregulation of p53 involves freeing it from negative regulation imposed by Mdm2 and MdmX (Mdm4). Accumulating evidence indicates that phosphorylation of Mdm proteins by different stress-activated kinases such as ATM or c-Abl significantly impacts p53 functions. We have previously shown that ATM phosphorylation of Mdm2 Ser394 is required for robust p53 stabilization and activation following DNA damage. This dissertation describes in vivo examination of the mechanism by which Mdm2 Ser394 phosphorylation impacts p53 activities and its contribution to suppression of oncogene and DNA damage-induced tumors. We determine that phosphorylation of Mdm2 Ser394 regulates p53 activity by modulating Mdm2 stability and paradoxically delays Myc-driven lymphomagenesis while increasing lymphomagenesis in sub-lethally irradiated mice. c-Abl phosphorylates the residue neighboring Mdm2 Ser394, Mdm2 Tyr393. This dissertation describes the generation of a novel Mdm2Y393F mutant mouse to determine if c-Abl phosphorylation of Mdm2 regulates p53-mediated DNA damage responses or tumor suppression in vivo. Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes. The studies presented herein further our understanding of the mechanisms by which DNA damage-associated kinases stabilize and activate p53, and influence p53-dependent responses and tumor suppression. A better understanding of the in vivo effects of Mdm2 phosphorylation may facilitate the development of novel therapeutics capable of stimulating p53 anti-tumor activity or alleviating p53- dependent toxicities in non-malignant tissues.