付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24520号 / 医博第4962号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Nikola Jaščevská Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effects of topoisomerase II beta on the sensitivity of the cancer cells to the antineoplastics Topoisomerase II (TOP II) is a cellular enzyme responsible for solving topological problems of double-stranded DNA. Alpha and beta isoforms of TOP II are different gene products having similar catalytic activities. The expression of TOP IIα is cell-cycle dependent, peaking in G2/M phase, while TOP II isoform is expressed constitutively throughout the cell cycle. It is therefore present also in non-proliferating differentiated cells. Anthracycline antibiotics are an old class of anticancer drugs, belonging to TOP II poisons. Although their clinical usefulness is high, the incidence of side effects (especially myelotoxicity and cardiotoxicity) may limit the therapy. The key role of TOP II inhibition, which is present also in cardiomyocytes, has been increasingly discussed. Dexrazoxane, the only clinically used cardioprotective, leads to depletion of TOP II in cardiomyocytes, which may explain its cardioprotection. Although TOP II was previously shown to be dispensable for cellular proliferation, its possible...

Inhibitors of topoisomerase II (topo II) are clinically effective in the management of hematological malignancies and solid tumors. The efficacy of anti-tumor drugs targeting topo II is often limited by resistance and studies with in vitro cell culture models have provided several insights on potential mechanisms. Multidrug transporters that are involved in the efflux and consequently reduced cytotoxicity of diverse anti-tumor agents suggest that they play an important role in resistance to clinically active drugs. However, in clinical trials, modulating the multidrug-resistant phenotype with agents that inhibit the efflux pump has not had an impact. Since reduced drug accumulation per se is insufficient to explain tumor cell resistance to topo II inhibitors several studies have focused on characterizing mechanisms that impact on DNA damage mediated by drugs that target the enzyme. Mammalian topo IIα and topo IIβ isozymes exhibit similar catalytic, but different biologic, activities. Whereas topo IIα is associated with cell division, topo IIβ is involved in differentiation. In addition to site specific mutations that can affect drug-induced topo II-mediated DNA damage, post-translation modification of topo II primarily by phosphorylation can potentially affect enzyme-mediated DNA damage and the downstream cytotoxic response of drugs targeting topo II. Signaling pathways that can affect phosphorylation and changes in intracellular calcium levels/calcium dependent signaling that can regulate site-specific phosphorylation of topoisomerase have an impact on downstream cytotoxic effects of topo II inhibitors. Overall, tumor cell resistance to inhibitors of topo II is a complex process that is orchestrated not only by cellular pharmacokinetics but more importantly by enzymatic alterations that govern the intrinsic drug sensitivity.

<p>The relatively recent discoveries of bacterial small RNAs (sRNAs) and their important regulatory functions prompted us to conduct a genome wide survey for sRNAs in <em>Streptomyces coelicolor</em>. We used a combined bioinformatics and experimental approach to identify and characterize six sRNAs. sRNA expression profiles were determined throughout <em>S. coelicolor</em> development, including vegetative and reproductive growth, during growth on minimal and rich media. Additionally, we also tested sRNA expression in various <em>S. coelicolor</em> developmental mutants. Two sRNAs were expressed exclusively during growth on one medium type and all but one were expressed constitutively throughout growth apart from the late sporulation timepoint. One of the identified sRNAs, scr1906, appeared to be closely associated with development. scr1906 was only expressed in nutrient limiting conditions just prior to aerial development and sporulation. Expression of scr1906 was abolished in a mutant that was defective in sporulation (due to a mutation in the sporulation sigma factor gene, <em>whiG</em>); however, expression was detected in mutants of both known σ^WhiG target genes, <em>whiH</em> and <em>whiI</em>, which encode sporulation transcription factors. Intriguingly, <em>in silico</em> analysis predicted <em>whiH</em> to be a direct target for scr1906-mediated regulation based on potential nucleotide binding sites. The effects of deletion and overexpression of <em>scr1906</em> on WhiH levels were tested, but require further experimentation.</p> <p>In a separate line of investigation, we sought to characterize a novel actinobacterial-specific protein named sIHF. The <em>sIHF</em> mutant strain revealed that sIHF influenced DNA compaction and segregation during <em>S. coelicolor</em> sporulation and also affected antibiotic production. sIHF associated with the nucleoid, and <em>in vitro</em>, it bound to DNA non-specifically in a length dependent manner, although it was determined to have a preference for three distinct DNA motifs. Like most nucleoid-associated proteins, sIHF affected gene expression indicating the potential for an additional role as a transcription factor. Interestingly, sIHF impacted the activity of topoisomerase. Leveraging information that we have gained from the sIHF-DNA co-crystal complex, studies aimed at characterizing the sIHF regions that are important for DNA interaction and topoisomerase modulation are currently underway.</p> / Doctor of Philosophy (PhD)

La sclérose systémique est une maladie autoimmune dont l’une des complications majeures est la fibrose. La DNA topoisomérase I (topo) est l’un des principaux autoantigènes associés à cette maladie. Toutefois, aucun lien n’a encore pu être établi entre la présence des anti-topo et le développement de la fibrose. Les travaux antérieurs du laboratoire d’accueil ont montré une interaction directe de la topo avec la surface des fibroblastes et la matrice extracellulaire. Nous avons voulu caractériser ces interactions du point de vue moléculaire. La topo a donc été exprimée sous forme de 5 fragments, déterminés à partir de ses principaux domaines structuraux et de ses épitopes majeurs, chez E. coli. Les fragments purifiés ont été analysés pour leur interaction avec l’héparine, représentant les héparane sulfates de la surface des fibroblastes, et avec des protéines purifiées de la matrice extracellulaire. Nous avons montré que le fragment topo-N est le principal responsable de l’interaction avec l’héparine, ce qui suggère donc l’implication potentielle de ce domaine dans l’interaction de la topo avec la surface des fibroblastes. Le fragment topo-DIDII est responsable de l’interaction avec la plupart des protéines de la matrice extracellulaire étudiées, alors que le fragment topo-H15 n’interagit qu’avec la vitronectine. Aucune interaction des fragments topo-DIII et topo-C n’a été décelée. Ces résultats pourront maintenant servir à mieux comprendre le rôle potentiel de la topo et des autoanticorps circulants anti-topo dans la fibrose présente chez les personnes atteintes de sclérose systémique en contribuant à l’identification de la cible de la topo sur les fibroblastes. / Systemic sclerosis is an autoimmune disease in which one of the major complications is fibrosis. DNA topoisomerase I (topo) is a major autoantigen associated with this disease. However, no link has yet been established between the presence of anti-topo and the development of fibrosis. Previous work of the host laboratory showed a direct interaction of the topo with the surface of fibroblasts and extracellular matrix. We wanted to characterize these interactions at the molecular level. Topo was expressed in 5 fragments, determined from its main structural domains and its major epitopes, in E. coli. The purified fragments were analyzed for their interaction with heparin, representing heparan sulfates on the surface of fibroblasts, and with purified proteins of the extracellular matrix. We have shown that the topo-N fragment is responsible for interaction with heparin, suggesting hence, potential involvement of this domain in the interaction of topo with the surface of fibroblasts. The topo-DIDII fragment is responsible for the interaction with most proteins of the extracellular matrix studied, whereas the topo-H15 fragment only binds to vitronectin. No interaction of fragments topo-DIII and topo-C was found. These results can now be used to better understand the potential role of topo and circulating anti-topo autoantibodies in the fibrosis present in patients with systemic sclerosis in helping to identify the target of topo on fibroblasts.

INTRODUÇÃO: A endometriose, uma doença benigna, tem características invasivas com potencial proliferativo. O desenvolvimento das lesões pode ocorrer em decorrência de crescimento celular glandular e/ou estromal ou de alterações na cinética celular. Cinética celular refere-se ao equilíbrio entre a morte celular, ou apoptose, e a proliferação celular, que pode ser avaliada pela expressão de fatores de crescimento como, por exemplo, a topoisomerase 2-alfa (TOP2A). Também influenciam a cinética celular oncoproteínas como p53 e c-erB2, conhecidas por interferir na apoptose, podendo resultar em oncogênese. OBJETIVOS: O objetivo principal deste estudo foi comparar a cinética celular da endometriose infiltrativa de retosigmoide com a do endométrio eutópico de pacientes sem endometriose. Para tanto, foi avaliada a expressão de apoptose e de TOP2A bem como das oncoproteínas p53 e c-erB2. MÉTODOS: Foram obtidas amostras de lesões de endometriose envolvendo o reto-sigmoide de 60 mulheres com a doença e amostras de endométrio eutópico de 20 mulheres sem endometriose. A expressão de TOP2A e das proteínas p53 e c-erB2 foram quantificadas por técnica imuno-histoquímica. Método TUNEL foi utilizado para analisar os padrões de apoptose, que resultaram em índice de apoptose (IA). Índice de proliferação celular (IP) foi determinado a partir do nível de expressão de TOP2A. Índice de renovação celular (IRC) foi calculado pela razão entre IP e IA. As análises imuno-histoquímicas foram realizadas tanto no tecido endometrial como um todo, quanto nos componentes estromal e glandular separadamente. Coeficiente de Correlação de Spearman foi aplicado para identificar eventuais correlações entre variáveis clínicas, morfológicas (tamanho, quantidade e nível de invasão das lesões) e experimentais. RESULTADOS: Na análise da amostra do tecido como um todo, não foram evidenciadas diferenças entre os grupos experimental e controle em relação ao IA (p = 0,389). Por outro lado, o IP foi significativamente maior nas amostras-controle (p < 0,001). Na avaliação em que se sepaxii raram as células estromais dos componentes glandulares, tanto o IP quanto o IRC foram significativamente maiores no grupo-controle em comparação com o grupo experimental (IP estromal: p = 0,006; IP glandular: p = 0,001; IRC estromal: p =0,032; IRC glandular: p = 0,007). Nas pacientes com endometriose, foi encontrada correlação entre IP e IRC glandular e o número de lesões (p = 0,003). Também foi observada correlação entre o IRC glandular e o tamanho das lesões (p = 0,006). Não houve diferença entre os grupos no que se refere à expressão de p53 e cerB2. CONCLUSÕES: A cinética celular se mostrou alterada em pacientes com endometriose do reto-sigmoide, conforme demonstrado pela redução nos níveis e na frequência de TOP2A, e pelos IP e IRC mais baixos; entretanto, apoptose e as expressões de p53 e c-erB2 se mostraram inalteradas / BACKGROUND: Endometriosis, a benign disease, has invasive features with its proliferative potential. Development of lesions may occur due to stromal and/or glandular cell growth and to alterations in cellular kinetics. Cellular kinetics involves a balance between the regulation of cell death, or apoptosis, and cell growth, that can be evaluated by the expression of growth factors, such as topoisomerase 2- alpha (TOP2A). Oncoproteins, such as p53 and c-erB2, known to affect apoptosis resulting in oncogenesis, also influence cellular kinetics. OBJECTIVES: The main objective of this study was to compare the cellular kinetics in deep endometriosis involving the recto-sigmoid to eutopic endometrium from patients without endometriosis. Apoptosis and TOP2A expression were primarily evaluated, as well as p53 and c-erB2 expression. METHODS: Study samples were obtained from endometriosis lesions involving the recto-sigmoid in 60 women, and control samples were obtained from eutopic endometrium from 20 women without endometriosis. The expression of TOP-2A, p53 and c-erB2 proteins were quantified using immuno-histochemistry. TUNEL method was used in the analysis of apoptosis patterns, and the apoptosis index (AI) was derived. The proliferation index (PI) was derived from the level of expression of TOP-2A. Cellular renew index (CRI) was calculated from the ratio of the PI and AI. Immunohistochemical analyses were performed in two ways: on the tissue collectively, and on the stromal and glandular components separately. Spearmans correlation coefficient was used to identify the correlation between clinical, morphological (size, number and level of invasion of lesions) and the study variables. RESULTS: When looked at collectively, there was no difference in the AI between study and control groups (p = 0.389). PI, however, was noted to be significantly higher in the control samples (p < 0.001). When evaluating the stromal cells separately from the glandular components, the PI and CRI were both significantly xiv higher in the control group compared to the study group (Study stromal PI vs control stromal PI; p = 0.006; Study glandular PI vs study glandular PI; p = 0.001; Study stromal CRI vs control stromal CRI; p = 0.032; study glandular CRI vs control glandular CRI; p = 0.007). In patients with endometriosis, a correlation was found between glandular PI, CRI and number of lesions (p = 0.003). The same result was observed in the analysis of stromal CRI and lesion size (p = 0.006). There was no difference in expression of p53 and c-erB2 between groups. CONCLUSIONS: Cellular kinetics is altered in endometriosis of the recto-sigmoid, as shown by the decrease in the levels and frequency of TOP2A expression, and lower PI and CRI; however, apoptosis and p53 and c-erB2 expression were unaffected

INTRODUÇÃO: A endometriose, uma doença benigna, tem características invasivas com potencial proliferativo. O desenvolvimento das lesões pode ocorrer em decorrência de crescimento celular glandular e/ou estromal ou de alterações na cinética celular. Cinética celular refere-se ao equilíbrio entre a morte celular, ou apoptose, e a proliferação celular, que pode ser avaliada pela expressão de fatores de crescimento como, por exemplo, a topoisomerase 2-alfa (TOP2A). Também influenciam a cinética celular oncoproteínas como p53 e c-erB2, conhecidas por interferir na apoptose, podendo resultar em oncogênese. OBJETIVOS: O objetivo principal deste estudo foi comparar a cinética celular da endometriose infiltrativa de retosigmoide com a do endométrio eutópico de pacientes sem endometriose. Para tanto, foi avaliada a expressão de apoptose e de TOP2A bem como das oncoproteínas p53 e c-erB2. MÉTODOS: Foram obtidas amostras de lesões de endometriose envolvendo o reto-sigmoide de 60 mulheres com a doença e amostras de endométrio eutópico de 20 mulheres sem endometriose. A expressão de TOP2A e das proteínas p53 e c-erB2 foram quantificadas por técnica imuno-histoquímica. Método TUNEL foi utilizado para analisar os padrões de apoptose, que resultaram em índice de apoptose (IA). Índice de proliferação celular (IP) foi determinado a partir do nível de expressão de TOP2A. Índice de renovação celular (IRC) foi calculado pela razão entre IP e IA. As análises imuno-histoquímicas foram realizadas tanto no tecido endometrial como um todo, quanto nos componentes estromal e glandular separadamente. Coeficiente de Correlação de Spearman foi aplicado para identificar eventuais correlações entre variáveis clínicas, morfológicas (tamanho, quantidade e nível de invasão das lesões) e experimentais. RESULTADOS: Na análise da amostra do tecido como um todo, não foram evidenciadas diferenças entre os grupos experimental e controle em relação ao IA (p = 0,389). Por outro lado, o IP foi significativamente maior nas amostras-controle (p < 0,001). Na avaliação em que se sepaxii raram as células estromais dos componentes glandulares, tanto o IP quanto o IRC foram significativamente maiores no grupo-controle em comparação com o grupo experimental (IP estromal: p = 0,006; IP glandular: p = 0,001; IRC estromal: p =0,032; IRC glandular: p = 0,007). Nas pacientes com endometriose, foi encontrada correlação entre IP e IRC glandular e o número de lesões (p = 0,003). Também foi observada correlação entre o IRC glandular e o tamanho das lesões (p = 0,006). Não houve diferença entre os grupos no que se refere à expressão de p53 e cerB2. CONCLUSÕES: A cinética celular se mostrou alterada em pacientes com endometriose do reto-sigmoide, conforme demonstrado pela redução nos níveis e na frequência de TOP2A, e pelos IP e IRC mais baixos; entretanto, apoptose e as expressões de p53 e c-erB2 se mostraram inalteradas / BACKGROUND: Endometriosis, a benign disease, has invasive features with its proliferative potential. Development of lesions may occur due to stromal and/or glandular cell growth and to alterations in cellular kinetics. Cellular kinetics involves a balance between the regulation of cell death, or apoptosis, and cell growth, that can be evaluated by the expression of growth factors, such as topoisomerase 2- alpha (TOP2A). Oncoproteins, such as p53 and c-erB2, known to affect apoptosis resulting in oncogenesis, also influence cellular kinetics. OBJECTIVES: The main objective of this study was to compare the cellular kinetics in deep endometriosis involving the recto-sigmoid to eutopic endometrium from patients without endometriosis. Apoptosis and TOP2A expression were primarily evaluated, as well as p53 and c-erB2 expression. METHODS: Study samples were obtained from endometriosis lesions involving the recto-sigmoid in 60 women, and control samples were obtained from eutopic endometrium from 20 women without endometriosis. The expression of TOP-2A, p53 and c-erB2 proteins were quantified using immuno-histochemistry. TUNEL method was used in the analysis of apoptosis patterns, and the apoptosis index (AI) was derived. The proliferation index (PI) was derived from the level of expression of TOP-2A. Cellular renew index (CRI) was calculated from the ratio of the PI and AI. Immunohistochemical analyses were performed in two ways: on the tissue collectively, and on the stromal and glandular components separately. Spearmans correlation coefficient was used to identify the correlation between clinical, morphological (size, number and level of invasion of lesions) and the study variables. RESULTS: When looked at collectively, there was no difference in the AI between study and control groups (p = 0.389). PI, however, was noted to be significantly higher in the control samples (p < 0.001). When evaluating the stromal cells separately from the glandular components, the PI and CRI were both significantly xiv higher in the control group compared to the study group (Study stromal PI vs control stromal PI; p = 0.006; Study glandular PI vs study glandular PI; p = 0.001; Study stromal CRI vs control stromal CRI; p = 0.032; study glandular CRI vs control glandular CRI; p = 0.007). In patients with endometriosis, a correlation was found between glandular PI, CRI and number of lesions (p = 0.003). The same result was observed in the analysis of stromal CRI and lesion size (p = 0.006). There was no difference in expression of p53 and c-erB2 between groups. CONCLUSIONS: Cellular kinetics is altered in endometriosis of the recto-sigmoid, as shown by the decrease in the levels and frequency of TOP2A expression, and lower PI and CRI; however, apoptosis and p53 and c-erB2 expression were unaffected

La sclérose systémique est une maladie autoimmune dont l’une des complications majeures est la fibrose. La DNA topoisomérase I (topo) est l’un des principaux autoantigènes associés à cette maladie. Toutefois, aucun lien n’a encore pu être établi entre la présence des anti-topo et le développement de la fibrose. Les travaux antérieurs du laboratoire d’accueil ont montré une interaction directe de la topo avec la surface des fibroblastes et la matrice extracellulaire. Nous avons voulu caractériser ces interactions du point de vue moléculaire. La topo a donc été exprimée sous forme de 5 fragments, déterminés à partir de ses principaux domaines structuraux et de ses épitopes majeurs, chez E. coli. Les fragments purifiés ont été analysés pour leur interaction avec l’héparine, représentant les héparane sulfates de la surface des fibroblastes, et avec des protéines purifiées de la matrice extracellulaire. Nous avons montré que le fragment topo-N est le principal responsable de l’interaction avec l’héparine, ce qui suggère donc l’implication potentielle de ce domaine dans l’interaction de la topo avec la surface des fibroblastes. Le fragment topo-DIDII est responsable de l’interaction avec la plupart des protéines de la matrice extracellulaire étudiées, alors que le fragment topo-H15 n’interagit qu’avec la vitronectine. Aucune interaction des fragments topo-DIII et topo-C n’a été décelée. Ces résultats pourront maintenant servir à mieux comprendre le rôle potentiel de la topo et des autoanticorps circulants anti-topo dans la fibrose présente chez les personnes atteintes de sclérose systémique en contribuant à l’identification de la cible de la topo sur les fibroblastes. / Systemic sclerosis is an autoimmune disease in which one of the major complications is fibrosis. DNA topoisomerase I (topo) is a major autoantigen associated with this disease. However, no link has yet been established between the presence of anti-topo and the development of fibrosis. Previous work of the host laboratory showed a direct interaction of the topo with the surface of fibroblasts and extracellular matrix. We wanted to characterize these interactions at the molecular level. Topo was expressed in 5 fragments, determined from its main structural domains and its major epitopes, in E. coli. The purified fragments were analyzed for their interaction with heparin, representing heparan sulfates on the surface of fibroblasts, and with purified proteins of the extracellular matrix. We have shown that the topo-N fragment is responsible for interaction with heparin, suggesting hence, potential involvement of this domain in the interaction of topo with the surface of fibroblasts. The topo-DIDII fragment is responsible for the interaction with most proteins of the extracellular matrix studied, whereas the topo-H15 fragment only binds to vitronectin. No interaction of fragments topo-DIII and topo-C was found. These results can now be used to better understand the potential role of topo and circulating anti-topo autoantibodies in the fibrosis present in patients with systemic sclerosis in helping to identify the target of topo on fibroblasts.

Introdução. O receptor epidérmico humano 2 (Her-2) e a topoisomerase-IIα (T2A) são dois marcadores biológicos importantes, ambos tendo um valor prognóstico e preditivo potencial em pacientes com tumores sólidos. A amplificação dos genes Her- 2 e T2A são eventos independentes, embora o último seja mais frequente em tumores com amplificação do Her-2 (34-90%), do que em tumores sem amplificação do Her-2 (5-10%). Existe uma melhor correlação entre amplificação e superexpressão do Her-2 no câncer de mama (CM) do que em outros tumores. No entanto, no CM, a correlação entre amplificação e superexpressão da T2A tem sido inconsistente, e existe uma carência de tais dados em outros tipos de tumores. A expressão da proteína T2A tem mostrado uma boa correlação com o índice de proliferação tumoral, particularmente no CM. Objetivos. Artigo 1: Sintetizar o conhecimento atual sobre a importância dos marcadores Her-2 e T2A nos tumores sólidos. Artigo 2: Investigar a prevalência de amplificação e superexpressão do Her-2 e da T2A, a correlação entre estas variáveis e a correlação entre as variáveis e estágio clínico, em amostras de câncer de ovário (CO) fixadas em parafina. Artigo 3: Investigar a prevalência de amplificação da T2A, assim como a correlação entre esta variável e a expressão da proteína T2A e do marcador de proliferação celular Ki-67, em amostras de CM fixadas em parafina, mostrando uma amplificação do Her-2. Métodos. Artigo 1: Os dados foram identificados através de busca em bases de dados eletrônicas (medline), livros de resumos de congressos e referências de artigos de revisão e originais. Artigo 2: 73 amostras de CO foram testadas para amplificação e superexpressão do Her-2 e T2A, por hibridização in situ fluorescente (FISH) e imuno-histoquímica (IHC), respectivamente. Artigo 3: 103 amostras de CM, com amplificação do Her-2, foram testadas para amplificação do gene T2A (por FISH) e superexpressão das proteínas T2A e Ki-67 (por IHC). Resultados. Artigo 2: Com base nos pontos de corte >1.5 e >2 (relação cópias/CEP17), as taxas de amplificação do Her-2 foram 15/64(23.4%) e 8/64(12.5%), versus 16/64(25%) e 5/64(7.8 %) para a T2A. Encontramos somente 3/72(4.2%) casos de superexpressão do Her-2(3+), contra 15/70(21.4%) para a T2A (marcagem em >10% das células). Foi observada uma modesta correlação entre amplificação e superexpressão da T2A (p= 0.01) e uma forte correlação entre amplificação da T2A e do Her-2, quando analisados como variáveis contínuas (p<0.001). A amplificação da T2A correlacionou-se com estágio FIGO avançado (p= 0.02). Artigo 3: Uma amplificação do gene T2A foi observada em 36.9%(38/103) dos casos. Os níveis de amplificação do Her-2 (número de cópias) não se correlacionaram com a amplificação da T2A. A porcentagem média de células positivas para a T2A (por IHC) foi de 5% e 10%, para casos T2A não-amplificados e amplificados, respectivamente. Uma correlação fraca, mas ainda significativa, foi observada entre amplificação do gene T2A e porcentagem de células T2A-positivas por IHC (Spearman=0.23, p=0.02); a correlação entre estas duas variáveis foi mais forte em tumores Ki-67 positivos. Conclusões. Artigo 2 : A avaliação da amplificação e da superexpressão do Her-2 e da T2A, por FISH e IHC, respectivamente, é realizável em amostras de CO. Foi observada uma boa correlação entre a amplificação dos genes Her-2 e T2A, mas a correlação entre amplificação do gene e superexpressão da proteína foi fraca para ambos marcadores. As taxas de amplificação dos genes Her-2 e T2A são mais elevadas quando não é realizada correção para o número de cópias do CEP17. Parece existir uma boa correlação entre amplificação da T2A e estágio clínico avançado. Estudos adicionais serão necessários para determinar o melhor ponto de corte para estes marcadores. Artigo 3: Contrariamente ao Her-2, a amplificação do gene T2A não parece necessariamente levar à superexpressão da proteína no CM. Outros fatores, como o índice de proliferação celular, podem interferir na síntese da proteína T2A. Embora a maioria dos casos de aberrações do gene T2A ocorram em tumores Her-2 positivos, os níveis de amplificação do Her-2 não se correlacionaram com a amplificação do gene T2A. / Background. The human epidermal receptor 2 (Her-2) and topoisomerase-IIα (T2A) are two important biomarkers, with potential prognostic and predictive value in patients with solid tumours. Her-2 and T2A gene amplification are separate events, although the latter is more frequently seen in Her-2 amplified (34-90%) than in Her-2 non-amplified (5-10%) tumours. There is a better correlation between Her-2 amplification and protein overexpression in breast cancer (BC) than in other tumour types. Nevertheless, there is a doubtful correlation between T2A amplification and overexpression in BC, with virtually no data available in other tumour types. In BC, the expression of the T2A protein has shown a good correlation with tumour proliferation rate. Objectives. Article 1: To summarise the available literature on Her-2 and T2A in solid tumours. Article 2: To investigate the prevalence of Her-2 and T2A amplification and overexpression, the correlation between these variables and with clinical stage, in paraffin-embedded samples of ovarian cancer (OC). Article 3: To investigate the prevalence of T2A amplification, as well as the correlation between this variable and the expression of T2A protein and the proliferation marker Ki-67, in paraffinembedded samples of Her-2 amplified BC. Methods. Article 1: The data were identified through search in electronic databases (medline), abstract books and references from review and original articles. Article 2: 73 samples of OC were tested for Her-2 and T2A amplification and overexpression, by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. Article 3: 103 samples of Her-2 amplified BC were tested for T2A amplification (by FISH) and overexpression (by IHC), and Ki-67 expression (by IHC). Results. Article 2: Based on cut-offs of ≥1.5 and ≥2 (ratio copies/CEP17), amplification rates for Her-2 were 15/64(23.4%) and 8/64(12.5%) versus 16/64(25%) and 5/64(7.8%) for T2A. We found only 3/72(4.2%) cases of Her-2 overexpression(3+) versus 15/70(21.4%) for T2A (staining in >10% of the cells). There was a modest correlation between T2A amplification and overexpression (p=0.01) and a strong correlation between T2A and Her-2 amplification when these markers were analysed as continuous variables (p<0.001). T2A amplification significantly correlated with advanced FIGO stage (p=0.02). Article 3: T2A gene amplification was observed in 36.9%(38/103) of the Her-2 amplified samples. Her-2 amplification level (i.e. copy number) was not predictive of T2A amplification. The median percentage of T2A positive cells for T2A non-amplified and amplified cases were 5% and 10%, respectively. A weak but still significant correlation was observed between T2A gene amplification level and percentage of positively stained cells (Spearman=0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Conclusions. Article 2: The assessment of Her-2 and T2A amplification and overexpression by FISH and IHC, respectively, is feasible in OC samples. There was a good correlation between Her-2 and T2A gene amplification, but the correlation between gene amplification and protein overexpression was poor for both markers. Amplification rates were higher in the absence of correction for the number of copies of the CEP17. Finally, we found a good correlation between T2A amplification and advanced disease stage. Further studies should aim to determine the optimal cut-offs for these markers. Article 3: Contrary to Her-2, T2A gene amplification does not always lead to protein overexpression in BC. Other factors, especially tumour proliferation rate, may interfere with the T2A protein status. Although the majority of the cases of T2A gene aberrations are seen in Her-2 positive tumours, the level of Her-2 amplification does not predict for T2A amplification.

La 20(S)-camptothécine (CPT) est un alcaloïde pentacyclique doté d'une activité anticancéreuse remarquable, agissant comme inhibiteur de la topoisomérase I (topo I). Le problème majeur de ce composé (et de la plupart de ses dérivés) est la fragilité de son cycle E qui s'hydrolyse rapidement à pH physiologique pour conduire au carboxylate correspondant inactif. L'une des approches permettant de pallier ce problème d'hydrolyse consiste à modifier le cycle E. Ce travail de thèse porte sur la synthèse totale de deux nouveaux analogues de la CPT modifiés au niveau du cycle E. Chacune de ces synthèses fait appel à une cycloaddition [3+2] afin de préparer l'hydroxypyridone de départ (cycles C et D), à un réarrangement de Claisen permettant de mettre en place le cycle E et à une condensation de Friedländer pour installer le motif quinoléinique (cycles A et B). Le premier analogue synthétisé, la (±) 17 norcamptothécine (17-norCPT), possède une α-hydroxy γ-lactone à la place de l'α-hydroxy δ-lactone de la CPT. Ce composé a été obtenu en neuf étapes avec un rendement de 4,4% à partir de l'hydroxypyridone de départ. Le test d'inhibition de la topo I a été réalisé, mais cette molécule s'est révélée totalement inactive. Cependant, une étude de la cinétique d'hydrolyse de la 17-norCPT, réalisée par spectroscopie de fluorescence, a permis de montrer que cet analogue était très instable en milieu aqueux. Le second composé préparé est en fait un homologue de la 17 norCPT possédant un méthylène entre l'oxygène et le carbonyle de la lactone. Cette molécule, comportant un motif céto éther, est donc un isomère de la CPT. Elle a été obtenue par deux voies de synthèse différant l'une de l'autre par l'ordre des réactions mises en œuvre. Chacune de ces approches permet d'obtenir le composé souhaité en neuf étapes à partir de l'hydroxypyridone de départ, avec un rendement de 16% et 10% respectivement. Les tests biologiques sur le composé final sont actuellement en cours. / 20(S)-Camptothecin (CPT) is a pentacyclic alkaloid endowed with remarkable anticancer activity, acting as an inhibitor of topoisomerase I (topo I). The major problem with this compound (and many of its derivatives) is the fragility of its E-ring, which suffers rapid hydrolysis at physiological pH to yield the inactive corresponding carboxylate. One of the approaches to overcome this problem of hydrolysis consists in modifying the E-ring. This thesis focuses on the total synthesis of two new E-ring modified CPT analogs. Each of the syntheses involves a [3+2] cycloaddition to prepare the starting hydroxypyridone (rings C and D), a Claisen rearrangement to realize the E-ring, and a Friedländer condensation to install the quinoline system (rings A and B). The first analog synthesized, (±) 17 norcamptothecin (17-norCPT), has an α-hydroxy γ-lactone in place of the α-hydroxy δ-lactone of CPT. This compound has been obtained in nine steps with a yield of 4.4% from the starting hydroxypyridone. Unfortunately, though, this molecule has proved to be totally inactive in the topo I assay. A kinetic hydrolysis study of 17-norCPT, carried out by fluorescence spectroscopy, has demonstrated, however, that this analog is, in fact, unstable in aqueous media. The second compound prepared is a homolog of 17-norCPT with a methylene between the lactone oxygen and carbonyl, thus a CPT keto ether isomer. It has been obtained through two synthetic routes that differ from each other in the order of the steps. Each of the approaches yields the desired compound in nine steps from the starting hydroxypyridone, with yields of 16% and 10% for the two syntheses. Biological tests on the final compound are currently underway.