Rôle des kinésines mitotiques Eg5 et MKLP-2 dans l’angiogenèse physiologique etpathologique.L’angiogenèse est un phénomène biologique complexe qui correspond à la formation de nouveauxvaisseaux à partir de vaisseaux préexistants. Ce processus essentiel est régulé par des nombreuxfacteurs, dont le plus puissant est le facteur de croissance de l’endothélium vasculaire (VEGF).Des inhibiteurs du VEGF sont actuellement utilisés dans le traitement de nombreux cancerssolides. Leur efficacité est constatée dans plusieurs études mais des résistances contre cesmolécules sont fréquemment observées. Afin d’identifier de nouvelles cibles thérapeutiques dansla voie de signalisation de VEGF, nous avons utilisé le modèle de la membrane chorioallantoïdienne(CAM) de l’embryon de poulet. Les CAM traitées au VEGF pendant 24hdéveloppent de nombreux vaisseaux. Ces tissus ont été isolés pour effectuer une analysetranscriptomique. En dehors des gènes endothéliaux déjà connus pour être régulés par le VEGF,de nouveaux gènes ont été identifiés. Nous avons focalisé notre recherche sur des gènes codantpour les kinésines mitotiques KIF11/Eg5 et KIF20A/MKLP-2 qui ont été fortement induites.Nous avons démontré qu’Eg5 et MKLP-2 sont fortement exprimées au niveau de l’endothéliumdans des tissus sains et dans des cancers solides. Des inhibiteurs chimiques spécifiques d’Eg5(dimethylenastron et ispinesib mesylate) et MKLP-2 (paprotrain) bloquent les étapes clés de laformation des vaisseaux sanguins (prolifération, adhérence et migration des cellules endothéliales),la prolifération des cellules tumorales ainsi que la formation de néo-vaisseaux dans des culturesd’anneaux aortiques. De plus, sur la CAM et chez la souris, l’inhibition de cette même kinésinediminue significativement la croissance et la vascularisation des modèles tumoraux utilisés lors dece projet (le glioblastome et le carcinome rénal). En conclusion, Eg5 et MKLP-2 pourraient descibles potentielles dans les thérapies anti-angiogéniques.Mots clés : Eg5, MKLP-2, angiogenèse, kinésine, ispinesib, dimethylenastron, glioblastome,cancer rénal / Role of the mitotic kinesins Eg5 and MKLP-2 in physiologic and pathologic angiogenesis.Angiogenesis is a complex biological phenomenon which corresponds to the formation of newblood vessels from pre-existing vessels. This process is regulated by a plethora of differentmolecules with vascular endothelial growth factor (VEGF) being one of the most important ones.VEGF inhibitors are currently used in the treatment of numerous solid cancers. Even though theefficacy of such treatment is prouven by numerous studies, resistance to anti-angiogenic therapy isa common feature. To identify new therapeutic targets downstream of VEGF, we modelized itsaction on the chick chorioallantoic membrane (CAM). VEGF-treated CAMs develop a densevascular network 24h after application. We used chick microarrays to monitor global geneexpression changes in VEGF-induced CAMs. Beside a consistent number of genes alreadydescribed to be regulated by VEGF, numerous unknown genes have been identified. We havefocused our work on the characterization of Eg5/KIF11 and MKLP-2/KIF20A, members of thekinesin family, both strongly upregulated by VEGF.We demonstrated that Eg5 and MKLP-2 are strongly expressed by blood vessels in normal andcancer tissue sections. KIF20A is involved in the proliferation and migration of endothelial cellsin vitro. We showed that chemical inhibitors specific for KIF11/Eg5 (dimethylenastron andispinesib mesylate) affect key steps in the formation of blood vessels (proliferation, adhesion andmigration of endothelial cells) and proliferation of tumor cells (glioma and renal cancer).Furthermore, in experimental glioblastoma and renal cell carcinoma models (CAM and orthotopicimplantation in mice), anti-Eg5 treatment strongly reduces tumor angiogenesis and growth. Inconclusion, Eg5 and MKLP-2 could be potential targets in anti-angiogenic therapies.Keywords: Eg5, MKLP-2, angiogenesis, kinesin, ispinesib, dimethylenastron, glioblastoma, renalcell cancer

L’angiogenèse est un processus majeur intervenant au cours du remodelage du microenvironnement tumoral, induit par l’hypoxie et le VEGF. La lysyl oxydase like-2 (LOXL2) appartient à la famille des lysyl oxydases, impliquée dans le pontage de constituants matriciels. Notre équipe a montré que l’induction de LOXL2 par l’hypoxie conduit à sa sécrétion par les cellules endothéliales et son accumulation dans la matrice extracellulaire endothéliale. Nous avons montré que LOXL2 joue un rôle dans l’angiogenèse au cours du développement. Des études ont montré que LOXL2 est surexprimée dans de nombreux cancers et que l’inhibition de LOXL2 extracellulaire empêche la formation d’un microenvironnement tumoral. Mon travail de thèse a porté sur l’étude du rôle de LOXL2, exprimée par les cellules endothéliales et tumorales, au cours de l’angiogenèse dans le carcinome du rein à cellules claires (ccRCC) humain. Nous avons montré que LOXL2 est exprimée à la fois par les cellules stromales et tumorales dans le ccRCC humain, mais aussi que LOXL2 pourrait jouer un rôle spécifique en fonction de son origine cellulaire dans ces tumeurs. L’étude de la contribution de LOXL2 endothéliale nous a permis de démontrer que LOXL2 promeut l’angiogenèse in vitro et in vivo dans le ccRCC, avec une implication partielle de son activité catalytique dans ce processus. Nous avons montré que LOXL2 sécrétée par les cellules tumorales stimule l’angiogenèse in vitro et in vivo, avec la participation de son activité catalytique, notamment en modulant la prolifération des cellules endothéliales. LOXL2 endothéliale et tumorale promeuvent l’angiogenèse dans le ccRCC, dépendamment ou non de son activité catalytique. / Angiogenesis is a major process in microenvironment remodeling which is mainly induced by hypoxia and VEGF. Lysyl oxidase like-2 (LOXL2) belongs to lysyl oxidase family involved in extracellular matrix crosslinking. Our team previously described that LOXL2 is a hypoxia-target, which is secreted by endothelial cells and accumulated into endothelial extracellular matrix. We also demonstrated that LOXL2 stimulates developmental angiogenesis. Moreover, several studies showed that LOXL2 is overexpressed in many cancers and inhibition of extracellular LOXL2 impedes the formation of a tumor microenvironment. My PhD work focused on the contribution of LOXL2 secreted by stromal cells (endothelial cells and cancer associated fibroblasts) and tumor cells in clear cell renal cell carcinoma (ccRCC) angiogenesis. ccRCC is a highly vascularized and metastatic tumor. We showed that LOXL2 is expressed both by stromal and tumor cells in ccRCC and might play a specific role depending on its cellular origin in these tumors. Then we demonstrated that LOXL2 secreted by endothelial cells promotes angiogenesis in vitro and in vivo with a partial contribution of its catalytic activity. We also demonstrated that LOXL2 secreted by tumor cells stimulates angiogenesis in vitro and in vivo and that LOXL2 catalytic activity is involved in this process, notably modulating endothelial cells proliferation. Moreover, we showed that endothelial and tumor LOXL2 regulate several signaling pathways implicated in different steps of the angiogenic process.Both tumor and endothelial LOXL2 are involved in angiogenesis of ccRCC, in a dependent or independent catalytic activity manner.

Neste estudo foram avaliados os efeitos antitumorais da fosfoetanolamina sintética (FS) em modelos de tumores experimentais e as vias de sinalizações envolvidas nesta atividade. In vitro a FS foi citotóxica para as linhagens de células tumorais de melanoma humano, SK-MEL-28, carcinoma renal murino RENCA e para as células do carcinoma de pulmão de não pequenas células NSCLC. Alterações ultraestruturais como a condensação da cromatina, formação de blubes de membranas e mitocôndrias eletrodensas foi acompanhada pela redução do potencial elétrico mitocondrial. Aumento da ativação da caspase 3 e 8 e da expressão da proteína Bax, além da redução da CDK9 e CDK4/6, também foram observados. A redução da migração e proliferação das células endoteliais HUVEC induzida pela FS foi associada com a modulação da expressão do VEGF-A. resultando na inibição in vitro da formação do tubo endotelial. In vivo a FS foi capaz de inibir o crescimento dos tumores sólidos do melanoma e do carcinoma renal murino de forma superior aos quimioterápicos Dacarbazina (DITC) e ao Sunitinib. No modelo de metástase pulmonar utilizando as células RENCA, a FS reduziu o número de nódulos metastáticos pulmonares e aumentou a taxa de sobrevida dos animais. Os efeitos terapêuticos da FS também foram avaliados no modelo de leucemia promielocítica aguda (LPA) transplantada em camundongos NOD/Scid. O tratamento com a FS reduziu o número de blastos periféricos e infiltrados na medula óssea e nos parênquimas, esplênico e hepático. De forma superior a Daunorrubicina (DA) e ao Ácido all-trans retinóico (ATRA), a FS induziu apoptose nos clones malignos que expressão CD34+, bem como nas células CD117+/Gr-1+. Este conjunto de resultados mostra que a FS é um composto promissor na terapêutica contra neoplasias / In this study, we evaluated the antitumor effects of synthetic phosphoethanolamine (FS) in an experimental tumor model and the signaling pathways involved in this activity. In vitro, FS was cytotoxic to tumor cell lines of human melanoma, SK- MEL-28, renal carcinoma murine, Renca, and for non-small cell lung cancer, NSCLC. Ultrastructural changes such as chromatin condensation, blubes formation on membranes and electrodense mitochondria were accompanied by a reduction of the mitochondrial electric potential. Increased activation of caspase-3, 8 and Bax protein expression, in addition to a reduction of CDK9 and CDK4/6 levels, was also observed. The reduction of migration and proliferation of endothelial cells, HUVEC, induced by FS was associated with the modulation of VEGF-A-expression, resulting in the in vitro inhibition of tubulogenesis. In vivo, FS was able to inhibit the growth of solid tumors of melanoma and renal carcinoma more potently than Dacarbazine (DITC) and Sunitinib. In the lung metastasis model, using RENCA cells, FS reduced the number of metastatic lung nodules and increased the survival rate of the animals. The therapeutic effects of FS were also evaluated in the model of acute promyelocytic leukemia transplanted in mice NOD/Scid. The treatment with FS reduced the number of peripheral blasts and infiltrates in the blood marrow, splenic and hepatic parenchyma. Fs was more potent than Daunorrubicine (DA) and all-trans retinoic acid (ATRA) in the induction of apoptosis in the malignant clones expressing CD34+ as well as CD117+/Gr-1+ cells. Taken together, theses results show that FS is a promising compound for the therapy against tumors

Neste estudo foram avaliados os efeitos antitumorais da fosfoetanolamina sintética (FS) em modelos de tumores experimentais e as vias de sinalizações envolvidas nesta atividade. In vitro a FS foi citotóxica para as linhagens de células tumorais de melanoma humano, SK-MEL-28, carcinoma renal murino RENCA e para as células do carcinoma de pulmão de não pequenas células NSCLC. Alterações ultraestruturais como a condensação da cromatina, formação de blubes de membranas e mitocôndrias eletrodensas foi acompanhada pela redução do potencial elétrico mitocondrial. Aumento da ativação da caspase 3 e 8 e da expressão da proteína Bax, além da redução da CDK9 e CDK4/6, também foram observados. A redução da migração e proliferação das células endoteliais HUVEC induzida pela FS foi associada com a modulação da expressão do VEGF-A. resultando na inibição in vitro da formação do tubo endotelial. In vivo a FS foi capaz de inibir o crescimento dos tumores sólidos do melanoma e do carcinoma renal murino de forma superior aos quimioterápicos Dacarbazina (DITC) e ao Sunitinib. No modelo de metástase pulmonar utilizando as células RENCA, a FS reduziu o número de nódulos metastáticos pulmonares e aumentou a taxa de sobrevida dos animais. Os efeitos terapêuticos da FS também foram avaliados no modelo de leucemia promielocítica aguda (LPA) transplantada em camundongos NOD/Scid. O tratamento com a FS reduziu o número de blastos periféricos e infiltrados na medula óssea e nos parênquimas, esplênico e hepático. De forma superior a Daunorrubicina (DA) e ao Ácido all-trans retinóico (ATRA), a FS induziu apoptose nos clones malignos que expressão CD34+, bem como nas células CD117+/Gr-1+. Este conjunto de resultados mostra que a FS é um composto promissor na terapêutica contra neoplasias / In this study, we evaluated the antitumor effects of synthetic phosphoethanolamine (FS) in an experimental tumor model and the signaling pathways involved in this activity. In vitro, FS was cytotoxic to tumor cell lines of human melanoma, SK- MEL-28, renal carcinoma murine, Renca, and for non-small cell lung cancer, NSCLC. Ultrastructural changes such as chromatin condensation, blubes formation on membranes and electrodense mitochondria were accompanied by a reduction of the mitochondrial electric potential. Increased activation of caspase-3, 8 and Bax protein expression, in addition to a reduction of CDK9 and CDK4/6 levels, was also observed. The reduction of migration and proliferation of endothelial cells, HUVEC, induced by FS was associated with the modulation of VEGF-A-expression, resulting in the in vitro inhibition of tubulogenesis. In vivo, FS was able to inhibit the growth of solid tumors of melanoma and renal carcinoma more potently than Dacarbazine (DITC) and Sunitinib. In the lung metastasis model, using RENCA cells, FS reduced the number of metastatic lung nodules and increased the survival rate of the animals. The therapeutic effects of FS were also evaluated in the model of acute promyelocytic leukemia transplanted in mice NOD/Scid. The treatment with FS reduced the number of peripheral blasts and infiltrates in the blood marrow, splenic and hepatic parenchyma. Fs was more potent than Daunorrubicine (DA) and all-trans retinoic acid (ATRA) in the induction of apoptosis in the malignant clones expressing CD34+ as well as CD117+/Gr-1+ cells. Taken together, theses results show that FS is a promising compound for the therapy against tumors

O carcinoma renal de células claras é o tumor renal maligno mais frequentemente diagnosticado nos adultos. Uma série de defeitos genéticos tem sido observada no tecido tumoral renal e tais achados podem estar envolvidos na gênese ou progressão desses tumores. Alterações metabólicas e genéticas da mitocôndria são fatores que contribuem para muitas doenças humanas, incluindo o câncer. Objetivo: Estabelecer método para extração mitocondrial sem contaminação pelo DNA nuclear; verificar se existe contaminação por inserções nucleares de origem mitocondrial (NUMTs) no sequenciamento do genoma mitocondrial no carcinoma renal de células claras. Métodos: Para o estudo foram selecionados quatro pacientes portadores de carcinoma renal de células claras. Após a cirurgia obtivemos de cada paciente um fragmento de tumor e um fragmento de parênquima renal sem comprometimento neoplásico, as amostras de cada paciente foram extraídas de forma que ao final pudéssemos obter quatro amostras de DNA, sendo duas com isolamento da mitocôndria e duas sem o isolamento da mitocôndria. As amostras obtidas foram submetidas às seguintes análises genéticas: Sequenciamento completo do mtDNA; Reação em cadeia da polimerase para avaliação da contaminação do mtDNA obtido com isolamento da organela por DNA nuclear; avaliação do número de cópias do mtDNA (depleção) e patogenicidade das mutações. Resultados: Com os resultados obtidos neste estudo podemos afirmar que é possível a realização da extração do DNA mitocondrial sem a contaminação do genoma nuclear; e que o DNA mitocondrial extraído de maneira clássica do DNA total não apresentou contaminação por inserções nucleares de origem mitocondrial (NUMTs). / Clear cell renal carcinoma is the most frequently diagnosed malignant renal tumor in adults. A number of genetic defects have been observed in renal tumor tissue and such findings may be involved in the genesis or progression of these tumors. Metabolic and genetic changes in mitochondria are contributing factors to many human diseases, including cancer. Objective: To establish a method for mitochondrial extraction without nuclear DNA contamination; check for contamination by nuclear inserts of mitochondrial origin (NUMTs) in the sequencing of the mitochondrial genome in clear cell renal carcinoma. Methods: Four patients with clear cell renal carcinoma were selected for the study. After surgery, we obtained from each patient a tumor fragment and a renal parenchyma fragment without neoplastic involvement, the samples from each patient were extracted so that in the end we could obtain four DNA samples, two with mitochondrial isolation and two without the mitochondria isolation of mitochondria. The obtained samples were submitted to the following genetic analyzes: Complete sequencing of mtDNA; Polymerase chain reaction to evaluate the contamination of mtDNA obtained with organelle isolation by nuclear DNA; evaluation of mtDNA copy number (depletion) and pathogenicity of mutations. Results: With the results obtained in this study we can affirm that it is possible to perform mitochondrial DNA extraction without contamination of the nuclear genome; and that the mitochondrial DNA extracted from the classical DNA of the total DNA was not contaminated by nuclear inserts of mitochondrial origin (NUMTs).

Le cancer du rein compte parmi les 10 types de cancers les plus fréquents chez l’Homme. Il n’existe aujourd’hui aucun marqueur biomoléculaire dans ce type de cancer, et dans le cas d’un cancer métastatique, l’arsenal thérapeutique aujourd’hui disponible manque d’efficacité. Les différents processus mis en jeu lors de la progression tumorale sont encore mal connus. La connaissance de ces processus pourrait permettre de mettre en évidence de nouvelles cibles thérapeutiques, ainsi que des marqueurs biomoléculaires pronostiques ou diagnostiques de la maladie. Dans un premier projet, et afin de mieux comprendre les mécanismes de la progression tumorale et d’identifier de nouvelles cibles thérapeutiques potentielles et de nouveaux marqueurs biomoléculaires dans le cancer du rein, un nouveau modèle innovant a été généré à partir d’une lignée tumorale de RCC murine. Ce modèle de réimplantations successives de cellules tumorales issues de tumeur primaire ou de métastases a permis de générer différentes lignées cellulaires montrant une agressivité accrue au cours des passages. En utilisant une stratégie de biologie des systèmes, ce modèle pourra permettre de mettre en évidence des cibles d’études prometteuses qui pourraient être de nouvelles cibles thérapeutiques ou de nouveaux marqueurs biomoléculaires dans le RCC. L’interleukine-34 est l’exemple d’une cible d’étude d’ores et déjà été sélectionnée, mettant en évidence la puissance du modèle généré. Dans un second projet, les rôles de certains membres de la matrice extracellulaire tumorale ont été évalués en utilisant cette même lignée de RCC murine (collagène de type I, fibronectine, matrigel). Cette étude a permis de mettre en évidence le potentiel pro-invasif et pro-métastatique du dépôt de collagène de type I dans les tumeurs. Des récepteurs activés par le collagène sont proposés comme potentiellement impliqués dans les effets induits par le collagène de type I dans le modèle. Ces deux projets permettent et permettront de mieux comprendre certains mécanismes de la progression tumorale, ainsi que de mettre en évidence des marqueurs biomoléculaires et de nouvelles cibles thérapeutiques. / Kidney cancer is one of the 10 commonest human cancers. To date, no biomolecular markers are available in this type of cancer, and in the case of metastatic cancer, the therapeutic arsenal is still inefficient. The different processes involved in cancer progression are still poorly understood. Understanding those processes could highlight new therapeutic targets, and new prognostic or diagnostic biomolecular markers of this disease. For a first project, a new innovative model has been generated from a murine RCC cell line as a tool to understand cancer progression mechanisms and to identify new therapeutic target and new biomolecular markers in kidney cancer. This model of sequential reimplantation of cancer cells isolated from primary tumours or metastases allowed us to generate different cell lines showing increased aggressiveness after passages. Using a systems biology strategy, this model will allow us to identify new potential therapeutic targets and new biomolecular markers in RCC. Interleukin-34 is an example of an already selected target, showing the power of the model generated. For a second project, the role of some members of extracellular matrix (collagen type I, fibronectin, matrigel).was studied using this same murine RCC cell line. This study demonstrated the potential pro-invasive and pro-metastatic roles of collagen type I deposition in tumors. Collagen-activated receptors are proposed as mediators of the effect induced by collagen type I in this model. Those two projects have and will continue to contribute to a better understanding of cancer progression mechanisms, and will bring out new biomolecular markers and new therapeutic targets.

Le carcinome à cellules rénales (CCR) se caractérise par une résistance importante aux thérapies. Notre hypothèse était que des voies signalétiques prolifératives, anti-apoptotiques et/ou angiogéniques sont mises en jeu dans la résistance aux thérapies. Il s’agissait de mesurer la sensibilité de lignées cellulaires de CCR humain à différentes classes thérapeutiques in vitro et in vivo. Une étude pilote a été réalisée sur la base de xénogreffes de la lignée A498 chez la souris nude, puis exploitée pour des analyses sur biopuces à protéines afin d’identifier les voies de signalisation induites par le sunitinib. In vitro, les lignées cellulaires de CCR se sont révélées sensibles aux thérapies indépendamment du statut VHL. In vivo, la lignée A498 est apparue résistante au sunitinib. L’approche par biopuces a montré que plusieurs protéines de l’angiogenèse sont modulées sous l'effet du traitement, notamment l’angiogénine. Il n’y a pas de modification de l’expression des protéines de l’apoptose testées. Les formes phosphorylées d’Akt sont également augmentées dans les tumeurs traitées, de même que Lim1 alors que la forme phosphorylée de NFκB est diminuée. Ce travail a ainsi identifié de potentielles cibles impliquées dans les mécanismes de résistance et devraient permettre de définir de nouvelles options thérapeutiques dans le cancer du rein. / The renal cell carcinoma is characterized by a high resistance to therapies. Our working hypothesis was that proliferative signaling pathways, anti-apoptotic and / or angiogenic are involved in resistance to therapies. Thus, as part of this thesis, we measured the sensitivity to chemotherapy and targeted therapies in kidney cancer cell lines in vitro as well in vivo.A pilot study was conducted on the basis of the A498 cell line xenografts in nude mice, and then used for analysis on proteome arrays to identify the signaling pathways induced by sunitinib. In vitro, the cell lines of RCC were sensitive to therapy regardless of the VHL status. In vivo, the line A498 appeared resistant to sunitinib. The approach using the proteome array has shown that several angiogenesis proteins are modulated as a result of treatment, including angiogenin. There was no change in the expression of proteins of apoptosis. Phosphorylated forms of Akt were also increased in the treated tumors, as well as Lim1 whereas the phosphorylated form of NFkB was reduced. This work has identified potential targets involved in resistance mechanisms and should define new therapeutic options in renal cancer.

O carcinoma renal de células claras é o tumor renal maligno mais frequentemente diagnosticado nos adultos. Uma série de defeitos genéticos tem sido observada no tecido tumoral renal e tais achados podem estar envolvidos na gênese ou progressão desses tumores. Alterações metabólicas e genéticas da mitocôndria são fatores que contribuem para muitas doenças humanas, incluindo o câncer. Objetivo: Estabelecer método para extração mitocondrial sem contaminação pelo DNA nuclear; verificar se existe contaminação por inserções nucleares de origem mitocondrial (NUMTs) no sequenciamento do genoma mitocondrial no carcinoma renal de células claras. Métodos: Para o estudo foram selecionados quatro pacientes portadores de carcinoma renal de células claras. Após a cirurgia obtivemos de cada paciente um fragmento de tumor e um fragmento de parênquima renal sem comprometimento neoplásico, as amostras de cada paciente foram extraídas de forma que ao final pudéssemos obter quatro amostras de DNA, sendo duas com isolamento da mitocôndria e duas sem o isolamento da mitocôndria. As amostras obtidas foram submetidas às seguintes análises genéticas: Sequenciamento completo do mtDNA; Reação em cadeia da polimerase para avaliação da contaminação do mtDNA obtido com isolamento da organela por DNA nuclear; avaliação do número de cópias do mtDNA (depleção) e patogenicidade das mutações. Resultados: Com os resultados obtidos neste estudo podemos afirmar que é possível a realização da extração do DNA mitocondrial sem a contaminação do genoma nuclear; e que o DNA mitocondrial extraído de maneira clássica do DNA total não apresentou contaminação por inserções nucleares de origem mitocondrial (NUMTs). / Clear cell renal carcinoma is the most frequently diagnosed malignant renal tumor in adults. A number of genetic defects have been observed in renal tumor tissue and such findings may be involved in the genesis or progression of these tumors. Metabolic and genetic changes in mitochondria are contributing factors to many human diseases, including cancer. Objective: To establish a method for mitochondrial extraction without nuclear DNA contamination; check for contamination by nuclear inserts of mitochondrial origin (NUMTs) in the sequencing of the mitochondrial genome in clear cell renal carcinoma. Methods: Four patients with clear cell renal carcinoma were selected for the study. After surgery, we obtained from each patient a tumor fragment and a renal parenchyma fragment without neoplastic involvement, the samples from each patient were extracted so that in the end we could obtain four DNA samples, two with mitochondrial isolation and two without the mitochondria isolation of mitochondria. The obtained samples were submitted to the following genetic analyzes: Complete sequencing of mtDNA; Polymerase chain reaction to evaluate the contamination of mtDNA obtained with organelle isolation by nuclear DNA; evaluation of mtDNA copy number (depletion) and pathogenicity of mutations. Results: With the results obtained in this study we can affirm that it is possible to perform mitochondrial DNA extraction without contamination of the nuclear genome; and that the mitochondrial DNA extracted from the classical DNA of the total DNA was not contaminated by nuclear inserts of mitochondrial origin (NUMTs).

The feasibility of FTIR-based spectroscopy as a tool to measure cellular response to therapeutics was investigated. Fourier transform mid-infrared spectroscopy has been used in conjunction with multivariate analysis (MVA) to assess the chemistry of many clinically relevant biological materials; however, the technique has not yet found its place in a clinical setting. One issue that has held the technique back is due to the spectral distortions caused by resonant Mie scattering (RMieS), which affects the ability to confidently assign molecular assignments to the spectral signals from biomaterials. In the light of recently improved understanding of RMieS, resulting in a novel correction algorithm, the analytical robustness of corrected FTIR spectra was validated against multi-discipline methods to characterise a set of renal cell lines which were selected for their difference in morphology.After validation of the FTIR methodology by discriminating different cell lines, the second stage of analyses tested the sensitivity of FTIR technique by determining if discrete chemical differences could be highlighted within a cell population of the same origin. The renal carcinoma cell line 2245R contains a sub-population to contain a sub-population of cells displaying 'stem-cell like' properties. These stem-like cells, however, are difficult to isolate and characterise by conventional '-omic' means. Finally, cellular response to chemotherapeutics was investigated using the established renal cell lines CAKI-2 and A-498. For the model, 5-fluorouracil (5FU), an established chemotherapeutic agent with known mechanisms of action was used. Novel gold-based therapeutic compounds were also assessed in parallel to determine their efficacy against renal cell carcinoma. The novel compounds displayed initial activity, as the FTIR evidence suggested compounds were able to enter the cells in the first instance, evoking a cellular response. The long-term performance, tracked with standard proliferation assays and FTIR spectroscopy in the renal cancer cell model, however, was poor. Rather than dismissing the compounds as in-active, the compounds may simply be more effective in cancer cell types of a different nature. The FTIR-based evidence provided the means to suggest such a conclusion. Overall, the initial results suggest that the combination of FTIR and MVA, in the presence of the novel RMieS-EMSC algorithm can detect differences in cellular response to chemotherapeutics. The results were also in-line with complimentary biological-based techniques, demonstrating the powerful potential of the technique as a promising drug screening tool.

A Síndrome de von Hippel-Lindau é uma doença hereditária multissistêmica, causada por mutações germinativas no gene VHL que predispõe o portador a manifestações benignas e malignas em diversos órgãos. Entre esses eventos, o carcinoma de células claras renais (CRC) é o de pior prognóstico, com uma penetração média de 25% e sendo a principal causa de morte nestes pacientes. Os CRCs são tumores agressivos, pouco responsivos à quimioterapia e imunoterapia, e muitas vezes são diagnosticados em estágios avançados. Podem estar associados a síndromes hereditárias como o VHL ou apresentar a forma esporádica. Caracteristicamente, o CRC é provocado pela inativação dos dois alelos do gene VHL. Nos casos associados ao VHL, um alelo do gene VHL sofre uma mutação germinativa e um segundo evento mutacional somático nas células do tumor. Por outro lado, na forma esporádica, o CRC é resultado de dois eventos somáticos adquiridos, que incluem uma combinação de metilação do promotor, mutações pontuais que afetam a sequência de leitura aberta (ORF) e rearranjos cromossômicos, principalmente perda de heterozigosidade (LOH). Embora os eventos somáticos nos CRCs esporádicos já tenham sido explorados em outros estudos, os mecanismos de inativação somáticos do gene VHL nos CRCs associados à síndrome ainda não foram bem descritos. Este estudo avaliou os eventos somáticos no gene VHL em CRCs retirados em procedimentos cirúrgicos de pacientes portadores da síndrome. Os eventos somáticos em vários tumores de um mesmo paciente foram comparados a fim de verificarmos se essas mutações são independentes e não clonais. Oito pacientes com amostras CRCs previamente armazenadas no BNT tiveram sua mutação germinativa no gene VHL caracterizada por sequenciamento ou MLPA. Todas as amostras foram submetidas a uma revisão da patologia e macrodissecadas sempre que necessário. Para a análise das manifestações somáticas do gene VHL, o DNA foi extraído de 30 CRCs conservados em RNA latter ou formaldeído (parafina). As amostras foram analisadas quanto à metilação da região promotora do gene pelo método MS-PCR e para mutações pontuais por sequenciamento. Fomos capazes de detectar a mutação somática em 25 dos 30 tumores, incluindo uma mutação pontual e dois tumores diferentes de um mesmo paciente, nenhuma microdeleção e 23 grandes deleções. Em contraste com a literatura, nenhum dos tumores apresentou metilação no promotor do VHL. Devido ao grande número de achados LOH e da resolução limitada da técnica de MLPA para avaliar a extensão dos rearranjos cromossômicos em 3p, não foi possível concluir a análise de clonalidade dos tumores. Um estudo exploratório para caracterizar ganhos e perdas genômicas utilizando a técnica CNV array está em andamento em nosso laboratório. / The von Hippel-Lindau syndrome (VHL) is a multissystemic hereditary disease, caused by germline mutations in the VHL gene that predisposes the carrier to benign and malignant manifestations in different organs. Among these events, the clear cell renal carcinoma (RCC) is the most fearful, with an average penetration of 25% being the leading cause of death in these patients. RCCs are aggressive tumors, poorly responsive to chemo- and immunotherapy that are often diagnosed in advanced stages. They can be associated with hereditary syndromes such as VHL or present in a sporadic form. Characteristically, RCCs carrier the inactivation of the two alleles of VHL gene. In cases associated with VHL, one allele of the VHL gene is mutated in the germline, and the second mutational event occurs in the somatic cells of the tumor. On the other hand, in the sporadic form, RCCs results of two acquired somatic events, which includes a combination of methylation of the promoter, point mutations affecting the ORF, and rearrangements mainly loss of heterozigosity (LOH). Although somatic events in sporadic RCC have been explored before by others, the mechanisms of somatic VHL gene inactivation in VHL-associated RCCs have been poorly characterized. This study evaluated the somatic mutational events in the VHL gene of RCCs removed from VHL patients in therapeutic surgical procedures. The somatic events in multiple tumors from the same patient were compared in order to analyze whether these mutations are independent and not clonal. Eight patients with RCCs samples previously stored at BNT had their germline VHL gene mutation characterized by sequencing or MLPA. All samples were submitted to a pathology review and macrodissected whenever necessary. For the analysis of somatic events of VHL gene, DNA from 30 RCCs were extracted from either RNA later or archival formalin-fixed, paraffin-embedded tissue sections. Samples were analyzed for VHL gene promoter methylation by MS-PCR, and for point mutation in the coding DNA by sequencing. We were able to detect the somatic mutation in 25 of the 30 tumors, including one point mutations in two different tumors of the same patient, no micro-deletions, and 23 large deletions. In contrast to the literature, none of the tumors have shown methylation on the VHL promoter. Because of the large number of LOH findings, and the limited resolution of MLPA to evaluate the extension of 3p chromosomal rearrangements, we could not conclude the analysis of tumor clonality. An exploratory study to characterize genomic gains and losses using CNV-array technique are ongoing in our laboratory.