Orientadores: Laura Sterian Ward, Márcio José da Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T06:32:04Z (GMT). No. of bitstreams: 1 Silva_AlineCarolinaDeNadaida_M.pdf: 2484854 bytes, checksum: 084b0bdeea2ea5baa391d3e9acfc9841 (MD5) Previous issue date: 2012 / Resumo: O câncer diferenciado de tiroide (CDT) é a malignidade endócrina mais comum e a quarta mais frequente entre as mulheres Brasileiras. O carcinoma papilífero da tiroide (CPT) representa 80-90% de todas as malignidades tiroidianas. As mais importantes vias envolvidas na formação e progressão do CDT são as vias MAPK e PI3K/AKT. O gene RAS possui como uma de suas vias efetoras a via PI3K/AKT, a qual tem um papel importante na sobrevivência e na proliferação celular. A mutação Q61R do gene NRAS é a mais frequentemente encontrada em CDT. O gene BRAF possui mutações pontuais que são identificadas em 40-45% nos CPT, sendo a V600E a mais comum encontrada nesta neoplasia e responsável por manter e promover a progressão a tumores mais agressivos do CPT. O gene AKT possui um papel importante na via de sinalização PI3K, regulando a sobrevivência, proliferação e crescimento celular, sendo que a isoforma AKT1 possui um papel de pró-motilidade em CDT. Para investigar a utilidade clínica das vias MAPK e PI3K/AKT no diagnóstico e prognóstico do CDT foi realizada a genotipagem da mutação dos genes NRAS, HRAS e BRAF e o seqüenciamento automático do éxon 3 do gene AKT1 em 281 pacientes com nódulos tiroidianos. Destes, 190 eram tumores benignos, incluindo 121 hiperplasias e 69 adenomas foliculares, e 91 eram tumores malignos, sendo 60 carcinomas papilíferos variante clássica, 26 carcinomas papilíferos variante folicular, 01 carcinoma papilífero variante células altas, 02 carcinomas foliculares e 02 carcinomas anaplásicos. Todos os pacientes foram tratados e seguidos de acordo com um protocolo padrão por 12 a 87 meses (38,06±19,9 meses). A genotipagem do códon 61 do gene NRAS mostrou que indivíduos com genótipo CT possuem maior risco de desenvolverem o CPT de variante folicular (p=0,025). Já a mutação de HRAS não teve nenhuma utilidade clínica. A presença do genótipo heterozigoto da mutação de BRAF teve associação com o desenvolvimento do CPT (p<0,001). Observamos alterações genéticas no éxon 3 do gene AKT1 em 114 (41,76%) de 273 pacientes. Foram encontradas 3 alterações intrônicas (rs2494738, rs3730368, rs3730358) e uma exônica (L52R). A presença da alteração rs2494738 mostrou associação com ausência de invasão tumoral (p=0,004) e a rs3730368 como fator de proteção no desenvolvimento de tumores malignos (p=0,046), enquanto que o genótipo selvagem se relacionou com o desenvolvimento de tumores benignos menores de 2cm (p=0,033). A presença da alteração rs3730358 se associou com tumores malignos menores de 2 cm (p=0,032). Já a presença da alteração L52R apareceu em tumores malignos (p=0,004) e encapsulados (p=0,006), em tumores benignos menores de 2cm (p=0,029) e de 2-4cm (p=0,0387). Portanto, podemos concluir que existem alterações genéticas importantes que possam influenciar no desenvolvimento do CDT, sugerindo que estas alterações possam servir como possíveis marcadores de diagnóstico. Porém neste estudo estas alterações não tiveram relação com prognóstico, talvez pelo baixo tempo de seguimento destes pacientes / Abstract: Differentiated thyroid cancer (DTC) is the most common endocrine malignancy and the fourth most frequent among Brazilian women. Papillary thyroid carcinoma (PTC) represents 80-90% of all thyroid malignancies. The most important pathways involved in the formation and progression of DTC are the MAPK and PI3K/AKT pathways. The RAS gene has as one of its effectors the PI3K/AKT pathway, which plays an important role in the survival and proliferation. The mutation Q61R of NRAS gene is most often found in DTC. The BRAF gene mutations that have been identified in 40-45% in the CPT, and the V600E found this the most common malignancy and is responsible for maintaining and promoting the progression to more aggressive tumors of the PTC. The AKT gene has a role in signaling fosfotidilinositol-3 kinase (PI3K) pathway regulating the survival, proliferation and cell growth, and the AKT1 isoform plays a pro-motility in DTC. To investigate the clinical utility of the MAPK and PI3K/AKT pathways in the diagnosis and prognosis of DTC was performed by genotyping of the mutations of NRAS, HRAS and BRAF genes and sequencing of exon 3 of the AKT1 gene in 281 patients with thyroid nodule. Of these, 190 were benign, including 121 hyperplasias and 69 follicular adenomas, and 91 were malignant tumors, including 60 classic variant papillary carcinomas, 26 follicular variant papillary carcinomas, 01 tall cell variant of papillary carcinoma, 02 follicular carcinomas and 02 anaplastic carcinomas. All patients were treated and followed according to a standard protocol for 12 to 87 months (38.06 ± 19.9 months). Genotyping of codon 61 of NRAS gene showed that patients with CT genotype have increased risk of PTC developed the follicular variant (p=0.025). Already HRAS mutation had no clinical utility. The presence of the heterozygous genotype of the BRAF mutation was associated with the development of PTC (p <0.001). We observed genetic alterations in exon 3 of the AKT1 gene in 114 (41.76%) of 273 patients. We found three intronic changes (rs2494738, rs3730368, rs3730358) and one exonic (L52R). The genetic alteration rs2494738 showed no association with tumor invasion (p=0,004) and the rs3730368 as a protective factor in the development of malignant tumors (p=0,046), whereas the wildtype genotype associated with benign tumors smaller than 2 cm (p=0,033). The presence of the alteration rs3730358 was associated with malignant tumors smaller than 2 cm (p=0,032). The presence of the alteration L52R appeared in malignant tumors (p=0,004) and encapsulated (p=0,006), and in benign tumors smaller than 2 cm (p=0,029) and 2-4cm (p=0,0387). Therefore, we conclude that there are important genetic alterations that may influence the development of the CDT, suggesting that these changes may serve as potential diagnostic markers, but in this study these changes did not correlate with prognosis perhaps by the low follow-up of these patients / Mestrado / Clinica Medica / Mestra em Clínica Médica

Les plantes ont longtemps été connues pour leur arsenal naturel, servant d'une source importante de substances nutritives et de composants thérapeutiques. Depuis il y a environ 600 000 ans, les hommes ont utilisé des plantes comme médicaments. Aujourd'hui, les médicaments issus des plantes sont largement présents dans le monde entier où environ 80 % de la population mondiale utilisent des plantes comme médicaments primaires. Cette valeur médicinale est principalement attribuée au fait que les plantes sont riches en composés phytochimiques bioactifs. Le Liban, en raison de son emplacement géographique et des caractéristiques environnementales importantes, est doté d'une flore riche. Des centaines de plantes libanaises ont été définies en termes de composition chimique et de valeur médicinale. Dans cette étude, nous avons caractérisé le contenu phytochimique de deux espèce de plantes libanaises, Crataegus azarolus L et Ephedra campylopoda. Des feuilles fraîches tirées de chaque espèce de plante, ont été dissoutes dans trois solvants différents : eau, éthanol et méthanol. La composition phytochimique des extraits a été examinée en utilisant la chromatographie liquide à haute performance (HPLC) et le contenu d'huile essentielle a été déterminé par la chromatographie gazeuse (GC) couplée avec la spectrométrie de masse (MS). Le radical DPPH a été utilisé pour évaluer le potentiel antioxydant. Le potentiel anti-inflammatoire a été évalué en mesurant les quantités sécrétées de la prostaglandine E2 (PGE2) en utilisant la technique ELISA, aussi bien qu'en déterminant les niveaux d’expression d’ARNm de cytokines pro-inflammatoires (IL-, IL-ß et IL-6), de chimiokines (CCL3 et CCL4) et de COX-2 et iNOS par RT-PCR quantitative (qRT-PCR). Un essai de viabilité cellulaire par le test XTT a été effectué pour déterminer l'effet antiprolifératif de chaque extrait. Nous avons observé un contenu phytochimique important à partir des extraits alcooliques. En parallèle, nous avons mis en évidence des activités biologiques significatives avec ces extraits alcooliques exerçant in vitro des effets antioxydants, anti-inflammatoires et antiprolifératifs.En résumé, nos observations suggèrent un potentiel prometteur pour Crataegus azarolus L et Ephedra campylopoda pour le traitement de maladies associées au stress oxydatif, à un processus inflammatoire ou à une prolifération cellulaire non contrôlée. Cependant, in vivo, la caractérisation de ces effets est indispensable. / Plants have long been known for their natural arsenal, serving as an important source of nutrients and therapeutic components. Since about 600,000 years ago, humans used plants as medicines. Plant medicines correspond to the preparations issued from those plants. Today, plant medicines are widely worldwide where about 80% of the world's population uses herbs as primary medicines. This medicinal value is mainly attributed to the fact that plants are rich inbioactive phytochemicals. Lebanon, due to its geographical location and important environmental characteristics, is endowed with a rich flora. Hundreds of Lebanese plants have been defined in terms of their chemical composition and medicinal value where many other species are yet to be characterized.In this study, we aimed at characterizing the phytochemical content and therapeutic value of two Lebanese plant species, Crataegus azarolus L and Ephedra campylopoda. Fresh leaves, derived from each plant species, were dissolved in three different solvents distilled water, ethanol, and methanol. The phytochemical composition of different extracts issued from the two plant species was examined using high performance liquid chromatography (HPLC) and the essential oil content was determined by gas chromatography (GC) coupled with mass spectrometry (MS). DPPH radical scavenging and Fe2+ chelating activity assays were used to assess the antioxidant potential. Anti-inflammatory potential was evaluated by measuring the secreted amounts of the pro-inflammatory mediator PGE2 using ELISA technique, as well as by assaying the mRNA levels of the pro-inflammatory cytokines (IL-α, IL-β and IL-6), chemokines (CCL3 and CCL4) and inflammation-sensitive COX-2 and iNOS using quantitative RT-PCR (qRT-PCR). XTT viability assay was carried out to determine the anti-proliferative effect of each extract. For both plant species, we observed an important phytochemical content with thealcoholic (methanol and ethanol) extracts being more rich in bioactive molecules. In parallel, the two plant species exhibited significant biological activities with the alcoholic extracts exerting important, in vitro, antioxidant, anti-inflammatory and anti-proliferative effects.Collectively, our observations suggest a promising potential for Crataegus azarolus L and Ephedra campylopoda during treatment of diseases associated with oxidative stress, aberrant inflammatory responses or uncontrolled cell proliferation. However, further in vivo characterization of these effects is indispensable.

Thrombosis and colorectal cancer have a bi-directional relationship. The presence of a colorectal malignancy results in an increased risk of developing a thrombosis and the presence of a thrombosis results in a worse cancer prognosis. The physiology causing this is at present unclear but it is proposed that proteins from the tissue factor (TF) pathway may be the instigator of this bi-directional relationship. The in-vitro studies have shown that in colorectal cancer TF impairs that action of colorectal cancer stem cells as demonstrated by reduced cancer sphere formation and also lower expression of the stem cell marker ALDH. The ability for a colorectal cell to avoid anoikis is impaired by a reduced TF level. Proliferation is affected by the level of expression of TF with a significant increase in proliferation with additional expression of TF. The increase in proliferation is further increased by the presence of TF’s ligand factor VIIa. Paradoxically reduced expression of TF also increases colorectal cancer expression. The ERK1/2 pathway offers a possible method by which TF and factor VIIa may exert their proliferative effects. In the prospective clinical cohort study (CHAMPion) abnormal expression of TF pathway proteins (TF, PAR1, PAR2 and thrombin) by both malignant epithelial and cancer associated stromal cells has been demonstrated. The stromal expression was independent of the epithelial expression and was only in stroma in close contact (0.1mm) with epithelial cells suggesting that the TF pathway proteins may have a role in stromal/epithelial communication. There was no link between the expression of TF pathway proteins and clinicopathological markers of a poor prognosis. The plasma expression of markers of TF pathway activation did not demonstrate any role as a biomarker for colorectal cancer or prognosis. The CHAMPion study has demonstrated that 7% of patients undergoing surgery for colorectal cancer have asymptomatic pre-operative DVTs present. A further 6% who were DVT free pre-operatively developed a DVT in the peri-operative period despite receiving venous thromboprophylaxis in line with current national guidelines. Pre-operative d-dimer may have the potential to identify those patients at risk of a post-operative VTE.This thesis establishes the role that TF has in promoting proliferation and anoikis resistance. It also confirms the abnormal expression of TF pathway proteins by colorectal cancer epithelial cells and for the first time demonstrates abnormal expression by the cancer associated stroma. The interaction between the stroma and epithelial cells, combined with the cellular effects of TF suggests that targeting this interaction may have a therapeutic role. The incidence of DVTs pre-operatively suggests that screening patients for the asymptomatic presence of a DVT may have an impact on their clinical outcome. The development of DVTs despite prophylaxis suggests that the level of anticoagulation is insufficient and current guidelines need to be revisited.

>Magister Scientiae - MSc / Tobacco consumption and exposure to tobacco smoke is one of the biggest contributing factors to a growing epidemic of non-communicable diseases (NCDs), primarily cancers, diabetes, cardiovascular and chronic lung diseases which account for 63% of all deaths worldwide (WHO, 2011). An increased concern is in pregnant women who smoke. They not only expose themselves to nicotine, but also their unborn child. Cigarette smoking during pregnancy is associated with many developmental and growth complications. There are critical periods within the “program” that directs normal growth and development, during which the fetus is vulnerable to the effects of external factors. During these critical periods of development the program can be changed to increase the susceptibility of the fetal organs to disease and increased risk of adverse health consequences in adulthood. Health care professionals have tried to reduce the consumption of tobacco smoke by prescribing nicotine replacement therapy (NRT) to pregnant females as an alternative to smoking, without considering the effects of nicotine on the developing embryo and the health risk that might arise after birth. It is known that nicotine induces oxidant formation with resulting oxidative effects. This induces an overload of oxidants in the fetus and a decrease in the antioxidant capacity thereof. This may interfere with normal lung development.

The prevalence of milder forms of HIV-associated neurocognitive disorders (HAND) is rising despite combination antiretroviral therapy (cART). Efavirenz (EFV) is among the most commonly used antiretroviral drugs globally, but causes neurological symptoms that may interfere with adherence and reduce tolerability, and may have central nervous system (CNS) effects that contribute in part to HAND in patients on cART. Thus we evaluated a commonly used EFV containing regimen: EFV/zidovudine (AZT)/lamivudine (3TC) in murine N2a cells transfected with the human “Swedish” mutant form of amyloid precursor protein (SweAPP N2a cells) to assess for promotion of amyloid-beta (Aβ) production (Chapter 3). Treatment with EFV or the EFV containing regimen generated significantly increased soluble Aβ, and promoted increased β-secretase-1 (BACE-1) expression while 3TC, AZT, or, vehicle control did not significantly alter these endpoints. Further, EFV or the EFV containing regimen promoted significantly more mitochondrial stress in SweAPP N2a cells as compared to 3TC, AZT, or vehicle control. We next tested the EFV containing regimen in Aβ - producing Tg2576 mice combined or singly using clinically relevant doses. EFV or the EFV containing regimen promoted significantly more BACE-1 expression and soluble Aβ generation while 3TC, AZT, or vehicle control did not. Finally, microglial Aβ phagocytosis was significantly reduced by EFV or the EFV containing regimen but not by AZT, 3TC, or vehicle control alone. These data suggest the majority of Aβ promoting effects of this cART regimen are dependent upon EFV as it promotes both increased production, and decreased clearance of Aβ peptide. Further (Chapter 4), there is evidence that neural stem cells (NSCs) can migrate to sites of brain injury such as those caused by inflammation and oxidative stress, which are pathological features of HAND. Thus, reductions in NSCs may contribute to HAND pathogenesis. Since the HIV non-nucleoside reverse transcriptase inhibitor EFV has previously been associated with cognitive deficits and promotion of oxidative stress pathways, we examined its effect on NSCs in vitro as well as in C57BL/6J mice. Here we report that EFV induced a decrease in NSC proliferation in vitro as indicated by MTT assay, as well as BrdU and nestin immunocytochemistry. In addition, EFV decreased intracellular NSC adenosine triphosphate (ATP) stores and NSC mitochondrial membrane potential (MMP). Further, we found that EFV promoted increased lactate dehydrogenase (LDH) release, activation of p38 mitogen-activated protein kinase (MAPK), and increased Bax expression in cultured NSCs. Moreover, EFV reduced the quantity of proliferating NSCs in the subventricular zone (SVZ) of C57BL/6 mice as suggested by BrdU, and increased apoptosis as measured by active caspase-3 immunohistochemistry. If these in vitro and in vivo models translate to the clinical syndrome, then a pharmacological or cell-based therapy aimed at opposing EFV-mediated reductions in NSC proliferation may be beneficial to prevent or treat HAND in patients receiving EFV. 1 Portions of this abstract have been previously published (Brown LAM, et al., 2014; Jin, J, et al, 2016) and are utilized with permission of the publisher.1

Il est connu qu’une consommation modérée de vin rouge protégerait contre de nombreuses pathologies, du fait notamment de ses polyphénols. Nous avons étudié en particulier l’effet de ces polyphénols sur le cancer colorectal, l’un des cancers les plus fréquents dans les pays industrialisés. Certains polyphénols purifiés de vin rouge, comme le resvératrol et la quercétine, montrent des effets antiprolifératifs contre les cellules de cancer colorectal.Notre première approche a consisté à utiliser des extraits polyphénoliques de vins rouges de Bourgogne. Nous avons alors étudié d’une part, leurs effets in vitro sur la prolifération cellulaire de lignées tumorales colorectales humaines SW480 et les interactions entre ces polyphénols et le transport et le métabolisme du resvératrol ; et d’autre part, les effets in vivo chez la souris CF-1 d’une consommation régulière de ces polyphénols dans l’alimentation sur la survenue de lésions pré-néoplasiques chimio-induites par l’azoxyméthane au niveau du colon.Notre deuxième approche s’est orientée vers les modifications de la structure chimique du resvératrol. Par l’ajout de groupements méthoxyles et hydroxyles, et par l’isomérisation de sa structure (cis ou trans), nous avons cherché à dégager des relations structures-fonctions du resvératrol et à créer des dérivés plus bioactifs.Nous montrons que les polyphénols du vin rouge inhibent la prolifération de cellules cancéreuses coliques, en bloquant le cycle cellulaire. Certains polyphénols du vin tel que la quercétine sont capables d’augmenter la capacité des cellules SW480 à accumuler le resvératrol. [Nous montrons que] chez les souris, la prise régulière de polyphénols de vin rouge dans l’alimentation diminue le nombre global de lésions pré-néoplasiques, et surtout les lésions de grande taille dont dérivent les polypes intestinaux. Les modifications chimiques de la structure du resvératrol montrent que les analogues méthoxylés du cis-resvératrol sont de puissants antiprolifératifs par blocage de la mitose, alors que le mécanisme d’action des dérivés du trans-resvératrol bloque le cycle cellulaire au niveau de la phase S.Ce travail supporte l’idée que le recours aux polyphénols du vin dans la prévention des adénocarcinomes coliques est possible et que ce champs de recherche est une voie prometteuse. Concernant des visées thérapeutiques, la recherche d’analogues du resvératrol présentant une biodisponibilité accrue est une piste à poursuivre / It is known that moderate consumption of red wine may protect against many diseases, in particular because of its polyphenols. We studied in particular the effect of these polyphenols on colorectal cancer, one of the most common cancers in industrialized countries. Some purified polyphenols from red wine, like resveratrol and quercetin, showed antiproliferative effects against colorectal cancer cells.Our first approach was to use polyphenolic extracts from Burgundy red wine. We then studied their in vitro effects on human colorectal tumor cell lines SW480’s proliferation and the interactions between the polyphenols and the transport and metabolism of resveratrol; and secondly, the effects on CF-1 mice of regular consumption of these polyphenols in the diet on the occurrence of chemically induced pre-neoplastic lesions by azoxymethane in the colon.Our second approach was directed towards changes in the chemical structure of resveratrol. By the addition of hydroxyl and methoxyl groups, and the isomerization of its structure (cis or trans), we wanted to identify structure-function relationships of resveratrol and create more bioactive derivatives.We show that the red wine polyphenols inhibit proliferation of colon cancer cells by blocking the cell cycle. Some wine polyphenols such as quercetin are able to increase the ability of SW480 cells to accumulate resveratrol. [We show that] in mice, the regular intake of red wine polyphenols in the diet reduces the overall number of pre-neoplastic lesions, especially large lesions which may become intestinal polyps. The chemical changes in the structure of resveratrol show that the methoxylated analogues of cis-resveratrol are potent antiproliferatives by blocking mitosis, while the mechanism of action of derivatives of trans-resveratrol blocks the cell cycle at the S phase.This work supports the idea that the use of wine polyphenols in the prevention of colon adenocarcinomas is possible and that this field of research is a promising way. Concerning therapeutic purposes, the search for analogues of resveratrol with improved bioavailability is a way to pursue

Orientadores: João Agostinho Machado Neto, Sara Teresinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T20:37:02Z (GMT). No. of bitstreams: 1 MachadoNeto_JoaoAgostinho_D.pdf: 13204649 bytes, checksum: a00ab26c9097cb10817c14fe2bdb2356 (MD5) Previous issue date: 2015 / Resumo: A identificação de genes/proteínas diferencialmente expressos em neoplasias hematológicas e a investigação funcional destas proteínas no fenótipo neoplásico são essenciais para o entendimento da biologia da doença. ANKHD1 é altamente expressa em células de leucemia aguda e tem potencial para regular vários processos celulares através de seus domínios de repetição de anquirina. Nosso grupo de pesquisa havia previamente identificado a interação entre ANKHD1 e SIVA através de ensaio de duplo híbrido. Outras proteínas potencialmente relacionadas à ANKHD1 que foram de interesse deste trabalho foram a Stathmin 1 que interage com SIVA e a YAP1 que interage com ANKHD1 em células não hematológicas. O objetivo principal do presente trabalho foi investigar a participação funcional de ANKHD1 e proteínas relacionadas em neoplasias hematológicas. Utilizamos modelos de linhagens leucêmicas humanas e células hematopoéticas primárias provenientes de indivíduos normais (n=52) e de pacientes com diagnóstico de síndrome mielodisplásica (SMD; n=65), neoplasia mieloproliferativa (NMP; n=82), leucemia mieloide aguda (LMA; n=60) e leucemia linfoide aguda (LLA; n=19). Técnicas para avaliação de expressão gênica (qPCR), expressão e interação proteica (western blotting), ensaios funcionais de migração (ensaio transwell), proliferação (MTT e clonogenicidade in vitro, formação de tumor in vivo) e apoptose (Anexina V/IP, TUNEL), e inibição de proteínas de interesse através do uso de inibidores farmacológicos ou ferramentas de terapia gênica foram utilizados. A interação entre ANKHD1 e SIVA foi confirmada por ensaios de coimunoprecipitação. Em linhagens celulares leucêmicas U937 e Jurkat, o silenciamento de ANKHD1 reduziu a proliferação celular, migração e o crescimento tumoral, enquanto que a inibição de SIVA aumentou a migração e o crescimento tumoral e reduziu a sensibilidade à apoptose induzida por radiação UV de células U937. A inibição de ANKHD1 reduziu a atividade de Stathmin 1 e a interação entre SIVA/Stathmin 1. Grande parte das linhagens leucêmicas e amostras primárias de pacientes com neoplasias hematológicas não apresentou a expressão de YAP1, e o silenciamento de ANKHD1 não modulou a atividade de YAP1 em células U937. A expressão de Stathmin 1 foi significativamente elevada em amostras de células hematopoéticas de pacientes com neoplasisa hematológicas (SMD AREB-1/AREB-2, LMA, LLA e mielofibrose primária) quando comparada às amostras de doadores saudáveis. O silenciamento de Stathmin 1 reduziu a proliferação celular e clonogenicidade em células leucêmicas U937, Namalwa e células HEL JAK2V617F. Especificamente em células HEL JAK2V617F, a inibição de Stathmin 1 ou o tratamento com o agente estabilizador de microtúbulos paclitaxel teve um efeito potencializador ao tratamento com ruxolitinib, inibidor de JAK1/2, na indução de apoptose. Em conclusão, propomos que a ANKHD1 participa do fenótipo neoplásico de células leucêmicas através de sua interação com SIVA, inibição de SIVA e ativação de Stathmin 1. Os nossos resultados indicam que a função de ANKHD1 na leucemogênese independe de sua interação com YAP1. Em células humanas com ativação constitutiva da via JAK2/STAT, identificamos a relevância funcional da participação de Stathmin 1 no fenótipo neoplásico / Abstract: The identification of genes/proteins differentially expressed in hematopoietic neoplasms and the functional investigation of these proteins in the neoplastic phenotype are essential for the understanding of disease biology. ANKHD1 is highly expressed in acute leukemia cells and has a potential role in regulating many cellular processes through its ankyrin repeat domains. Our research group has previously identified the interaction between ANKHD1 and SIVA through two-hybrid assay. Other proteins potentially related to ANKHD1 that were of interest of this research are Stathmin 1 that interacts with SIVA, and YAP1 that interacts with ANKHD1 in non hematologic cells. The aim of this study was to investigate the functional role of ANKHD1 and its related-proteins in hematologic malignancies. We used human leukemia cell lines and primary hematopoietic cells from healthy donors (n=52) and patients with myelodysplastic syndromes (MDS; n=65), myeloproliferative neoplasms (MPN; n=82), acute myeloid leukemia (AML; n=60) and acute lymphoid leukemia (ALL; n=19). Techniques for the evaluation of gene expression (qPCR), protein expression and interaction (western blotting), functional assays of migration (transwell assay), proliferation (in vitro MTT and clonogenicity, in vivo tumor formation) and apoptosis (Annexin V/PI, TUNEL), and inhibition of proteins of interest through pharmacologic agents or gene therapy tools were used. The interaction between ANKHD1 and SIVA was confirmed by co-immunoprecipitation assays. In leukemia cell lines U937 and Jurkat, ANKHD1 silencing reduced cell proliferation, migration and tumor growth, while SIVA inhibition increased migration and tumor growth, and reduced sensitivity to UV-induced apoptosis of U937 cells. Inhibition of ANKHD1 reduced Stathmin 1 activity and the interaction between SIVA/Stathmin 1. A large proportion of leukemia cell lines and primary samples from patients with hematologic malignancies did not present YAP1 expression, and ANKHD1 silencing did not modulate YAP1 activity in U937 cells. Stathmin 1 expression was significantly higher in primary hematopoietic cells from patients with hematological malignancies (MDS RAEB-1/RAEB-2, AML, ALL and primary myelofibrosis) compared to samples from healthy donors. Stathmin 1 silencing reduced cell proliferation and clonogenicity of U937 and Namalwa leukemia cells, and HEL JAK2V617F cells. Specifically in HEL JAK2V617F cells, Stathmin 1 silencing or treatment with the microtubule-stabilizing agent paclitaxel had a potentiating effect to treatment with the JAK1/2 inhibitor ruxolitinib on apoptosis induction. In conclusion, we propose that ANKHD1 participates in the leukemia phenotype through its interaction with SIVA, SIVA inhibition, and Stathmin 1 activation. Our results indicate that ANKHD1 plays a role in leukemogenesis independently of its interaction with YAP1. In human cells with a constitutive activation of the JAK2/STAT pathway, we identified the relevant role of Stathmin 1 in the malignant phenotype / Doutorado / Clinica Medica / Doutor em Clínica Médica

Os hormônios glicocorticóides (GCs) têm sido amplamente empregados como agentes antiinflamatórios e anti-tumorais. Sua ação ocorre via receptores nucleares (GR) sendo dependente da remodelação da estrutura da cromatina. As proteínas Brm e BRG1, componentes essenciais de um complexo regulador global da transcrição (SWI/SNF), por remodelamento da cromatina, exercem um papel-chave na ação de GR. Para estudar o mecanismo de ação de GCs, foram utilizadas as linhagens celulares ST1 e P7, derivadas da linhagem celular C6, de glioma de rato. P7 é insensível ao tratamento com GC, enquanto ST1 apresenta reversão fenotípica tumoral&#8594;normal, gerando um bloqueio específico na fase G1. Um anti-soro policlonal específico para Brm e BRG1, foi gerado através da inoculaçâo de coelha com a proteína hBRG1 recombinante. Este antisoro foi utilizado para análisar os níveis destas proteínas nas duas linhagens celulares, sob ação de GC. Enquanto em ST1, Brm é induzida por GC, em células P7, o nível basal de Brm é relativamente alto, mantendo-se inalterado na presença de GC. A possíbilidade de existirem mutações no gene brm de células P7, foi investigada através de amplificação do DNA, por PCR, e seqüenciamento. A superexpressão de brm e BRG1 em células P7 mostrou que clones isolados apresentavam, de um modo geral, achatamento celular, diminuição da taxa de crescimento e da eficiência de plaqueamento em substrato sólido e semi-sólido. Alguns destes clones passaram a responder ao tratamento com GC, porém não tão drasticamente como as células ST1. Co-imunonoprecipitação mostrou algumas diferenças entre os complexos SWI/SNF de células ST1 e P7. / Glucocorticoid hormones (GCs) have been used as anti-inflammatory and anti-tumor agents, acting via nuclear receptors (GR) and being dependent on remodeling of the chromatin structure. As components of the global chromatin remodeling transcription complex (SWI/SNF), Brm and BRG-1 proteins play a key role in the action of GR. In order to study the mechanisms of action of GCs, we have been using the ST1 and P7 cell lines, derived from the C6, a rat glioma cell line. P7 is insensitive to the GC treatment, while ST1 displays a complete phenotypic reversion from tumoral to normal, including a G1-specific block in the cell cycle. A Brm and BRG1-specific polyclonal antiserum was generated, in rabbit, using recombinant hBRG1 protein as antigen. This antiserum was used to analyze the levels of Brm and BRG1 in these two cell lines, under GC treatment. While Brm is induced by GC, in ST1 cells, the basal level of Brm, in P7 cells, is relatively high, remaining unchanged under GC treatment. The possibility of brm mutations occurring in the P7 cells, was analyzed by DNA sequencing. Overexpression of brm and BRG1 in P7 cells led to morphological alterations (cell flattening) and decreased colony formation in agarose suspension and in solid substrate. Some of these clones became partially responsive to GC, when compared to the ST1 cell line. Co-immunoprecipitation assays revealed some differences in the SWI/SNF complex between ST1 and P7 cells.

Gastrópodes terrestres secretam muco pela superfície corporal, quando se locomovem, para proteção do corpo contra injúria mecânica, dessecação ou contato com substâncias nocivas. O muco de moluscos tem sido estudado como fonte de novos compostos naturais com diversas atividades biológicas, entre elas a capacidade de induzir proliferação celular. O presente trabalho propôs o estudo do muco produzido pelas lesmas terrestres Phyllocaulis boraceiensis como agente indutor de proliferação celular e síntese de colágeno em cultura de fibroblastos humanos normais, células endoteliais e no reparo de processos cicatriciais no modelo de ferida cirúrgica da derme de camundongos. Fibroblastos tratados com proteínas do muco de P. boraceiensis em concentrações menores que 0,012 <font face=\"Symbol\">mg/µl apresentaram aumento nas taxas de proliferação avaliadas pelo método colorimétrico do MTT e em citometria de fluxo CSFE, mostrando um efeito dose-dependente. Os resultados obtidos mostraram que fibroblastos humanos normais e células endoteliais tratados com 0,012 g/<font face=\"Symbol\">ml de proteínas do muco, aumentaram significativamente a produção e secreção de elementos da matriz extracelular, como as fibras de colágeno. Houve a redução da produção de radicais livres lipídicos poli-insaturados. O ensaio de cicatrização da derme lesionada de camundongos Balb-c tratados com proteínas do muco de P. boraceiensis na concentração de 0,012 <font face=\"Symbol\">mg/µl induziu o reparo da cicatrização com maior eficácia e em menor tempo quando comparado ao grupo controle e aos tratados com a concentração de 0,18 <font face=\"Symbol\">mg/µl. Esses resultados corroboram a premissa de que o muco de P. boraceiensis, assim como o de outros gastrópodes, apresenta propriedade proliferativa das células envolvidas nos processos de cicatrização. / Mucus of molluscs has been studied as a source of new natural compounds with diverse biological activities, as ability to induce cell proliferation. The aim of this study is the potential use of mucus produced by land slugs Phyllocaulis boraceiensis as a promoter of cell proliferation and collagen synthesis in human fibroblasts, endothelial cells and in repair processes of healing wound skin of mice. Fibroblasts treated with P. boraceiensis mucus at concentrations below 0.012 <font face=\"Symbol\">mg/µl have high rates of proliferation as evaluated by the MTT and CFSE flow cytometer assays, proliferative effect was dose-dependent. Fibroblasts and endothelial cells treated with 0.012 <font face=\"Symbol\">mlowg/µl mucus induced a significant increase in production and secretion of extracellular matrix such as collagen fibers. There was a reduction in polyunsaturated lipid production. Healing assay of lesions in mice treated with mucus at 0.012 <font face=\"Symbol\">mg/µl induce repair of the scar more effectively and in less time when compared to the control group and those treated 0.18 <font face=\"Symbol\">mg/µl. These findings support the premise that the P. boraceiensis mucus demonstrates proliferative properties in cells involved in the healing process.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23380号 / 医博第4749号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 木村 剛, 教授 YOUSSEFIAN Shohab / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM