Orientador: André Almeida Schenka / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T13:11:13Z (GMT). No. of bitstreams: 1 Souza_PhilipiCoutinhode_M.pdf: 7492039 bytes, checksum: 04ef7a018050c98ae0be37a7e79c79e7 (MD5) Previous issue date: 2013 / Resumo: O câncer de mama é a neoplasia maligna mais prevalente e a principal causa de óbito entre mulheres, no mundo. A despeito de avanços substanciais no entendimento da biologia da doença, nos métodos de detecção precoce, e em sua farmacoterapia, a sobrevida geral não se modificou significantemente nas últimas décadas. Portanto, pode se dizer que um dos deveres primordiais das Universidades Públicas engajadas com pesquisa básica e aplicadas consiste em contribuir para o desenvolvimento de novas estratégias de tratamento sistêmico desta neoplasia. Nesse contexto, um dos alvos estratégicos mais promissores no desenvolvimento de novos fármacos antineoplásicos é representado pela célula-tronco neoplásica (CTN). As CTNs têm sido associadas em inúmeros estudos à capacidade de algumas neoplasias malignas de resistir às principais modalidades terapêuticas antineoplásicas, especialmente à: radio-, quimio-, hormônio- e imunoterapias. Em resumo, na atualidade, a detecção de CTNs constitui uma ferramenta clínica bastante promissora enquanto alvo terapêutico, fator prognóstico e preditivo de resposta terapêutica. O objetivo deste trabalho foi descrever e discutir as potencialidades e limitações do modelo de carcinogênese mamária pelo DMBA, após reclassificação das neoplasias mamárias segundo os critérios diagnósticos da OMS (2003, 2012), subtipagem molecular e quantificação de imunomarcadores prognósticos, preditivos e de CTN. Após a aplicação do protocolo experimental de indução química pelo DMBA e a eutanásia dos animais controle e experimental, suas linhas mamárias (contendo ou não tumores) foram ressecadas e avaliadas quanto à morfologia e a imunoexpressão para marcadores de CTNs. Após 13 semanas, 100% dos animais desenvolveram neoplasias macroscópicas e histologicamente compatíveis com os critérios de avaliação indicados pela OMS. Os tumores foram classificados em carcinoma ductal, carcinoma papilífero, carcinoma lobular, carcinoma mioepitelial e tumor filóide, sendo o tipo mais frequente, o ductal. Poucos marcadores imunoistoquímicos mostraram correlação com variáveis de comportamento biológico. Mesmo assim, o grau de correlação não foi elevado. A grande heterogeneidade morfológica intratumoral e interanimal, associada à presença de subtipos histológicos bifásicos (tumor filóide), tumores com diferenciação mioepitelial/basal e à grande positividade para marcadores CTN clássicos (tanto em termos de frequência na amostra como em termos de distribuição média nas neoplasias) demonstram a participação clara de CTNs no modelo, o que o torna um importante referencial como modelo in vivo para o teste de drogas anti-CTN específicas. O modelo reproduz os principais subtipos histológicos e moleculares de câncer mamário, o que significa que poderá ser utilizado no estudo pormenorizado de drogas indicadas especificamente para cada subcategoria molecular (e.g., agentes hormonais que são indicados especificamente para os tipos luminais e trastuzumab, indicado nos tumores do subtipo HER2), bem como no desenvolvimento de novas drogas com maior especificidade para a classe molecular de interesse / Abstract: Breast cancer is the most common malignancy and the leading cause of death from cancer among females worldwide. Despite all the research and all the progress, methods of early detection, and its pharmacotherapy, overall survival has not changed significantly in recent decades. Therefore, the Public University has been engaged in basic and applied research is too contributed to the development of new strategies for systemic treatment of this malignancy. In this context, one of the most promising strategic targets in the development of the anticancer drugs is represented by neoplastic stem cell (NSC). Neoplastic stem cell has been linked in various studies to the capacity of some malignancies to resist major antineoplastic therapeutic modalities, especially: radio-, chemo-, hormone- and immunotherapies. In summary, the detection of NSCs is a clinical tool very promising while therapeutic target and prognostic factor predictive of therapeutic response. The aim of this study was to describe and discuss the strengths and limitations of the model of mammary carcinogenesis by DMBA, after reclassification of breast cancer according to the diagnostic criteria of the WHO (2003, 2012), and quantification of molecular subtyping prognostic immunomarkers, predictive and NSC. After application of the experimental protocol of chemical induction by DMBA and euthanasia of experimental and control animals, the mammary lines (with or without tumors) were resected and evaluated the morphology and immunostaining for markers of NSCs. After 13 weeks, 100% of the animals developed macroscopic neoplasms and histologically consistent with the evaluation criteria of evaluation indicated by WHO. Tumors were classified as ductal carcinoma, papillary carcinoma, lobular carcinoma, myoepithelial carcinoma and phyllodes tumor, being the most common type, the ductal. Few immunohistochemical markers correlated with variable behavior biological. Nevertheless, the degree of correlation was not high. The morphological intratumoral heterogeneity and interanimal associated with the presence of histological biphasic subtypes (phyllodes tumor), tumors with myoepithelial/basal differentiation and the positivity for NSCs classic markers (both in terms of frequency in the sample and in terms of the average distribution neoplasias) demonstrate a clear involvement of NSCs in the model, making it an important reference as a model for in vivo testing of anti-specific NSC. The model reproduces the main molecular and histological subtypes of breast cancer, which means that could be used in detailed study drug indicated specifically for each subcategory molecular (eg, hormonal agents that are suitable specifically for the luminal type and trastuzumab indicated in tumors HER2 subtype), as well as to develop new drugs with higher specificity for the class of molecular interest / Mestrado / Farmacologia / Mestre em Farmacologia

Le neuropaludisme (NP) est la forme la plus mortelle du paludisme. C'est une complication neurologique observée uniquement dans les cas d'infection par Plasmodium falciparum, principalement chez les enfants de moins de 5 ans vivant en Afrique Sub-saharienne, et les adultes non-immuns, notamment les femmes enceintes et les touristes visitant les zones d'endémie. Les signes cliniques sont à présent bien décrits (prostration, convulsions répétées, difficultés respiratoires, coma,...), mais les mécanismes physiopathologiques conduisant au NP sont encore mal définis. Leur élucidation est rendue difficile par la localisation cérébrale de la pathologie du vivant des patients et la faible disponibilité des données nécropsiques. Bien que l'accès aux tissus humains soit limité en nombre, les résultats d'autopsie ont permis d'établir que le NP résulte d'une séquestration des globules parasités au niveau de l'endothélium intra-vasculaire, associée à une forte réaction immunitaire. La stratégie de prise en charge du NP combine un traitement étiologique à base de dérivés d'artémisinine, ou de quinine et un traitement adjuvant symptomatique destiné à pallier à la défaillance multiorganique qui est à l'origine de l'issue fatale souvent observée. Le modèle de NP expérimental actuellement le plus utilisé est le modèle souris infecté par P. berghei ANKA. La pertinence de ce modèle est toutefois remise en cause en raison notamment des différences histo-pathologiques observées par rapport à la forme humaine. En effet, les souris manifestant les symptômes du NP ne présentent que très rarement le phénomène de séquestration, caractéristique majeure du NP chez l'Homme. Par ailleurs, comparativement à la réponse immunitaire de la souris, le modèle rat s'est également révélé plus proche de la réaction de l'Homme, dans le cas d'une autre parasitose, la schistosomose. L'objectif de la première partie du projet thèse a donc été la mise en place et l'évaluation d'un modèle alternatif de NP chez le rat. Ainsi un modèle de NP chez le rat Sprague Dawley infecté par la souche murine P. berghei K173 a été caractérisé sur les plans clinique, biologique (paramètres hématologiques et biochimiques),histopathologique et du profil cytokinique (cytokines cérébrales et sériques). La forte similarité des symptômes et des lésions associées au NP du rat Sprague Dawley infecté par P. berghei K173 par rapport au NP humain permet de valider la pertinence de ce modèle pour l'étude de la physiopathologie du NP. L'objectif de la deuxième partie de mon projet de thèse a été d'évaluer les effets pharmacologiques d'un candidat-médicament, l'isoflurane, pour le traitement adjuvant du NP. Ce composé présente l'avantage d'être déjà utilisé chez l'Homme à d'autres fins thérapeutiques et dispose donc d'une autorisation de mise sur le marché. Les résultats obtenus montrent une efficacité nette de l'isoflurane avec une rémission totale des signes de paralysie pour 47.8% des rats traités ainsi qu'un gain de survie des rats NP traités de 2 à 10 jours par rapport aux rats NP non traités. Ce gain de survie des animaux traités pourrait permettre un allongement de la fenêtre du temps de traitement étiologique, améliorant ainsi sensiblement le pronostic du NP. L'isoflurane, dont le mécanisme d'action semble être la réversion de la séquestration des globules parasités, limite les complications neurologiques souvent responsables de séquelles liées au NP. Des études ultérieures permettront d'optimiser ce nouveau protocole de traitement adjuvant du NP. / Cerebral malaria (CM) is the most deadly form of malaria. It is a neurological complication observed only in cases of infection with Plasmodium falciparum that affects mainly children under five years living in Sub-Saharan Africa and non-immune adults including pregnant women and tourists visiting endemic areas. Although clinical signs are well described (prostration, respiratory distress convulsions, coma), the pathophysiological mechanisms leading to CM are still unclear. Their elucidation in vivo is made difficult by the cerebral location and the low availability of autopsy data. Instead of limited access to human tissues, autopsy results have shown that CM results from a strong immune response linked to sequestration of infected red blood cells in the intravascular endothelium. Cerebral malaria management combines an etiological treatment with artemisinin derivatives or quinine and adjunct treatment of the multi-visceral failures, responsible of fatal outcome. P. berghei ANKA-infected mouse is widely used as experimental murine model of CM. However the relevance of this model is still questioned because of the histopathologic differences from the human form. Indeed, CM mice rarely exhibit the red blood cell sequestration that is a major feature of human CM. Furthermore, compared to mouse, the rat displays a closer immune response to human in Schistosoma infection. This PhD research project first aimed to implement and assess an alternative rat model of CM. The clinical, biological, histo-pathological features as well as the cytokine profiling of an experimental model of CM were characterized in Sprague Dawley rats infected with P. berghei strain K173. The strong similarity of the symptoms and lesions observed in this model with those reported in human CM confirms its high relevance. The second objective of this thesis project was to assess the pharmacological effects of a drug-candidate in adjunct treatment of CM. Results demonstrated a strong efficacy of the molecule tested with 47.8% of the treated CM rats showing total remission. Moreover we observed a 2- to 10-day survival gain in the treated CM rats group compared to the non-treated CM rat group. Preliminary data suggest that this drug-candidate may reverse the endothelial sequestration of parasitized red blood cells and so limit the neurological sequels related to CM. It is anticipated that the gain in survival associated with this drug-candidate use will extend the window of the etiological treatment time, thus significantly improving the global prognosis of CM. Further studies are needed to optimize this adjunct CM treatment protocol.

On étudie les effets des nanoparticules (d'argent et de silice) manufacturées sur les métabolismes oxydatifs et inflammatoire. La première partie étudie la toxicité in vivo de l'ingestion de nanoparticules d'argent, pendant 11 semaines, sur un modèle animal - rat Sprague Dawley. Nous y avons mis en évidence l'action toxique des nanoparticules d'argent notamment une hausse de la production d'anion superoxyde par les NADPH oxydases hépatiques et cardiaques, des dyslipidémies, une cytolyse hépatique, une augmentation de cytokines pro-inflammatoires et une tendance à la baisse de l'activité d'enzymes antioxydantes. Ceci nous a conduit à aborder l'étude in vitro, sur des modèles cellulaires intestinaux (Caco-2) et cutanés (HaCaT). Au cours de cette étude, des nanoparticules de silice, fonctionnalisées ou non avec des antioxydants, ont été incubées pendant 24 H en présence des cellules. Nous montrons que la modification de la surface des nanoparticules réduit considérablement leur toxicité en limitant la production d'espèces radicalaires et la mortalité cellulaire. D’autre part, le couplage avec un antioxydant permet d’augmenter la stimulation de voie de signalisation du facteur Nrf2. Cette voie est impliquée dans la protection de l’organisme contre les troubles liés aux espèces radicalaires. En somme, ce travail met en avant les potentialités de la vectorisation d’antioxydants avec des nanoparticules à des fins thérapeutiques. / The purpose of this study is to explore the effects of nanoparticles (silver and silica) manufactured on oxidative and inflammatory metabolism. In the first part of this work, we explored the in vivo toxicity from ingestion of silver nanoparticles, for 11 weeks, in an animal model - Sprague Dawley rat. This enabled us to demonstrate the toxic properties of silver nanoparticles including superoxide anion production by hepatic and cardiac NADPH oxidases, dyslipidemia, hepatic cytolysis, an increase in proinflammatory cytokines and a downward trend the activity of antioxidant enzymes. This led us to address the in vitro study on intestinal cell models (Caco-2) and cutaneous (HaCaT). During this study, silica nanoparticles, functionalized or not with anti-oxidants, were incubated for 24 hours in the presence of the cells. We show that the modification of the surface of the nanoparticles significantly reduces their toxicity limiting the production of free radical species and cell death. Furthermore, the coupling with an anti-oxidant increases the stimulation of Nrf2 factor that involves the protection of the body against disorders associated with radical species. In summary, this work highlights the potential of vectorization of antioxidants with nanoparticles for therapeutic purposes.

Doctor of Philosophy / Department of Psychology / Mary E. Cain / Glutamate contributes to the neurological and behavioral changes that occur during differential rearing, and those that occur during conditioned hyperactivity and sensitization. Metabotropic glutamate receptor 5 (mGluR5) in particular contributes to the psychostimulant reward pathway, plasticity, and differential rearing. The present study examined the role of mGluR5 in conditioning and sensitization in differentially reared rats. Rats were reared in an enriched (EC), impoverished (IC), or social (SC) condition for 30 days, after which they received repeated amphetamine (0.3 mg/kg) or saline injections. Following training, rats received an injection of the mGluR5 antagonist MTEP or saline prior to undergoing conditioned hyperactivity and sensitization tests. Results showed that MTEP attenuated conditioned hyperactivity and sensitization in IC but not EC and SC rats, suggesting that glutamatergic changes occur during differential rearing that alter the effects of MTEP on amphetamine conditioning and sensitization. Additionally, results demonstrated that enrichment rearing has a protective effect against conditioned hyperactivity at low doses of amphetamine.

Master of Science / Department of Psychology / Mary E. Cain / Previous research has shown that both the environment and psychostimulant use influence dopamine levels via the mesolimbic dopamine pathway. C-fos expression has also been observed following exposure to novel environments and psychostimulants. The present study looked to determine the effects of acute amphetamine exposure on locomotor activity and c-fos expression in the basolateral and central nucleus of the amygdala, for rats raised in either an enriched condition (EC), impoverished condition (IC), or social condition (SC). Rats were reared in either the EC, IC, or SC for 30 days, after which they received an acute amphetamine injection (1.0 mg/kg) and locomotor activity was measured. Following the locomotor test rats were perfused and immunohistochemistry was used to measure c-fos levels in the basolateral and central nucleus of the amygdala. Results showed that EC amphetamine rats had significantly greater locomotor activity compared to EC saline rats. There were no significant group or treatment differences in c-fos expression in the ACe. In the BLA SC amphetamine rats had significantly greater c-fos expression than EC amphetamine rats. Overall, the current study revealed that environmental enrichment and amphetamine do significantly alter locomotor activity and c-fos expression in the BLA.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The non-alcoholic fatty liver disease (NAFLD) is the most common liver pathology in developed countries with an estimated prevalence of 20 to 30% in the American population. A typically benign and asymptomatic pathology, NAFLD is characterized by hepatic steatosis and abnormal levels of hepatic enzymes stemming from an increase in circulating free fatty acids originating from white adipose tissue lipolysis, an increased de novo lipogenesis, reduced fatty acid oxidation and decreased hepatic triglycerides secretion, all within an insulin resistance context. NAFLD has the potential to progress to the non-alcoholic steatohepatitis (NASH), a condition marked by inflammation, advanced oxidative stress and fibrosis. NASH is expected to be the leading cause of liver transplant by 2020 due to its complications (i.e.: cirrhosis, hepatocellular carcinoma and liver failure). Various xenobiotics such as pesticides have been shown to promote the apparition and development of NAFLD. Of interest to this study is the neonicotinoid imidacloprid, more contemporarily known for its suspected role in the colony collapse disorder of various anthophilae species. Imidacloprid has been shown to induce hepatic oxidative stress in rats, a significant factor in the development of NAFLD and its progression to NASH. Lifestyle modifications, namely physical exercise, is a current treatment which has been proven beneficial to prevent and treat NAFLD by reducing hepatic steatosis, oxidative stress and improving insulin sensitivity. The role of any neonicotinoid on the development of NAFLD has yet to be examine and few have looked at the role of exercise in the treatment of NAFLD brought about by pesticide contamination.

In this paper I introduce some background for subtraction games and explore the Sprague-Grundy functions defined on them. I exhibit some subtraction games where the functions are guaranteed to be strictly periodic. I also exhibit a class of subtraction games which have bounded range, and show there are uncountably many of these.

Thesis (M.S.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.