A asma é definida como uma doença inflamatória crônica de caráter multifatorial, caracterizada pela obstrução reversível das vias aéreas, denso infiltrado inflamatório e hiper-reatividade brônquica a estímulos externos. Clinicamente, a doença é marcada por sintomas episódicos de dispneia, sibilo, tosse seca e sensação de aperto no peito. A terapia convencional da asma compreende o uso de anti-inflamatórios e broncodilatadores. A budesonida é um glicocorticoide esteroide e é dos fármacos mais utilizados na terapêutica da asma. No entanto, a budesonida apresenta baixa biodisponibilidade oral e o uso prolongado pode levar a efeitos adversos graves como afinamento da pele e supressão adrenocortical. No desenvolvimento de novas formulações, a avaliação da toxicidade é de extrema importância. Por conseguinte, o uso de cultura celular é de grande valia no desenvolvimento de protocolos para avaliação da toxicidade de novas formulações. Adicionalmente, a nanotecnologia é uma ferramenta importante para resolver problemas de biodisponibilidade e para contornar efeitos adversos da terapêutica convencional. Desta forma, o objetivo desta tese foi desenvolver um novo sistema nanoestruturado na forma de pó seco para inalação (Dry powders inhalers – DPI), obtido por aspersão contendo budesonida encapsulada, visando o tratamento da asma aguda e crônica. Essa proposta foi baseada na obtenção de um sistema pulverulento nanoestruturado com tamanho reduzido e controlado, visando a entrega pulmonar da budesonida. Na etapa de pré-formulação foi realizado um estudo fatorial avaliando diferentes métodos de preparação das nanocápsulas e os adjuvantes de secagem utilizados. As análises de tamanho de partícula, da formulação selecionada (nanocápsulas contendo budesonida e secas por aspersão com leucina) mostraram um tamanho reduzido e adequado para a administração pulmonar (2,7 μm). A morfologia demonstrou que estas partículas possuem um tamanho reduzido, forma esférica e superfície irregular, características importantes para a administração pulmonar. Quando analisada a distribuição pulmonar in vitro, em Impactador de Andersen, a formulação apresentou uma fração de partículas finas (Fine Particle Fraction – FPF) de 28%. Analisando os resultados dos experimentos em modelos de asma aguda e crônica induzidos por ovalbumina, os resultados da mecânica respiratória e função pulmonar mostraram uma diminuição na resistência e na elastância pulmonar, quando a budesonida nanoencapsulada foi utilizada, quando comparada com uma formulação comercial de budesonida, nas duas doses utilizadas (0,5 e 1,0 mg/Kg). Esse tratamento com nanocápsulas também mostrou eficiência na redução da inflamação, pela redução do número de leucócitos totais no fluido de lavagem bronco alveolar (Broncho Alveolar Lavage Fluid – BALF) e, principalmente, redução significativa no número dos eosinófilos no infiltrado pulmonar. Corroborando esses resultados, a quantificação da eotaxina – 1 e das citocinas pró-inflamatórias foram reduzidas, quando comparadas ao tratamento comercial. A análise histopatológica mostrou que quando o tratamento com as nanocápsulas foi utilizado, a produção de muco foi reduzida, bem como a produção de fibrose sub-epitelial, sugerindo um possível efeito sobre o remodelamento tecidual. Os resultados de toxicidade utilizando linhagem celular epitelial pulmonar (H441) mostrou uma redução na toxicidade da budesonida, quando encapsulada nas nanopartículas, tanto na forma de suspensão como na forma pulverulenta. Essa redução da toxicidade foi de 75% e de 50%, na dose de 100 μg/mL, para a suspensão e para o DPI, respectivamente. O conjunto dos resultados obtidos mostrou a potencial aplicabilidade da budesonida nanoencapsulada para o tratamento da asma, utilizando esse novo sistema DPI. / Asthma is characterized as a chronic inflammatory disease developed by multifactorial aspects such as genetic predisposition and exposure to environmental factors such as pollution, smoke and microorganisms. The conventional asthma therapy comprises the use of bronchodilators and anti-inflammatory. Budesonide is a glucocorticoid and is the most frequently used therapy in the treatment of asthma. However, this drug has low oral bioavailability and long term use may lead to adverse effects such as skin thinning and adrenal suppression. The evaluation of the toxicity of new formulation has critical role in the pharmaceutical development. The use of cell culture experiments can help this aspect. Additionally, nanotechnology is an important tool to solve problems regarding bioavailability and to circumvent adverse effects of conventional therapy. The aim of this work was to develop a nanostructured system as dry powder inhaler (DPI) containing budesonide loaded, obtained by spray-drying, targeting the treatment of acute and chronic asthma. This proposal was based on obtaining a nanostructured powder system with reduced and controlled size, aiming an alternative to treatment of asthma. A factorial study comparing different methods to produce the nanocapsules as well as the type of drying adjuvants was performed. The particle size of the selected formulation was 2.7 μm, an adequate reduced size suitable for pulmonary administration. The morphology of these particles showed a small size, spherical shape and irregular surface. All these characteristics are important for pulmonary administration. When analyzed the in vitro pulmonary distribution of the DPI, using an Andersen Cascade Impactor, showed a fine particle fraction (FPF) of 28%. Analyzing the results of the biological experiments, the mechanical respiratory and pulmonary function showed a decrease in lung elastance and resistance when budesonide was used nanoencapsulated compared with a commercial formulation of budesonide in two doses (0.5 and 1.0 mg / kg). Both treatments also showed nanocapsules efficiency in reduction of inflammation by reducing the total of leukocytes in the bronchial alveolar lavage fluid (BALF) and especially significant reduction in eosinophil infiltration in the lung tissue. Corroborating with these results, the quantification of eotaxin - 1 and proinflammatory cytokines was reduced when compared to commercial budesonide treatment. Histopathological analysis showed that when treatment with the nanocapsules was used, mucus production was reduced and reversed the phenomena of airway remodeling. The cytotoxicity assay by Alamar blue using the bronchial epithelium cell line (H441) showed a reduction on the toxicity of budesonide when the nanocapsules were used even in suspension or in the DPI. The cytotoxicity reduction were 75 and 50%, at 100 μg/mL, for the suspension and the DPI, respectively. All these results show that budesonide-loaded nanocapsules in dry powder inhaler is a promising approach for the treatment of asthma.

In this study, we show that removal of a quorum sensing subtype of stromal macrophage expands the support capacity of ex vivo bone marrow culture. Notably, this system maintains much of the remaining paracrine signaling of the organ, unlike traditional macrophage ablation or cytokine supplemented media and does not place undue stress on the HSPC itself. Recent studies have independently identified alternatively activated macrophages that suppress hematopoiesis in in vitro culture. We have identified for the first time, a small molecule capable of preferentially killing those cells, thus providing a method to both culture unaltered HSPC ex vivo for long periods of time and significantly expand transient progenitor cells to assist transplantation efficiency. Our culture system in unique in its ability to maintain cultured HSPC in the physiological micro-environment of the bone marrow We found the small molecule “999” capable of expanding hematopoietic capacity of stroma culture by selectively eliminating an MHCII-Hi subpopulation of stromal macrophages that suppress HSPC growth. Removal of these macrophages enables long-term HSC ex vivo stability and massive expansion of the MPP and its progeny. Cultures expanded in this manner have increased engraftment potential and behave physiologically normal upon transplantation. This investigation has also helped to uncover the role of TGFB in bone marrow quiescence signaling. The MHCII-HI target cells express TGFB and through it, signal quiescence to the HSPC, likely as a form of quorum sensing. Targeted acute elimination of that signal leads to unabashed expansion of MPP. Furthermore, macrophage polarization in the tumor microenvironment has also been show to promote tumor formation and often leads to poor prognosis. Molecular tools such as 999 that have the ability to alter macrophage polarization ratios may prove to be valuable synergistic tools for oncologists in conjunction with current therapies.

Cells sense and interact with their microenvironment to retrieve signals which include cell-matrix and cell-cell contacts. These signals account for the influence of culturing conditions and often control the local cellular phenotype and global functions of tissues. Here, I sought to understand if there is any information processed by cells in guiding cellular phenotype given the control of cell shapes and substrate rigidities. If there is, would these phenotypic changes achieve biomedical purposes? What is the strategy to engineer platforms that can handle the longstanding challenges in those fields? In this dissertation, the first chapter serves as an introduction which involves the origin of motivations, which mainly came from current challenges in biomedical researches of kidney podocytes. I have attempted to understand if it is possible to control podocyte differentiation through cell shape control which mimics their in vivo morphology. On the other hand, I have tried to reveal if it is possible that tissue stiffness can affect podocyte phenotype as a result of stiffness sensing. These two topics were rarely investigated for kidney podocytes, which is the critical component of human filtration barrier to perform renal functions. The effort that addresses the question how shape and substrate rigidity as in- formation repositories affect kidney podocytes phenotype has profound meaning in the understanding of renal physiological system and pathological mechanisms. The second chapter will focus on the methods to achieve successful long-term shape control on cells. Engineered cell-device interface using cross-linking biomaterial SU-8 plays a key role in this study. Compared with other previously used approaches summarized in this chapter, SU-8 provides various advantages both in the fabrication of micro- pattern architecture as well as its sustaining effectiveness in controlling cell shape. This approach has been proved very efficient and economic to facilitate single cell level manipulation. The chapter will describe in details the interface micro-fabrication and encountered technical challenges. The results that kidney podocytes were in good compliance with the micro-pattern proved the successful application of this technique. The third chapter will then transfer from micro-fabrication to biological experiments, which discusses in details how in intro kidney podocytes responded to their shapes by enforcing protein localization which characterizes a phenotype found in vivo. This phenotype among in vitro podocytes was further verified that it may contribute to podocytes differentiation and physiological functions. The information processed by shape was proved independent of tension-related processes and thus shape and tension could be regarded as separate contributors in cellular development. The interpretation of shape’s contribution could be referred to my previous publication in the journal of Cell: ”Decoding Information in Cell Shape”. In this study, the motifs of research were applied to other cell lines (Human vascular smooth muscle cell) as a step to generalize the ubiquity of shape’s contribution to cell differentiation. The study here was to differentiate shape and tension through investigating the difference between two major mechanosensors: β3 and β1 integrin receptors. The difference in cell phenotypes through integrin inhibition experiments demonstrated critical but unique role of integrin-based shape sensing in vitro. This chapter in dissertation covers most of the content in a previously submitted paper to Nature Cell Biology. In the fourth chapter, I further carried out a study that investigated if stiffness sensing can influence kidney podocyte phenotype. The fourth chapter will basically review the techniques in the fabrication of hydrogel-based cell culture platforms. In a similar manner to previous study using biomimetic shape for podocytes and find its phenotype, the target of this analysis was to use hydrogel-based biomimetic substrate with renal physiological stiffness and find if there is a differentiation phenotype. Since numerous materials have been reported in hydrogel studies, I will focus on the introduction to representative ones that have been most widely used. Their characteristics will be compared with the demands of kidney podocyte reasearch. Methodologies were the key to a successful research, and in this chapter I will describe in details what choices I made in choosing experimental methods that improved the efficiency and quality of cell culture platforms. A natural enzyme (microbial transglutaminase) cross-linked gelatin hydrogel was adopted here to provide ideal substrate rigidity control for podocytes. This method has demonstrated high efficiency and stability in making large cell culture surface. Moreover, it provides the hydrogel platform with an ideal range of elastic moduli suitable for renal tissue culture. The results will be discussed in detail in the fifth chapter. I successfully found a differentiation phenotype for podocytes cultured on the hydrogel platforms with a physiological stiffness. Similar phenotype, on the contrary, were not found in podocytes on platforms which were either too soft or too stiff. These resutls have formed one of my submitted paper to Scientific Report. The differentiation phenotype for kidney podocytes was characterized by up-regulation of differentiation markers. These findings were in a similar manner to a series of stem cells differentiation guided by regulated substrate stiffnesses. This phenotype of kidney podocytes was verified by microarray technique which confirmed the stiffness sensing using transcription factors. The enrichment analysis of kinases also showed significant response of Src, Fyn etc, of which the activities have been shown critical for podocytes to preserve their physiological functions. These results have successfully suggested the close relations between stiffness changes of glomeruli basement membrane (GBM) and progressive podocyte dysfunction. In summary, this dissertation covers interdisciplinary researches that decoded the information processed by cells from two separate aspects: shape and stiffness sensing. The details in each chapter cover a broader scope than the content selected for publications. Through this dissertation, readers will get in touch with the technique developed for plat- form and their applications to biomedical researches. I wish this will help people new in the field to get my hands-on experience.

No presente estudo foi analisado o perfil eletroforético de 15 amostras de rotavírus, sendo 11 de bezerros, 02 de leitões e 02 de criança, que foram cultivadas até a sexta passagem em células da linhagem MA104, com o monitoramento realizado pela técnica de eletroforese em gel de poliacrilamida (PAGE). O objetivo foi comparar o perfil eletroforético de amostras fecais e após o isolamento As amostras leitões não revelaram mudanças de migração aparente, mas uma amostra apresentou um segmento adicional entre os segmentos 5 e 6, tanto no material anterior ao cultivo, quanto na amostra isolada. Em relação as amostras de crianças, uma apresentou diferença na velocidade de migração eletroforética. Além disso, uma amostra de bezerro apresentou um segmento adicional entre os segmentos 5 e 6. Estes dados ressaltam a importância da PAGE como uma técnica de triagem de amostras, que podem, posteriormente serem analisadas por técnicas moleculares mais especificas. As mudanças de comportamento na migração eletroforética dos segmentos genômicos do RNA viral podem ser sugestivas de alteração nas propriedades antigênicas, além do fato de que mudanças que ocorreram in vitro poderão, também, ocorrer in vivo. / In the present work, we have analyzed the electrophoretic profile of 15 samples of rotavírus, 11 of calves, 02 of pigs and 02 of children, that have been cultivated on MA,104 cell up to the sixth passage. The polyacrilamide gel electrophoresis(PAGE) was used to confirm the viral isolation. The aim of the work was to compare the electropherotic profile of fecal samples before and after isolation in MA104 cells. The samples from pigs did not reveal apparent changes in bands migration. However, an additional segment, betweem segments 5 and 6 was detected, in one fecal sample and after isolation. Furthermore, one child sample showed differences in the electrophoretic mobility and also an additional segment was detected between segments 5 - 6 in one sample from a calf. The results emphasize the importance of PAGE as a technique to screen samples that could be analyzed posteriorly by more specific molecular techniques. The changes in the electrophoretic profiles of genomic segments of viral RNA might be suggestive of changes in the antigenic properties, besides the fact that changes that occurred in vitro may also happen in vivo.

O estabelecimento e perfeito funcionamento da placenta são fatores dependentes da intensa vascularização ocorrida no órgão. Os processos de vasculogênese e angiogênese placentária são modulados por diversos fatores, incluindo o VEGF (fator de crescimento vascular endotelial) e bFGF (fator de crescimento fibroblástico básico). Apesar da importância do VEGF e bFGF durante a vascularização estar estabelecida, vários estudos indicam a participação desses fatores de crescimento como moduladores locais em outras funções fisiológicas, como por exemplo o controle da produção hormonal em tecidos esteroidogênicos. Animais clonados podem apresentar alterações na expressão de determinados genes durante seu desenvolvimento, o que pode alterar a função placentária. Os objetivos deste estudo são determinar a localização tecidual do VEGF, bFGF e seus receptores na placenta bovina e avaliar a influência destes fatores de crescimento sobre a produção de progesterona placentária em bovinos não clonados e clonados. Placentomas de 90, 150 e 210 dias de gestação foram obtidos em abatedouro e placentônios de gestações aos 270 dias provenientes de bovinos clonados e não clonados foram coletados após cesarianas. As amostras foram fixadas em formol tamponado 4%, desidratadas e incluídas em parafina. Cortes foram submetidos a imuno-histoquímica para posterior localização das proteínas do VEGF, bFGF e seus receptores. Sob condições assépticas, as células foram mecanicamente dispersas e cultivadas em placas de 96 cavidades. Os fatores foram adicionados em concentrações de 10 e 50 ηg/ml de bFGF e VEGF, respectivamente. Amostras de meio de cultura e as células dos grupos controle, bFGF, VEGF e VEGF mais bFGF foram coletadas 24, 48 e 96 horas após a adição dos fatores. A progesterona foi dosada por radioimunoensaio e o conteúdo protéico pelo método de Lowry. Os dados foram analisados utilizando-se o programa estatístico SAS (Statistical Analysis System), as diferenças estatísticas encontradas foram comparadas pelo teste de variação múltipla de Duncan. O VEGF, bFGF e seus receptores foram localizados em células do epitélio e estroma maternos e fetais e células endoteliais vasculares em bovinos não clonados e clonados. As células placentárias apresentaram diferentes capacidades de síntese de progesterona ao longo da gestação. Aos 90 e 210 dias de gestação o VEGF estimulou a produção de progesterona, enquanto aos 270 dias de gestação o fator inibiu a produção deste hormônio. O bFGF estimulou a produção de progesterona pelas células placentárias aos 90 dias de gestação. A adição dos dois fatores de crescimento conjuntamente determinou um estímulo na produção de progesterona aos 210 dias de gestação. A produção de progesterona pelas células de bovinos clonados foi semelhante àquela observada em células de bovinos não clonados na mesma idade gestacional e os fatores de crescimento não influenciaram essa produção. Conclui-se que o VEGF e bFGF, atuando localmente no tecido placentário, funcionam como moduladores do processo de esteroidogênese, influenciando de maneira tempo-dependente a produção de progesterona deste órgão. / Placental establishment and function are dependent on intense vascularization. Placental vasculogenesis and angiogenesis are modulated by several factors, including VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor). Although the role of VEGF and bFGF during vascularization is already well established, some studies have indicated the participation of these growth factors as local modulators in other physiological functions, such as control of hormonal production in steroidogenic tissues. Cloned animals may exhibit alterations in gene expression during development modifying placental function. The aims of this study are to determine the tissue localization of VEGF, bFGF and their receptors in the bovine placenta and to evaluate the influence of bFGF and VEGF on placental progesterone production in non-cloned and cloned bovines. Placentomes from days 90, 150 and 210 of pregnancy were obtained at local slaughterhouse and placentomes from cloned and non-cloned gestations at 270 days were obtained after cesarean sections. Samples were fixed in 4% buffered formol solution, dehydrated and included in paraffin. Sections were subimitted to immunohistochemistry for subsequent localization of VEGF, bFGF and their receptors proteins. Under aseptic conditions, cells were mechanically dispersed and then cultivated in a 96-well plate. Growth factors were added at concentrations of 10 and 50 ηg/ml for bFGF and VEGF, respectively. Samples of culture medium and cells from control, bFGF, VEGF and bFGF plus VEGF groups were collected 24, 48 and 96 hours after growth factor addition. Progesterone concentrations were assessed by radioimmunoassay and protein content was measured by Lowry?s method. Data were analyzed by SAS (Statistical Analysis System) program, significant differences were compared by Duncan?s range multiple test. VEGF, bFGF and their receptors were localized in maternal and fetal epithelial and stromal cells and vascular endothelial cells during pregnancy in non-cloned animals and in cloned bovine placenta at 270 days of pregnancy. Bovine placental cells were able to produce different amounts of progesterone during pregnancy. Growth factors were able to influence progesterone production in placental cells only after 24 hours in culture. At 90 and 210 days of pregnancy VEGF stimulated progesterone production, while at 270 days of pregnancy the growth factor inhibited production of this hormone. bFGF stimulated progesterone production in placental cells from 90 days of pregnancy. Both growth factors together determined an increase in progesterone production in placental cells from 210 days of pregnancy. Progesterone production in placental cells from cloned cattle is similar when compared with non-cloned placental cells at the same gestational age and growth factors did not influence progesterone production in these cells. VEGF and bFGF, acting locally in the placental tissue, are modulators of the steroidogenic process, influencing in a time-dependent manner the progesterone production in this organ.

Os modelos de reconstrução do microambiente são úteis para investigar as propriedades biológicas dos melanócitos humanos com a matriz e como plataforma para testes de novos fármacos. Existe uma demanda crescente para a utilização de pele e derme reconstruídas em laboratório, em ensaios in vitro de citotoxicidade, viabilidade celular, crescimento celular, irritabilidade e avaliação dos constituintes da matriz extracelular. Caracterizamos, em equivalente dérmico, alguns mecanismos de viabilidade e invasão de melanoma metastático humano quando na presença da matriz, a fim de ampliar o conhecimento a respeito de novas terapias contra o melanoma e entender os mecanismos que favorecem seu potencial invasivo, mimetizando com mais fidelidade o que ocorre in vivo. Através da validação deste modelo, constatamos que ele é essencial para resgatar a fisiopatologia do tumor, pois o melanoma, na presença de equivalente dérmico, torna-se capaz de evadir mecanismos de morte e aumentar a secreção de metaproteinases e citocinas que favorecerão a evolução tumoral. Avaliamos também as propriedades antineoplásicas do ácido clorogênico, que já foram relatadas em um grande número de tumores, porém os mecanismos que levam à sua ação antitumoral ainda não foram bem elucidados. Diante dos trabalhos enfatizando o efeito quimioprotetor dos polifenóis, referentes à sua ação antioxidante em células neoplásicas e com potencial metastático, não há na literatura estudos que comprovem a eficácia do ácido clorogênico sobre células de melanoma metastático humano. Portanto, este estudo visou recriar a estrutura dérmica in vitro e, a partir disso, comparar a resposta de fármacos em células de melanoma cultivada em equivalente dérmico, a fim de avaliar se há modulação diferencial nesse substrato, sugerindo, portanto, um protocolo mais eficaz para a prospecção de fármacos antimelanoma. / The microenvironment reconstruction models are useful for investigating the biological properties of human melanocytes onto the matrix and as platform for testing new drugs. There is a growing demand for the use of reconstructed skin and dermis in the laboratory for in vitro assays of cytotoxicity, viability, cell growth, irritability, and evaluation of the extracellular matrix. We characterize in dermal equivalent some mechanisms for cell viability and invasion of human metastatic melanoma in the presence of matrix, the order to increase knowledge about new therapies against melanoma and to understand the mechanisms that favor its invasive potential, to more closely mimic what occurs in vivo. By means of this model, we find that it is essential to rescue the tumor physiopathology as melanoma, in the presence of dermal equivalent, it is able to evade death mechanisms and increase the expression of metalloproteinases and cytokines which favor the tumor evolution. We also evaluated the antineoplastic properties of chlorogenic acid, that have been reported in a large number of tumors, but the mechanisms that lead to its antitumor action has not been well elucidated. Before the work emphasizing the chemoprotective effect of polyphenols related to its antioxidant action in neoplastic cells and with metastatic potential, there is no literature studies confirming the effectiveness of chlorogenic acid on human metastatic melanoma cells. Therefore, this study aims to rebuild the dermal structure in vitro, and from this, to compare the response to drugs in melanoma cells dermal equivalent cultured in order to evaluate whether modulation differential on the substrate, thus suggesting a protocol more effective for prospecting antimelanoma drugs.

Moléculas de origem vegetal foram há muito testadas como fonte de drogas antineoplásicas com sucesso promissor. Este trabalho trata dos efeitos antiangiogênicos do Metil Jasmonato. Este derivado hidrofóbico do ácido jasmônico foi demonstrado anteriormente como um agente de dano seletivo para a mitocôndria de células neoplásicas. In vitro, o Metil Jasmonato 1-10 mM promoveu a morte celular de células endoteliais humanas de cordão umbilical (HUVEC) e de melanoma murino (B16 -F10), enquanto concentrações micromolares foram inócuas. A inclusão do Metil Jasmonato em liposomos de fosfatidilcolina e em um nanocarreador hidrofílico baseado em açúcar mostrou efeitos diferenciais sobre a citotoxicidade. A interrupção do ciclo celular foi observada em concentrações citotóxicas, enquanto a diminuição na produção de VEGF e algum grau de autofagia foram sugeridos em concentrações micromolares. In vivo, Metil Jasmonato 1-10mM foi francamente tóxico, e reduziu a densidade de vasos em membranas corioalantóicas de embrião de galinha (CAM). Entretanto, concentrações entre 1-10 ?M produziram um efeito complexo. Ocorreu aumento no brotamento capilar, mas os novos vasos apresentaram-se frágeis e menos organizados que os controles correspondentes. Sugere-se que, além da toxicidade direta, a ação do Metil Jasmonato sobre a angiogênese seja relevante para seu efeito antineoplásico. / Molecular plant components have long been tested as sources for antineoplasic drugs with promising success. The present work deals with the anti-angiogenic effects of Methyl Jasmonate. This hydrophobic Jasmonate derivative was previously demonstrated to selectively damage the mitochondria of cancer cells. In vitro, 1-10 mM Methyl Jasmonate induced the cell death of the human umbilical vein endothelial cells (HUVEC) and the Murine melanoma cells (B16-F10), while micromolar concentrations were ineffective. Methyl Jasmonate inclusion in phosphatidylcholine liposomes and in an hydrophilic sugar based nanocarrier presented differential effects upon citotoxicity. Cell cycle arrest was observed in citotoxic concentrations, while VEGF withdrawn and some autophagy was suggested in the micromolar range. In vivo, 1-10mM concentrations were explicitly toxic and reduced the vessel density of the Chorioallantoic Membrane of the Chicken Embryo (CAM). However, 1-10 ?M concentrations produced a complex effect. There was increased capillary budding, but the new vessels were leakier and less organized than corresponding controls. It is suggested that not only direct toxicity, but also the drug effects upon angiogenesis are relevant to the antineoplasic effects of Methyl Jasmonate.

Um dos grandes problemas da saúde pública no país esta diretamente ligada à dengue, uma vez que aproximadamente 550 mil pessoas são infectadas anualmente, e cerca de 20 mil vêm a óbito. Esta doença é causada pelo vírus da família Flaviviridae, transmitido pela fêmea ao hospedeiro durante o repasto sanguíneo. Este projeto teve como objetivo isolar e caracterizar células indiferenciadas de ovos embrionados e do tubo digestivo de larvas em quarto instar de Aedes aegypti, como fonte de pesquisa na área de biologia celular e estrutural destes vetores. Mediante cultura celular, as células foram isoladas e recultivadas em meio de cultura DMEM-High, e temperatura de 28 ºC. As seguintes análises foram feitas: morfologia celular, ciclo celular, testes de imunofenotipagem pelo método de citometria de fluxo e analise de imunohistoquimica. As células de Aedes aegypti analisadas pela morfologia celular, apresentaram formato globóide, tamanho pequeno e população heterogênica. A análise das fases do ciclo Sub-G1, G0-G1, S e G2-M após cultura primaria de ovos embrionados atingiram (27,5%; 68%; 30,2% e 1,9%, respectivamente). Após 24 horas atingiram (10%; 92,4%; 6,2% e 0,6%, respectivamente). A análise de imunofenotipagem realizada pelo método de citometria de fluxo demonstrou em ovos embrionados, marcações positivas para pluripotencia, assim como baixa expressão para Oct 3/4, e alta expressão para o (Sox2). As células mesenquimais tiveram alta expressão para os marcadores (Stro-1, Vimentina e Nestin). O marcador de morte celular por apoptose (Caspase3), teve alta expressão nas células cultivadas, enquanto que os marcadores endossomicos específicos para células de insetos (Anti GM130 e Anti RAB-5) também foram altamente expressos. Na cultura celular derivada do tubo digestivo de larvas de Aedes aegypti, as analises dos marcadores para pluripotencia tais como Oct 3/4 não demonstrou expressão, entretanto, o marcador Sox2 foi altamente expresso na cultura. Para as células mesenquimais não houve expressão de nenhum marcador utilizado, ou seja, (Stro-1, Vimentina e Nestin). No entanto para o marcador de morte celular por apoptose (Caspase 3) as células em cultura demonstraram alta expressão, enquanto que para os marcadores endossomicos específicos de insetos os mesmos foram altamente marcados. O teste de imunohistoquímica realizado em ovos embrionados no final do período embrionário demonstrou que as células tinham potencial ploriferativo mediante marcação positiva para PCNA. As células pluripotentes foram pouco expressas pelos marcadores Nanog, Oct 3/4, e muito expressas pelo marcador Sox2. As células de origem mesenquimais foram altamente expressas pelos marcadores SSEA-4, Stro-1, Vimentina e Nestin assim como para os marcadores endossomicos específicos para células de insetos, os quais demonstraram alta expressão nas células de embriões de Aedes. Sendo assim, conclui-se que tanto ovos embrionados quanto larvas de quarto instar de Aedes aegypti possuem células indiferenciadas, com grande potencial para estudos com biologia molecular, visando o controle biológico deste vetor / A major problem of public health in the country is directly linked to dengue, as approximately 550 million people are infected annually, and about 20,000 have died. This disease is caused by the virus of the Flaviviridae family, transmitted by the female to the host during blood feeding. This project aimed to isolate and characterize stem cells from fertilized eggs and larvae gut fourth instar Aedes aegypti, as a source of research in cell biology and structural area of these vectors. Upon cell culture, the cells were isolated and recultured in DMEM-high culture temperature and 28 º C. The following analyzes were performed: cell morphology, cell cycle tests immunophenotyping by flow cytometry and immunohistochemistry analysis. The Aedes aegypti cells analyzed by cell morphology showed globular shape, small size and heterogenic population. The analysis of sub-G1 phase of the cycle, G0, G1, S and G2-M after primary culture reached embryonated eggs (27.5%, 68%, 30.2% and 1.9%, respectively). Achieved after 24 hours (10%, 92.4%, 6.2% and 0.6%, respectively). Immunophenotyping analysis by flow cytometry demonstrated in embryonated eggs, positive for pluripotency markers, as well as low expression of Oct 3/4 and high expression for the (Sox2). The mesenchymal cells had high expression for markers (Stro-1, vimentin and Nestin). The marker of cell death by apoptosis (Caspase3) had high expression in cultured cells, whereas the endosomal markers specific for insect cells (GM130 and Anti-Anti RAB 5) were also highly expressed. In cell culture derived from the gut of larvae of Aedes aegypti, the analysis of pluripotency markers such as Oct for 3/4 showed no expression, however, the marker Sox2 was highly expressed in the culture. For mesenchymal cells no expression of any label used, ie (Stro-1, Nestin and vimentin). However marker for cell death by apoptosis (caspase 3) cells in culture showed high expression, whereas for the specific endosomal markers insects they were highly labeled. The immunohistochemistry test on embryonated eggs at the end of the incubation period the cells have demonstrated potential ploriferativo by positive staining for PCNA. Pluripotent cells were slightly expressed by markers Nanog, Oct 3/4, Sox2, and expressed by the marker. The cells of mesenchymal origin were highly expressed by SSEA-4 markers, Stro-1, Nestin and vimentin as well as the specific endosomal markers for insect cells, which showed high expression in cells of Aedes embryos. Therefore, it is concluded that both embryonated eggs as room instar larvae of Aedes aegypti have undifferentiated cells, with great potential for studies of molecular biology, targeting the biological control of this vector

Paclitaxel is an anticancer natural product with several biomedical applications produced by Taxus species, with a demand exceeding its supply. We have developed cambial meristematic cells (CMCs) from Taxus cuspidata as high yield source of paclitaxel. The biosynthesis of paclitaxel is predominantly under transcriptional control. Thus, the identification of transcriptional regulators of paclitaxel biosynthesis and their subsequent manipulation may enable further yield enhancement in Taxus CMCs. Previously, Roche 454 sequencing was employed to establish the transcriptome of T. cuspidata CMCs treated with the plant immune activator methyl jasmonate (MeJA). The bioinformatic analysis identified 19 jasmonate related transcription factors (TFs), based on their differential expression. Results of the Arabidopsis thaliana transient assay screen identified two MYB TFs that constitute positive regulators for paclitaxel genes, named MYB3 and MYB4. In this thesis, MYB3 and MYB4 showed in vitro binding to the cis-elements in ten promoters of paclitaxel genes using the electrophoretic mobility shift assay (EMSA). Then, a Taxus CMC protoplasts transient assay demonstrated that the expression of MYB3 and MYB4 trans-activated all tested genes. Further, MYB4 was found to activate the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) gene, key in the mevalonate pathway and precursor of paclitaxel biosynthesis. MYB3 and MYB4 were capable of auto-regulating their own transcription, constituting an important control point for paclitaxel biosynthesis. A possible mechanism for the early activation of MYB3 and MYB4 after MeJA elicitation is proposed. Finally, preliminary results on the expression of MYB3 and MYB4 in unelicited T. baccata CMC protoplasts indicate that their transient expression was sufficient to increase accumulation of paclitaxel and the precursor, 10-deacetyl baccatin III, highlighting their utility for paclitaxel production.

A causa mais comum da osteoporose é o declínio do hormônio sexual feminino, o estrógeno, que ocorre após a menopausa. Esse hormônio regula a produção de citocinas que influenciam a proliferação dos osteoclastos, aumentando a reabsorção óssea. Os efeitos da cafeína no metabolismo ósseo são controversos e podem estar associados ao aumento significativo de doenças periodontais, fraturas, aumento do cálcio urinário e redução da densidade mineral óssea, por exercer efeitos inibidores sobre as funções dos osteoblastos. O principal objetivo deste estudo foi avaliar o efeito in vitro de diferentes concentrações de cafeína no metabolismo de células osteoblásticas da medula óssea de ratas osteoporóticas. Após aprovação pela Comissão de Ética no Uso de Animais, ratas Wistar foram divididas em dois grupos experimentais: controle (C) e submetidas à ovariectomia (OVX). Após 60 dias da cirurgia, as ratas foram sacrificadas para coleta dos fêmures e das células mesenquimais da medula óssea, que foram induzidas à diferenciação em osteoblastos com meio osteogênico com três concentrações de cafeína (1, 3 e 5 mM - grupos OVX1, OVX3 e OVX5) e cultivadas em placas de 24 poços (n = 5) para avaliação da proliferação celular, atividade de fosfatase alcalina (ALP) bioquímica e in situ, detecção e quantificação de nódulos mineralizados e análise da expressão de genes relacionados à atividade osteoblástica através de PCR em tempo real. Os ensaios foram realizados em triplicata e analisados por meio do software estatístico GraphPad Prism, com nível de significância fixado em 5%. A proliferação celular foi menor nos grupos osteoporóticos com adição de cafeína, tendo a menor queda o grupo com adição de 1mM. O método bioquímico de ALP não foi significante nos períodos analisados, entretanto, aos 10 e 14 dias, a atividade aumentou quando a concentração de cafeína era maior. Já na atividade de ALP in situ, o grupo OVX1 foi o que apresentou melhor resultado nos períodos avaliados (p < 0,05), com pico aos 14 dias. A quantificação de matriz mineralizada foi maior no grupo OVX comparado ao grupo C; entre as concentrações, a maior quantificação dos nódulos de cálcio se deu no grupo com 1mM de cafeína. Os resultados obtidos no PCR mostraram que o gene para fosfatase alcalina teve maior expressão no grupo OVX, seguido do grupo OVX1 aos 7 e 10 dias; a expressão gênica de osteocalcina foi maior para o grupo OVX1 aos 10 e 14 dias e esse grupo apresentou a maior expressão em todos os períodos para os genes Runx2 e RankL. No caso do Bmp4, o grupo OVX3 foi o mais expresso aos 10 e 14 dias. Os genes osteoprotegerina e osteopontina variaram sua expressão de acordo com o período e grupo avaliado. A expressão de osterix foi similar entre os grupos aos 7 e 10 dias, enquanto a expressão de Bsp, aos 7 e 14 dias, mostrou semelhança entre os grupos controle e OVX1. Frente aos resultados obtidos, sugere-se que a concentração de 1mM de cafeína pareceu ser a menos prejudicial ao metabolismo das células osteoblásticas neste modelo experimental de osteoporose. / The most common cause of osteoporosis is the decrease of estrogen after menopause. This hormone regulates the production of cytokines that influence osteoclast proliferation, increasing bone resorption. The effects of caffeine in bone metabolism are controversial and may be associated to periodontal disease, bone fractures, increase of urinary calcium levels and reduction of mineral bone density due to inhibition of osteoblast activities. Thus, the goal of this investigation was to evaluate the in vitro effect of different caffeine concentrations in the metabolism of bone marrow osteoblastic cells from osteoporotic rats (OVX). After Ethical Committee approval, wistar female rats were divided in two experimental groups: control (C) and submitted to ovariectomy (OVX). After 60 days of surgery, femurs were removed to isolate bone marrow mesenchymal cells, which were induced to osteoblastic differentiation in osteogenic medium along with three different concentrations of caffeine (1, 3 and 5 mM - OVX1, OVX3 e OVX5 respectively) and posteriorly seeded in 24-well plates (n = 5) to evaluate cell proliferation, alkaline phosphatase activity and its in situ detection, detection and quantification of mineralized nodules as well as assess quantitative expression of genes associated to osteoblastic activity by means of real time PCR. All the experiments were performed in triplicate and analyzed by means of the statistical software GraphPad Prism for p<0.05. Cell proliferation was diminished in the all osteoporotic groups that received caffeine, with group OVX1 being the less affected. Biochemical assay of ALP activity did not show differences among the groups in the periods analyzed; nevertheless there was a tendency to a higher activity proportional to the higher concentration of caffeine. The in situ detection of ALP showed better results in group OVX1 after 14 days of culture. Mineralized matrix quantification was higher in OVX groups when compared to control group; among the concentrations, the higher quantification of calcium nodules was in group OVX1. The results obtained with PCR showed that the gene for ALP had its highest expression in OVX group, followed by OVX1 at 7 and 10 days; expression of osteocalcin was higher in OVX1 after 10 and 14 days and this same group presented higher expression in all periods for genes Runx2 e Rankl. Analysis of Bmp4 gene showed that it was expressed in group OVX3 after 10 and 14 days. The genes that code for osteoprotegerin and osteopontin had different expression values in accordance to the period and group evaluated. The expression of osterix was similar between the groups after 7 and 10 days, whereas the expression of Bsp was similar between control and OVX1 groups after 7 and 14 days. The results suggest that the concentration of 1mM of caffeine is the most beneficial to the metabolism of osteoblastic cells in a model of osteoporosis.