A patogênese e o estadiamento do melanoma são clinicamente bem caracterizados, entretanto, muitas variações são observadas no que diz respeito à progressão e respostas terapêuticas. Mecanismos intrínsecos das células de melanoma contribuem para esse fenômeno, como a plasticidade fenotípica e a expressão de determinadas moléculas, como a galectina-3. Galectina-3 tem papel fundamental para o crescimento do tumor, metástase e montagem de uma rede vascular funcional. Dessa forma, o melanoma possui mecanismos alternativos de vascularização, onde células endoteliais e células tumorais formam a superfície luminal dos vasos e são expostas às forças fluídicas do sangue, o shear stress. Nós hipotetizamos que o shear promove a adaptação celular e células de melanoma se comunicam com (1) outras células tumorais para sustentar a progressão do tumor, ou (2) com células endoteliais para garantir a manutenção vascular. Para isso, comparamos dois modelos de geração do shear denominados pela agitação de placas concêntricas e rotação de um cone sobre placa, que é o modelo padrão dos estudos em células endoteliais. O modelo de placas concêntricas gerou resultados mais robustos, pois garantiu a viabilidade e a adesão celular em até 24h. Também investigamos o potencial angiogênico das células de melanoma após monitorar a ativação de células endoteliais em contato com o meio condicionado (MC) do shear. O shear aumentou significativamente a secreção de Angiopoietina-2, IL-8 e PLGF num contexto independente de galectina-3. Além disso, o shear diminuiu a taxa de crescimento das células que expressam galectina-3, o que não foi observado no tratamento com o MC. Já a capacidade de sobreviver em longo prazo foi maior quando as células tumorais são expostas ao MC derivado do shear, sem influência da expressão de galectina-3. Em relação ao efeito do MC liberado após shear para o remodelamento vascular, nós não observamos alteração na proliferação das células endoteliais. Entretanto, o MC do shear parece acelerar a formação de túbulos e favorecer a desorganização das fibras de actina, além de diminuir a fosforilação de ERK nas células endoteliais. Juntos, esses resultados mostram que o shear induz a secreção de moléculas pró-angiogênicas e pró-inflamatórias que promovem a sobrevivência e proliferação a longo prazo das células tumorais naive. Nas células endoteliais, o MC derivado do shear altera a sinalização de ERK e o rearranjo do citoesqueleto / Melanoma pathogenesis and staging are clinically well characterized, although a large variation is observed in progression and therapeutic responses. Intrinsic mechanisms of melanoma cells contribute to this phenomenon, such as phenotypic plasticity and the expression of certain molecules as galectin-3. Galectin-3 plays a prominent role in tumor growth, metastasis, and to build a functional vascular network. In this way, melanoma has alternative mechanisms of vascularization, in which both endothelial cells and tumor cells form the luminal surface of vessels and are in contact with flowing blood. Here, we postulated that shear stress promotes cell adaptation and melanoma cells communicate with (1) other tumor cells in order to support tumor progression; or (2) with endothelial cells to ensure vascular maintenance. We compared two methods for generating shear stress, namely revolution of concentric plates and stirring of a suspended cone on a plate, which is the gold standard method for studies on endothelial cells. The concentric plates model yielded more reliable results, since it ensured the greater viability and adhesion of cells up to 24h, as compared to the cone and plate system. We then investigated the angiogenic potential of melanoma cells by monitoring the activation of endothelial cells upon exposure to the conditioned medium (CM) from cells exposed to shear stress. Shear stress significantly increased the secretion of Angiopoietin-2, IL-8 and PLGF, in a galectin-3 independent manner. Also, shear stress decreased the growth rate of galectin-3 positive melanoma cells, but not when galectin-3 was down regulated. No such effect was observed with the shear-derived CM. Instead, we did observed long-term melanoma survival under low density upon treatment with the shear-derived CM. In this scenario, the expression of galectin-3 did not change the results. Regarding the effect of shear-derived CM to vascular remodeling, endothelial cells did not show any change in cell proliferation. However, the CM of melanoma cells upon shear accelerated the endothelial cell tube formation and disorganized the actin stress fibers. CM also decreased the phosphorylation of ERK. Taken together, these data indicate that the production of proangiogenic and proinflammatory molecules by melanoma cells under shear enables long-term survival and proliferation of naïve melanoma cells. To endothelial cells, shear-derived CM led to changes in ERK signaling and cytoskeletal rearrangement

O objetivo principal da presente Tese é expor um método de Tomografia de Estado Quântico desenvolvido para ser aplicado em sistemas de núcleos quadrupolares isolados. O espaço de Hilbert de tais sistemas pode ser usado para processar a informação quântica de um sistema equivalente constituído por vários q-bits. O método proposto baseia-se na aplicação de pulsos de radiofrqüência não-seletivos que possuem a propriedade de promover rotações globais do estado quântico do sistema. Utilizando uma descrição analítica dessas rotações foi possível generalizar o método proposto para núcleos quadrupolares com qualquer número quântico de spin. O método também pode ser adaptado para sistemas de núcleos acoplados, embora para esses casos seja necessário utilizar períodos de evolução sob a hamiltoniana de interação livre para determinar alguns dos elementos da correspondente matriz densidade. Como aplicação do método de Tomografia de Estado Quântico, utilizaram-se núcleos de 23Na dissolvidos em um cristal líquido liotrópico para obter os resultados experimentais das implementações do algoritmo de Deutsch e do algoritmo de busca de Grover, além da medida da dinâmica de relaxação de vários estados pseudo-puros. Também foram realizadas simulações do método proposto para o caso de um sistema quadrupolar de spin 7/2 e para três spins 1/2 homonucleares acoplados. / The main purpose of the present thesis is to propose a Quantum State Tomography method developed to be applied in quadrupolar isolated nuclei systems. The Hilbert space of such systems can be used to process the quantum information of an equivalent system formed by many qubits. The proposed method is based on the application of non-selective radiofrequency pulses that produce global rotations of the system quantum state. Using an analytical description of those rotations, it was possible to generalize the proposed method to quadrupolar nuclei with any spin quantum number. The method can also be adapted to coupled nuclear systems, although in such cases it is necessary the use of evolution periods under the free interaction hamiltonian in order to determine some of the density matrix elements. As an application of the method, 23Na nuclei dissolved in a lyotropic liquid crystal were used to obtain the experimental results of the Deutsch and Grover algorithms, together with the measurement of the relaxation dynamics of some pseudo-pure states. Simulations of the proposed method applied to the quadrupolar spin 7/2 nucleus and to three homonuclear coupled spin 1/2 were also obtained.

O objetivo principal da presente Tese é expor um método de Tomografia de Estado Quântico desenvolvido para ser aplicado em sistemas de núcleos quadrupolares isolados. O espaço de Hilbert de tais sistemas pode ser usado para processar a informação quântica de um sistema equivalente constituído por vários q-bits. O método proposto baseia-se na aplicação de pulsos de radiofrqüência não-seletivos que possuem a propriedade de promover rotações globais do estado quântico do sistema. Utilizando uma descrição analítica dessas rotações foi possível generalizar o método proposto para núcleos quadrupolares com qualquer número quântico de spin. O método também pode ser adaptado para sistemas de núcleos acoplados, embora para esses casos seja necessário utilizar períodos de evolução sob a hamiltoniana de interação livre para determinar alguns dos elementos da correspondente matriz densidade. Como aplicação do método de Tomografia de Estado Quântico, utilizaram-se núcleos de 23Na dissolvidos em um cristal líquido liotrópico para obter os resultados experimentais das implementações do algoritmo de Deutsch e do algoritmo de busca de Grover, além da medida da dinâmica de relaxação de vários estados pseudo-puros. Também foram realizadas simulações do método proposto para o caso de um sistema quadrupolar de spin 7/2 e para três spins 1/2 homonucleares acoplados. / The main purpose of the present thesis is to propose a Quantum State Tomography method developed to be applied in quadrupolar isolated nuclei systems. The Hilbert space of such systems can be used to process the quantum information of an equivalent system formed by many qubits. The proposed method is based on the application of non-selective radiofrequency pulses that produce global rotations of the system quantum state. Using an analytical description of those rotations, it was possible to generalize the proposed method to quadrupolar nuclei with any spin quantum number. The method can also be adapted to coupled nuclear systems, although in such cases it is necessary the use of evolution periods under the free interaction hamiltonian in order to determine some of the density matrix elements. As an application of the method, 23Na nuclei dissolved in a lyotropic liquid crystal were used to obtain the experimental results of the Deutsch and Grover algorithms, together with the measurement of the relaxation dynamics of some pseudo-pure states. Simulations of the proposed method applied to the quadrupolar spin 7/2 nucleus and to three homonuclear coupled spin 1/2 were also obtained.

Nossa proposta de investigar o discurso sobre o livro didático de inglês toca inevitavelmente na questão do discurso de verdade do livro didático. Direcionada pela hipótese de que o poder de discurso de verdade do livro didático de inglês (LDI) é construído pelos dizeres acerca do LDI, nossa pesquisa dedica-se a estudar o funcionamento desse que chamamos de discurso sobre o LDI e os efeitos de sentido que ele produz. Mais especificamente, estudamos os dizeres sobre o LDI adotado na escola pública brasileira a partir do Programa Nacional do Livro Didático, PNLD 2011. Apesar de o LDI já ter sido adotado localmente na rede pública por algumas escolas ou alguns professores, é através do PNLD 2011 que o governo federal, pela primeira vez, propõe títulos a serem adotados em âmbito nacional, patrocinando o custo desses materiais consumíveis, para o ensino de língua estrangeira (espanhol e inglês). Para esta investigação, apoiamos nossas observações nos conceitos pertinentes à linha pecheutiana de Análise do Discurso. Esses conceitos fundamentam a nossa análise dos dizeres do corpus de pesquisa, formado por três segmentos. O segmento oficial compreende os principais documentos referentes ao PNDL 2011. O segmento midiático foi composto por textos disponibilizados na internet, publicados por agências de notícias governamentais e não governamentais e por sites de escolas e associações. O segmento profissional contou com as respostas de professores de inglês do 6º ao 9º ano da rede pública de ensino. Essas respostas foram coletadas em dois momentos através de questionários de pesquisa. No primeiro momento, as coleções que compuseram o PNLD 2011 ainda não eram conhecidas pelos professores respondentes. No segundo momento, os professores que responderam ao segundo questionário já utilizavam as coleções de inglês do PNLD 2011. A análise do discurso sobre o livro didático de inglês nos possibilitou a percepção de efeitos que apontam ao funcionamento do LDI como verdade por ser representado como tal pelo discurso que fala sobre ele. Através da análise, observamos também efeitos característicos do funcionamento das sociedades de controle, como prevista por Michel Foucault ([1979] 2004) e discutida por Gilles Deleuze ([1990] 1992). No eixo do interdiscurso, percebemos nos enunciados os princípios moduladores, garantindo o adiamento do fim, demandando atualizações contínuas, motivando o indivíduo e assegurando o controle. No eixo do intradiscurso, a materialidade linguística mostrou-se frequentemente marcada pela modalização dos dizeres, suavizando-os e suscitando múltiplas possibilidades. Os efeitos percebidos parecem funcionar como os princípios moduladores, formulados por Deleuze ([1990] 1992), que marcam a relação saber-poder funcionando já nos moldes da sociedade de controle / Our aim to investigate the discourse about the English textbook inevitably touches on the issue of the textbook as a discourse of truth. Driven by the hypothesis that the power of discourse of truth, often attributed to the textbook, is built by the discourse which speaks about the textbook, our research aims to study the functioning mode and the effects of that discourse which we name discourse about the English textbook. Although English textbooks have been adopted in some courses by some public school teachers here and there, this is the first time that the Brazilian federal government has proposed the nation-wide adoption of foreign language textbooks (English and/or Spanish) through the National Textbook Program for the year of 2011 (PNLD 2011) by selecting titles for teachers to choose from and by bearing all the expenses involved in the process. Our research follows the conceptualization of Discourse Analysis as established by Michel Pêcheux. Those concepts guide the analysis of the excerpts in our research corpus, which is composed of three segments. The official discourse segment contains excerpts from the most relevant documents issued by the government on the PNLD 2011. The media discourse segment comprises online news and articles issued by governmental as well as non-governmental news agencies and by schools and associations sites. The professional discourse segment holds the answers provided by public school teachers of English (6th to 9th grades) to two questionnaires on the adoption of the English textbook. Their answers were collected in two distinct moments. The first questionnaire was answered by the teachers before textbook collections were pre-selected to integrate the PNLD 2011. The second questionnaire was answered by teachers who already used the PNLD 2011 textbooks. The analysis of the discourse about the English textbook led us to the perception of effects which confirm the functioning of the English textbook as a discourse of truth since it is represented as such by the discourse about it. Through discourse analysis, we have also observed effects which are typical of the functioning mode of societies of control, as briefly defined by Michel Foucault ([1979] 2004) and further contended by Gilles Deleuze ([1990] 1992). In the interdiscourse axis, modulating principles were observed in enouncements, as they ensure limitless postponements, demand continuous updating, motivate the individual and ensure control. In the intradiscourse axis, the linguistic materiality often presented excerpts marked by modalization, which softens the remarks and gives rise to multiple possibilities. The effects observed seem to work as modulating principles, as formulated by Deleuze ([1990] 1992), which confirms that the relationship between power and knowledge is already functioning in the modes of societies of control

INTRODUÇÃO: O crescimento e a progressão de diversos tipos de tumores dependem da angiogênese, isto é, a formação de novos vasos sanguíneos. Fatores da família do VEGF (VEGF-A, VEGF-C, Flk-1/KDR) são potentes mediadores da angiogênese e níveis elevados destes fatores podem ser detectados em vários tumores humanos. Outras proteínas como NDRG1, osteonectina, HIF-1 e galectina-3 encontram-se associadas, de modo indireto, à angiogênese. MÉTODOS: O objetivo deste estudo foi analisar a expressão imunoistoquímica de NDRG1, osteonectina, VEGF-A, VEGF-C, Flk-1/KDR, HIF-1 e galectina-3 em 265 lesões benignas e malignas da tireóide por tissue microarray (TMA), incluindo tireóide normal, bócio, adenoma folicular, carcinoma papilífero, carcinoma folicular e metástases de carcinoma papilífero e folicular da tireóide. Lâminas em diferentes níveis do bloco de TMA foram submetidas à reação imunoistoquímica utilizando-se anticorpos específicos contra NDRG1, osteonectina, VEGF-A, VEGF-C, Flk-1/KDR, HIF-1, galectina-3. Foi realizada uma avaliação semiquantitativa da expressão destas proteínas, analisando-se a intensidade de coloração e a porcentagem de células positivas para cada marcador. RESULTADOS: De um modo geral, a expressão de NDRG1, osteonectina, VEGF-A, VEGF-C, Flk-1/KDR, HIF-1 e galectina-3 foi maior nos carcinomas de tireóide em relação ao bócio e adenoma folicular. Dentre as proteínas estudadas, a galectina-3 foi aquela que apresentou melhor acurácia diagnóstica na distinção entre lesões benignas e malignas da tireóide, com uma sensibilidade 64,1% e especificidade de 94,4%. Nos carcinomas de tireóide, a expressão de NDRG1, VEGF-A, VEGF-C e HIF-1 apresentou correlação estatística significativa com estádio TNM mais avançado e alto risco pelo método AMES. A análise multivariada revelou que a idade 45 anos, o tamanho tumoral maior do que 4,0 cm, o estádio IV do TNM e a perda de expressão de galectina-3 foram fatores que contribuíram para a recorrência ou óbito em pacientes com carcinoma de tireóide. CONCLUSÕES: Nos carcinomas de tireóide, a expressão de NDRG1, VEGF-A, VEGF-C, HIF-1 se correlacionou com tumores mais avançados. Com exceção da galectina-3, nenhuma das proteínas estudadas apresentou impacto na sobrevida dos pacientes com carcinoma de tireóide. Nestes pacientes, a perda de expressão de galectina-3 foi um fator de risco para recorrência ou óbito. / INTRODUCTION: The growth and progression of different types of tumors depend on angiogenesis, i.e., the formation of new blood vessels. Members of the VEGF family (VEGF-A, VEGF-C, Flk-1/KDR) are potent mediators of angiogenesis and elevated levels of these factors can be detected in various human tumors. Other proteins such as NDRG1, osteonectin, HIF-1 and galectin-3 are indirectly associated with angiogenesis. METHODS: The objective of this study was to investigate the immunohistochemical expression of NDRG1, osteonectin, VEGF-A, VEGF-C, Flk-1/KDR, HIF-1 and galectin-3 in 265 benign and malignant thyroid lesions by tissue microarray (TMA), including normal thyroid, goiter, follicular adenoma, papillary carcinoma, follicular carcinoma, and papillary and follicular thyroid carcinoma metastases. Slides obtained from different levels of the TMA block were submitted to immunohistochemistry using specific antibodies against NDRG1, osteonectin, VEGF-A, VEGF-C, Flk-1/KDR, HIF-1, and galectin-3. The expression of these proteins was analyzed semiquantitatively, evaluating the intensity of staining and the percentage of positive cells for each marker. RESULTS: In general, the expression of NDRG1, osteonectin, VEGF-A, VEGF-C, Flk-1/KDR, HIF-1 and galectin-3 was higher in thyroid carcinomas when compared to goiter and follicular adenoma. Among the proteins studied, galectin-3 presented the best diagnostic accuracy in the distinction between benign and malignant thyroid lesions, with a sensitivity of 64.1% and specificity of 94.4%. In thyroid carcinomas, the expression of NDRG1, VEGF-A, VEGF-C and HIF-1 was significantly correlated with a more advanced TNM stage and AMES high risk. Multivariate analysis revealed that age 45 years, tumor size > 4.0 cm, TNM stage IV and loss of expression of galectin-3 were associated with recurrence or death in patients with thyroid carcinoma. CONCLUSIONS: In thyroid carcinomas, the expression of NDRG1, VEGF-A, VEGF-C and HIF-1 is correlated with more advanced tumors. Except for galectin-3, none of the proteins studied had an impact on the survival of patients with thyroid carcinoma. In these patients, loss of expression of galectin-3 was a risk factor for recurrence or death.

Abstract Primary intracerebral hemorrhage (pICH) is a severe, suddenly occurring disease involving high mortality and poor functional outcome. In the absence of curative treatment patient management is mainly supportive with the emphasis on preventing hematoma enlargement and complications. Better understanding of the factors predicting outcome are needed to define effective treatments. An unselected population-based registry study of 982 pICH patients admitted to Oulu University Hospital during the years 1993 to 2008 was conducted The study revealed that concomitant use of warfarin and serotonin-modulating antidepressants at the time of pICH increases the case fatality rate compared to patients with warfarin alone. An elevated C-reactive protein value on admission was an independent predictor of unfavorable outcome after pICH. This association was not explained by pre-existing heart disease, diabetes, severity of the bleeding, or infections. Patients undergoing surgical hematoma evacuation were observed to have improved 3-month survival compared to conservatively treated patients. Improved survival was noticed especially in patients with ≤70 years of age with ≥30ml supratentorial ICHs. Hematoma evacuation did not improve functional outcome. Earlier ischemic stroke was found to be an independent predictor of recurrent pICH. Diabetes seemed to increase and treated hypertension decrease the risk for fatal recurrence. Aspirin or serotonin-modulating antidepressants did not seem to increase the risk of recurrence. / Tiivistelmä Primääri aivoverenvuoto (pICH) on vakava, yhtäkkisesti alkava sairaus, johon liittyy korkea kuolleisuus ja vaikea vammautuminen. Parantavan hoidon puuttuessa on hoito lähinnä elintoimintoja tukevaa vuodon laajenemisen ja komplikaatioiden estämistä. Ennusteeseen vaikuttavien tekijöiden parempi tunteminen on ehto tehokkaiden hoitojen löytämiseksi. Väitöskirjatutkimustani varten kerättiin Oulun yliopistollisen sairaalan alueelta vuosien 1993-2008 aikana 982 aivoverenvuotoon sairastuneen potilaan väestöpohjainen aineisto. Tutkimus osoitti, että varfariinin ja selektiivisen serotoniinin takaisinoton estäjän (SSRI) yhteiskäyttö aivoverenvuodon aikana lisäsi kuolevuutta pelkkään varfariiniin nähden. Alkuvaiheen koholla oleva C-reaktiivinen proteiini oli itsenäinen aivoverenvuodon jälkeistä vammautuneisuutta ennustava tekijä. Yhteys ei selittynyt olemassa olevalla sydänsairaudella, diabeteksella, aivoverenvuodon vaikeudella tai infektioilla. Kirurginen aivoverenvuodon poistoleikkaus paransi kolmen kuukauden ennustetta verrattuna potilaisiin ilman leikkausta. Erityisesti leikkaus auttoi alle 70-vuotiaita potilaita, joilla oli yli 30 millilitran kokoinen pinnallisempi vuoto. Leikkaus ei parantanut fyysistä kuntoutumista. Aiempi sairastettu aivoinfarkti oli itsenäinen aivoverenvuodon uusiutumista ennustava tekijä. Diabetes saattaa lisätä ja hoidossa oleva verenpainetauti laskea riskiä tappavaan uusintavuotoon. Aspiriinin tai SSRI:n käyttö eivät lisänneet uusintavuodon riskiä.

Since ancient times, indigenous plants have been used by traditional healers for treating various ailments. Sutherlandia frutescens is one of the most commonly used medicinal plants of southern Africa. This widely distributed plant has been traditionally used to treat cancer and HIV patients; however scientific validation is still in high demand. This research aimed to phytochemically characterise the various extracts prepared and to determine if any chemotypes were present. Subsequent biological characterisation was carried out to preliminary ascertain whether this medicinal plant could have anti-cancer and/or immunemodulating properties and which compounds might be responsible for these actions. Various traditional and organic extracts were prepared. Extracts, fractions and compounds generated were analysed and chemical profiles obtained. Column chromatographic techniques were used to isolate and purify compounds and structure elucidation was carried out using various analytical techniques. Sulforhodamine B and cytometric bead array assays were performed to determine the biological activities of samples generated. / Life and Consumer Sciences / (M. Sc. (Life Sciences)

INTRODUÇÃO: O pênfigo foliáceo (PF) caracteriza-se pela síntese de autoanticorpos contra a desmogleína 1 (Dsg1) com acantólise na epiderme superior. As lesões na face e tronco podem evoluir para eritrodermia (PFE), cuja patogênese é pouco conhecida. Estudos prévios sugerem a participação do fator de crescimento endotelial vascular (VEGF) e da endoglina (Eng). OBJETIVOS: Avaliar dados demográficos, expressão tecidual e níveis séricos do VEGF e Eng, bem como o perfil dos imunocomplexos no PFE. PACIENTES, MATERIAIS E MÉTODOS: Foram selecionados pacientes de PFE (n=31) e indivíduos controles com pênfigo vulgar (PV; n=10), psoríase (PSO; n=10) e saudáveis (CS; n=14) com amostras de soro e pele armazenadas. Os níveis séricos do VEGF, receptor solúvel do VEGF (sVEGFR-1), anti-Dsg1 e -Dsg3 foram avaliados por meio da técnica de ELISA, e a presença do VEGF, Eng, e imunocomplexos in situ foi determinada por imuno-histoquímica (IH) em plataforma automatizada (n=19). Após digitalização das lâminas, a reatividade aos anticorpos supracitados foi classificada manualmente em: 0 (ausente), 1 (discreta-moderada) e 2 (intensa), e analisada por software. RESULTADOS: PFE ocorreu no início da doença em 25/31 pacientes (80,6%), com média de idade de 42,7 anos e predomínio feminino (23/31; 74,2%). Os pacientes foram hospitalizados em média por 41,2 dias; infecção bacteriana foi a principal complicação (30/31; 96,8%), com bacteremia em 10/31 (32,3%) causada por Staphylococcus aureus em 7/10 (70%) dos pacientes. Infecção pelo vírus do herpes simples (HSV) em 11/31 (35,5%) doentes determinou internação prolongada. Valores de imunofluorescência indireta, e anti-Dsg1 e VEGF séricos (ELISA) foram superiores no PFE vs. PF não-eritrodérmico (PFNE) (p < 0,05). Níveis do sVEGFR-1 foram semelhantes no PFE e PFNE, e correlacionaram-se fracamente com a anti-Dsg1 no PFNE (p=0,034). A avaliação manual da IH com anti-VEGF no PFE foi estatisticamente diferente do PFNE (p=0,042) e CS (p=0,004), e similar ao PV (p=0,667) e PSO (p=0,667). Já no PFNE, a expressão do VEGF foi estatisticamente diferente do PV (p=0,049) e PSO (p=0,049) e semelhante ao CS (p=0,247). A contagem automatizada dos vasos marcados com anti-Eng no PFE foi similar ao PFNE (p=0,700) e PSO (p=0,133), e diferente do PV (p=0,0009) e CS (p=0,0009). A avaliação de 6 espécimes de PFE mostrou depósitos de imunocomplexos: intercelulares intraepidérmicos com IgG e C3 (n=6), IgA (n=5) e IgM (n=1); nas células inflamatórias com IgG e C3 (n=6), IgM e IgA (n=1); nos vasos com IgG, C3 e IgA (n=6), e IgM (n=5); e nos anexos com IgG e C3 (n=6), IgA (n=3) e IgM (n=1). CONCLUSÕES: O PFE predomina no início da doença e em mulheres, com maior risco de infecção. O aumento do VEGF sérico e tecidual sugere uma resposta reparadora ao dano tecidual causado pelos níveis elevados de autoanticorpos no PFE. A menor expressão de Eng no PFE indica uma desregulação da angiogênese na eritrodermia. De forma pioneira, a IH automatizada demonstrou a presença de imunocomplexos intraepidérmicos e nas estruturas dérmicas / BACKGROUND: Pemphigus foliaceus (PF) is characterized by the production of autoantibodies against desmoglein 1 (Dsg1), triggering superficial acantholysis. Lesions on the face and trunk may evolve to erythroderma (PFE). The pathogenesis of PFE is not fully understood. Previous studies suggest the role of vascular endothelial growth factor (VEGF) and endoglin (Eng). OBJECTIVES: To evaluate demographic data, VEGF and Eng expression, and immune complexes deposition in patients with PFE. METHODS: This study included patients with PFE (n=31) and controls with pemphigus vulgaris (PV; n=10), psoriasis (PSO; n=10), and health individuals (CS; n=14) that had serum and skin samples stored. Serum levels of VEGF, soluble VEGF receptor (sVEGFR-1), anti-Dsg1 and Dsg3 were measured by ELISA, and the in situ expression of VEGF, Eng, and immune complexes was evaluated utilizing an automated immunohistochemistry (IH) platform (n=19). After digitalizing the slides, the reactivity was manually classified as 0 (negative), 1 (mild-to-moderate) and 2 (intense), and also analyzed by software. RESULTS: PFE occurred at the onset of the disease in 25/31 (80.6%) patients, with a mean age of 42.7 years and a female predominance (23/31; 74.2%). Patients were hospitalized with an average length of stay of 41.2 days. Bacterial infection was the main complication (30/31; 96.8%), with bacteremia in 10/31 (32.3%) due to Staphylococcus aureus in 7/10 (70%) patients. Herpes simplex virus infection in 11/31 (35.5%) PFE patients caused prolonged hospitalization. Indirect immunofluorescence titers and serum anti-Dsg1 and VEGF (ELISA) were increased in PFE vs. non-erythrodermic PF (PFNE) (p < 0.05). Serum levels of sVEGFR-1 were similar in PFE and PFNE, and weakly correlated with anti-Dsg1 in PFNE (p=0.0342). Manual analysis of anti-VEGF positivity in PFE was statistically different from PFNE (p=0.042) and CS (p=0.004), and similar to PSO (p=0.667) and PV (p=0.667). VEGF expression in PFNE was statistically different from PSO (p=0.049) and PV (p=0.049) and similar to CS (p=0.247). The automated positive vessel count with anti-Eng was similar between PFE and PFNE (p=0.700) and PSO (p=0.133), but different from PV (p=0.0009). Immune complex deposits were evaluated in 6 specimens obtained during PFE and exhibited: intraepidermal intercellular deposits with IgG and C3 (n=6), IgA (n=5) and IgM (n=1); reactivity to inflammatory cells with IgG e C3 (n=6), IgM and IgA (n=1); vascular deposits with IgG, C3 and IgA (n=6), and IgM (n=5); and adnexal positivity with IgG and C3 (n=6), IgA (n=3) and IgM (n=1). CONCLUSIONS: Erythroderma predominates at the onset of PF, especially in women, with higher infectious risk. Increased expression of serum and in situ VEGF suggests that healing processes are triggered in response to the tissue damage caused by high levels of circulating autoantibodies in PFE. The reduced expression of Eng in PFE demonstrates a dysregulated angiogenesis during erythroderma. To the best of our knowledge, this is the first study that showed intraepidermal and dermal deposits of multiple immune complexes utilizing automated IH analysis

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliais / The vasculature plays a central role in tumor progression and represents a therapeutic target of great interest. Inhibition of angiogenesis has the potential to slow down tumor progression and inhibit metastasis. As a result, anti-angiogenic therapies have been shown to be promising for the control of tumor growth. According to the literature, interferon ? (IFN?, activator of the innate and adaptive immune systems) and p19Arf (tumor suppressor and functional partner of p53) when studied individually alter tumor vasculature. Our group has constructed and used recombinant adenovirus vectors carrying the cDNAs of INFbeta and p19Arf and noted that the transfer of this combination of genes induced cell death and decreased tumor progression, as observed in the B16 murine model of in situ melanoma gene therapy as well as prophylactic and therapeutic vaccine approaches. In this study, we explore the idea that the combination of adenoviral vectors bearing INFbeta and p19Arf produce anti-angiogenic effects due to their impact on endothelial cells. To test this hypothesis, murine endothelial cells (tEnd) were transduced with adenoviral vectors, revealing that Ad-p19 vector confers inhibition of proliferation, tube formation, migration and induces increased expression of genes related to the p53 cell death pathway. The Ad-IFNbeta vector alone had no significant impact on these tests. Alternatively, influences on paracrine effects are evaluated on endothelial cells co-cultured with B16 cells that were previously transduced with adenoviral vectors. When the B16 cells were transduced with Ad-IFNbeta or co-transduced with Ad-IFNbeta + Ad-p19, co-culture resulted in the inhibition of proliferation of the endothelial cells. When B16 cells were transduced with Ad-p19 only, co-culture did alter endothelial cell behavior. Following the co-culture assay, we produce conditioned medium from B16 cells that were transduced with the vectors and applied the media on tEnd cells. We noted that conditioned medium derived from B16 transduced with Ad-IFN alone or in combination with Ad-19 decreased the viability and proliferation and induced cell death of tEnd. In this work, we show that inhibition of endothelial cells can be performed directly by transduction with Ad-19 or when such cells are exposed to the environment modulated by tumor cells transduced with Ad-IFNbeta. Even though the gene transfer of both IFNbeta and p19 was not found to be superior to the application of single genes, we observed that our approach may have an important impact on the inhibition of angiogenesis through endothelial cells

Droplet evaporation is ubiquitous to multitude of applications such as microfluidics, surface patterning and ink-jet printing. In many of the process like food processing tiny concentrations of suspended particles may alter the behavior of an evaporating droplet remarkably, leading to partially viscous and partially elastic dynamical characteristics. This, in turn, may lead to some striking mechanical instabilities, such as buckling and rupture. In this thesis, we provide a comprehensive physical description of the vaporization, self-assembly, agglomeration and buckling kinetics of sessile nanofluid droplet pinned on a hydrophobic substrate in various configurations. We have deciphered five distinct regimes of droplet lifecycle. Regime I-III consists of evaporation induced preferential agglomeration that leads to the formation of unique dome shaped inhomogeneous shell with stratified varying density liquid core. Regime IV involves capillary pressure initiated shell buckling and stress induced shell rupture. Regime V marks rupture induced cavity inception and growth. We provide a regime map explaining the droplet morphology and buckling characteristics for droplets evaporating on various substrates. Specifically, we find that final droplet volume and radius of curvature at buckling onset are universal functions of particle concentration. Furthermore, flow characteristics inside the heated and unheated droplets are investigated and found to be driven by the buoyancy effects. Velocity magnitudes are observed to increase by an order at higher temperatures with self-similar flow profiles. With an increase in the surface temperature, droplets exhibit buckling from multiple sites over a larger sector in the top half of the droplet. In addition, irrespective of the initial nanoparticle concentration and substrate temperature, hydrophobicity and roughness, growth of daughter cavity (subsequent to buckling) inside the droplet is found to be controlled by the solvent evaporation rate from the droplet periphery. The results are of great significance to a plethora of applications like DNA deposition and nanofabrication. In the next part of the thesis, we deploy the droplet in a rectangular channel. The rich physics governing the universality in the underlying dynamics remains grossly elusive. Here, we bring out hitherto unexplored universal features of the evaporation dynamics of a sessile droplet entrapped in a 3D confined fluidic environment. Increment in channel length delays the completion of the evaporation process and leads to unique spatio-temporal evaporation flux and internal flow. We show, through extensive set of experiments and theoretical formulations, that the evaporationtimescale for such a droplet can be represented by a unique function of the initial conditions. Moreover, using same theoretical considerations, we are able to trace and universally merge the volume evolution history of the droplets along with evaporation lifetimes, irrespective of the extent of confinement. These results are explained in the light of increase in vapor concentration inside the channel due to greater accumulation of water vapor on account of increased channel length. We have formulated a theoretical framework which introduces two key parameters namely an enhanced concentration of the vapor field in the vicinity of the confined droplet and a corresponding accumulation lengthscale over which the accumulated vapor relaxes to the ambient concentration. Lastly, we report the effect of confinement on particle agglomeration and buckling dynamics. Compared to unconfined scenario, we report non-intuitive suppression of rupturing beyond a critical confinement. We attribute this to confinement-induced dramatic alteration in the evaporating flux, leading to distinctive spatio-temporal characteristics of the internal flow leading to preferential particle transport and subsequent morphological transitions. We present a regime map quantifying buckling & non-buckling pathways. These results may turn out to be of profound importance towards achieving desired morphological features of a colloidal droplet, by aptly tuning the confinement space, initial particle concentration, as well as the initial droplet volume. These findings may have implications in designing functionalized droplet evaporation devices for emerging engineering and biomedical applications.