Microcistinas (MCs) são heptapeptídeos cíclicos produzidos por cianobactérias e possuem potente hepatotoxicidade e atividade promotora de tumor. Em intoxicações agudas induzidas por MCs ocorre infiltração leucocitária no foco inflamatório. Embora os mecanismos de hepatotoxicidade não são claros, o recrutamento de neutrófilos no fígado pode contribuir ao dano tecidual e desenvolvimento tumoral causados por xenobióticos. O objetivo dessa tese foi investigar os efeitos de três estruturalmente distintas MCs (MC-LA, MC-YR e MC-LR) nas seguintes funções de neutrófilos: síntese e expressão de moléculas de adesão, rolamento, adesão, migração e liberação de citocinas e de ROS. Nos ensaios de migração em bolsa de ar, as três MCs induziram similarmente a migração de leucócitos in vivo em tecido subcutâneo de ratos e diferencialmente a secreção de citocinas pró-inflamatórias (CINC, IL-1β, TNF-α, VEGF-α e MIP) no exsudato. Concentrações elevadas de CINC-2αβ foram encontradas nos exsudatos inflamatórios de animais após injeção de MC-LA, MC-LR ou MC-YR. MIP-2 elevou-se apenas em exsudatos de animais expostos a MC-LR. Não foram observadas alterações em secreção de IL-1β, TNF-α e VEGF-α. Estudos de microscopia intravital mostraram que apenas a aplicação tópica de MC-LR reforçou o rolamento e a adesão de leucócitos no endotélio de vênulas mesentéricas pós-capilares. Esses últimos resultados podem ser dependentes de aumento e expressão da síntese da L-selectina e β2-integrina nos neutrófilos, tal como avaliado por FACS e PCR-RT, respectivamente. Adicionalmente, em ensaios em câmara de Boyden in vitro, as três MCs promoveram locomoção direta de neutrófilos e aumentaram a migração dessas células em resposta ao fMLP, além do aumento de cálcio intracelular observado por microscopia confocal. Os efeitos das MC-LA, MC-YR e MC-LR em neutrófilos humanos e de ratos tiveram o mesmo padrão de resposta. A análise de viabilidade celular, fragmentação de DNA, despolarização de membrana mitocondrial e liberação de ROS intracelulares foram avaliadas pela técnica de FACS. A concentração de ROS extracelular foi medida por quimiluminescência amplificada por lucigenina. A produção de citocinas em células tratadas foram determinadas por ELISA. Observamos aumento da liberação de IL-8, CINC-2α β e da concentração de ROS extracelular por neutrófilos humanos e de ratos pela exposição dessas células com as MCs. Os resultados aqui obtidos mostram o efeito pró-inflamatório direto das MC-LA, MC-YR e MC-LR. A possibilidade de os neutrófilos migrarem e responderem localmente a MCs contribui para a toxicidade destas toxinas. / Microcystins (MCs) are a family of heptapeptide toxins produced by some genera of Cyanobacteria. MCs have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity induced by MCs are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The purpose of this thesis was to investigate the effects of three structurally distinct MCs (MC-LA, MC-YR and MC-LR) in the neutrophil functions: synthesis and expression of adhesion molecules, rolling, adhesion, migration and release of cytokines and ROS. In migration assays of the air pouch, the three MCs similarly induced the migration of leukocytes in vivo in subcutaneous tissue of rats and differentially the secretion of pro-inflammatory cytokines (CINC-2αβ, IL-1-β, TNF--α, VEGF- α and MIP-2) in exudates. Elevated concentrations of CINC-2αβ were found in the inflamed exudates from animals injected with MC-LA, MC-LR or MC-YR, although MIP-2 was only detected in the exudates from animals injected with MC-LR. There were no changes in the secretion of IL-1-β, TNF-α and VEGF--α. Intravital microscopic studies showed that topical application of MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression of L-selectin and -α2-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to fMLP, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. The effects of MC-LA, MC-YR and MC-LR in human neutrophils and mice had the same pattern of response. The analyses of cell viability, DNA fragmentation, mitochondrial membrane depolarization of and release of intracellular ROS were evaluated by the technique of FACS. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MCs increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2αβ (CINC-2αβ) and extracellular ROS levels in human and rat neutrophils. In conclusion, our results showed that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. This process can significantly contribute to the pathogenesis of hepatic damage due to generation of ROS by neutrophils as well as act on hepatocytes under such conditions and potentially increase injury processes induced by MCs.

Human embryonic stem cells are pluripotent cells that have indefinite replicative potential and ability to differentiate into derivatives of three germ layers. HESCs are conventionally derived and grown on mitotically inactivated mouse embryonic fibroblasts and there are some alternative feeder types of human origin that have been used to replenish hESCs while trying to prevent cross-species contamination. The trophic factors that are secreted by the feeders are found to be important for long-term pluripotency but there are also supportive culture systems for hESCs lacking feeder cells which might suggest that not only the interactions with the feeders affect the behaviour of hESCs but also the components of the niche may take part in the decision of self-renewal or differentiation. Extracellular matrix components are known to exert their stimulatory or inhibitory effects by localising cells into a specific microenvironment in natural niches but have been relatively little investigated for hESCs. The aim of this study was to investigate ECM components which might have a role in the maintenance of hESCs. I have first investigated human placental stromal fibroblasts and immortalised human placental stromal fibroblasts for the support hESC pluripotency as an anlternative feeder type to conventional mouse embryonic fibroblasts. Secondly, the matrices derived from hPSFs and ihPSFs were assessed for their ability to support hESC pluripotency. Tandem mass spectrometry was used to identify ECM components released by human feeders in order to characterise the range of extracellular matrix proteins that support the growth of self-renewing hESCs. The majority of the molecules was shared between the cell types irrespective of hPSF cell derived matrix was not being supportive for hESC pluripotency, with some ECM components being unique ihPSFs. Collagen VI, tenascin C and versican were tested for hESC attachment and as substrates for feeder-free culture system in order to develop an optimised feeder-free system. Furthermore, integrin receptor profile of different hESC lines was also determined in order to identify the mechanisms of substrate attachment. Integrin attachment was shown to be vital for hESC engagement to fibronectin and vitronectin in feeder-free systems. The components of the integrin signalling machinery were identified in hESCs and the significance of integrin-mediated signalling in hESC self-renewal was demonstrated by blocking integrin β1 on fibronectin and integrin aVβ5 on vitronectin. Moreover, intracellular signalling mediator c-Src was shown to involve in ECMregulated signalling by affecting the phosphorylation of Focal Adhesion Kinase. Inhibition of Src led to a decrease in the expression of pluripotency-associated markers. Finally, the effects of growth factor supplementation on the maintenance of pluripotency in defined feeder-free conditions were studied by withdrawal of growth factors and blocking FGF Receptors. FGF-2 was shown to be essential for long-term self-renewal while the effects on pluripotency deteriorated in the absence of both FGF-2 and Activin A. Taken together this project highlighted the importance of substrate attachment and growth factors on the regulation of hESC self-renewal.

Les Leucémies Aigues Myéloïdes (LAM) sont des hémopathies au pronostic sombre. A l’exception des Leucémies Aigues Promyélocytaires (LAP), un sous type minoritaire de LAM, la plupart des patients sont traités par une chimiothérapie d’induction composée de la cytarabine et d’une anthracycline. Cependant, une fraction importante (20-30%) des patients ne répond pas à la chimiothérapie d’induction et les taux de rechutes sont très élevés.Il n’existe actuellement pas d’outils disponibles au diagnostic permettant de prédire la réponse des patients aux chimiothérapies. De tels tests permettraient d’adapter les doses utilisées pour augmenter le taux de réponse et limiter la toxicité des chimiothérapies, très importante chez les personnes âgées.Les protéines de la famille de l’Ubiquitine (UbL), dont l’ubiquitine, SUMO (-1,-2,-3) et Nedd8 sont les membres les plus étudiés, sont des modificateurs post-traductionnels peptidiques conjugués de façon covalente sur des milliers de protéines. Elles sont impliquées dans la plupart des fonctions cellulaires et dérégulées dans de nombreuses pathologies. De nombreux travaux suggèrent que leur dérégulation est associée à la chimiorésistance des LAM.La première partie de nos travaux a consisté à identifier un ensemble de protéines dont le niveau de modification par l’ubiquitine, SUMO et NEDD8 pourrait constituer des biomarqueurs de la réponse des LAM aux drogues chimiothérapeutiques. Nous avons de plus développé un test permettant d’évaluer leur niveau de modification par des extraits de cellules de patients de manière simple et rapide, utilisable en clinique au diagnostic.Le ciblage des voies Ubiquitine, SUMO et Nedd8 est en train d’apparaître comme une stratégie thérapeutique prometteuse dans les cancers et plusieurs molécules ciblant ces voies ont été récemment décrites. Le développement clinique de ces molécules nécessite la mise au point de tests compagnons pour suivre leur efficacité sur leurs cibles moléculaires. Nous avons ainsi développé un test d’activité permettant de mesurer de façon simple et quantitative l’efficacité des molécules ciblant les modificateurs de la famille de l’ubiquitine,Enfin, compte tenu du potentiel thérapeutique du ciblage de la SUMOylation, nous avons débuté le développement d’un inhibiteur de la SUMOylation basé sur l’inhibition de l’interaction entre les enzymes E1 et E2 par des peptides agrafés. / Acute Myeloid Leukemias (AML) are severe haematological malignancies with a poor prognosis. Except Acute Promyelocytic Leukemia (APL), a minor subtype of AML, most patients receive an induction chemotherapy consisting of cytarabine and an anthracycline. However, a large number (20-30%) of patients do not respond to this induction chemotherapy, and relapses are frequent.There are currently no tools available at diagnosis to predict patient response to chemotherapy. Such tests would make it possible to adapt the doses used to increase the response rate and limit the toxicity of chemotherapies, which is significant in older patients.Ubiquitin-like modifiers (UbL), among which ubiquitin, SUMO (-1, -2, -3) and Nedd8 are the most studied members, are post-translational modifiers covalently conjugated to thousands of proteins. They are involved in most cellular pathways, and deregulated in several pathologies. Many studies suggest that their deregulation is associated with LAM chemoresistance.The first part of our work consisted in identifying a set of proteins whose level of modification by ubiquitin, SUMO and NEDD8 could constitute biomarkers of AMLs response to chemotherapeutic drugs. We have also developed a test to evaluate their level of modification by extracts of patient cells in a simple and rapid manner, usable in clinical diagnosis.Targeting of the Ubiquitin, SUMO and Nedd8 pathways is emerging as a promising therapeutic strategy in cancers and several molecules targeting these pathways have recently been described. The clinical development of these molecules requires the development of companion tests to monitor their efficacy on their molecular targets. We have developed an activity test to measure in a simple and quantitative way the efficacy of molecules targeting ubiquitin-like modifiers.Finally, given the therapeutic potential of targeting SUMOylation, we have started the development of a SUMOylation inhibitor based on the inhibition of the interaction between E1 and E2 enzymes by stapled peptides.

The budding yeast S. cerevisiae is widely used as a model organism to study biological processes that are conserved among eukaryotes. Di fferent genomic approaches have been applied successfully to interrogate the mode of action of small molecules and their combinations. In this thesis, these technologies were applied to di fferent sets of chemical compounds in the context of two collaborative projects. In addition to insight into the mode of action of these molecules, novel approaches for analysis of chemical-genetic pro files to integrate GO annotation, genetic interactions and protein complex data have been developed. The fi rst project was motivated by a pressing need to design novel therapeutic strategies to combat infections caused by opportunistic fungal pathogens. Systematic screens of 1180 FDA approved drugs identifi ed 148 small molecules that exhibit synergy in combination with uconcazole, a widely used anti-fungal drug (Wright lab, McMaster University, Canada). Genome-wide chemical-genetic profiles for 6 of these drugs revealed two di fferent modes of action of synergy. Five of the compounds a ffected membrane integrity; these chemical-genetic interactions were supported by microscopy analysis and sorbitol rescue assays. The sixth compound targets a distinct membrane-associated pathway, sphingolipid biosynthesis. These results not only give insight into the mechanism of the synergistic interactions, they also provide starting points for the prediction of synergistic anti-fungal combinations with potential clinical applications. The second project characterised compounds that aff ected melanocytes in a chemical screen in zebra fish (Patton lab, Edinburgh). Chemical-genetic screens in S.cerevisiae enabled us to show that melanocyte pigmentation reducing compounds do so by interfering with copper metabolism. Further, we found that defects in intracellular AP1 and AP3 trafficking pathways cause sensitivity to low copper conditions. Surprisingly, we observed that the widely-used MAP-kinase inhibitor U0126 a ffects copper metabolism. A nitrofuran compound was found to speci fically promote melanocyte cell death in zebrafi sh. This enabled us to study off -target eff ects of these compounds that are used to treat trypanosome infections. Nifurtimox is a nitrofuran prodrug that is activated by pathogen-specifi c nitroreductases. Using yeast and zebra fish we were able to show that nitrofurans are also bioactivated by host-specifi c aldehyde dehydrogenases suggesting that a combination therapy with an aldehyde dehydrogenase inhibitor might reduce side e ffects associated with nifurtimox.

Pluripotent embryonic stem cells (ESCs) possess an unlimited capacity for self-renewal. This property of ES cells is both defining and unique. Harnessing this potential of ESCs would provide tremendous opportunity in the field of regenerative medicine and its attempts to combat degenerative diseases such as Parkinson’s, muscular dystrophy, etc. In 2006, Shinya Yamanaka was able to demonstrate that the ectopic expression of four proteins could reverse the process of differentiation and provide somatic cells with the characteristics ESCs. One year later, James Thompson’s group proved the same feat could be accomplished in human somatic cells using a different set of four proteins, including Nanog. The prospect of converting one’s own cells into a stem cell which could subsequently differentiate and repopulate an area of the body afflicted by gross degeneration was revolutionary. In the years following Yamanaka’s and Thompson’s discoveries, however, there has been little insight gained into how these proteins are regulated post-translationally. In this study, four proteins which had previously been identified by Yamanaka as being ‘pluripotency factors’ were used as baits in order to ascertain a protein-protein interaction network. This network was subsequently interrogated using various chemical compounds and small molecules in order to dissect the signal transduction pathways feeding into pluripotency, as well as, post-translational modifications regulating the factors themselves. In this way, the chemical inhibitor H89 was found to decrease the presence of Nanog phosphorylation and possibly its dimerization resulting in the Nanog protein being destabilized and targeted for degradation. Inversely, the pan-cullin inhibitor MLN4924 was identified to increase the abundance of both phosphorylated Nanog and total Nanog protein. In an attempt to identify the Cullin Ring Ligase (CRL) responsible for the degradation of Nanog protein in ESCs, each cullin identified in the protein interaction network was inhibited using specific shRNAs. Quantitative fluorescence microscopy was performed and identified that inhibition of CUL3 increases Nanog protein levels, suggesting that a CUL3-based CRL may be responsible for the post-translation regulation of Nanog. Additionally, the quantitation of Sox2 protein levels in CUL4B shRNA cell line demonstrates that Sox2 protein levels may be regulated by a CUL4B-based CRL. Further studies will reveal whether or not CUL4A depletion also results in elevated Sox2 protein levels. If not, this would include the pluripotency factor Sox2 among the recently identified CUL4B-isoform-specific substrates for degradation and possibly provide the basis for a hypothesis of developmentally regulated substrate specificity. In addition to MLN4924, several other small molecules were identified as being able to increase phospho-Nanog protein levels in this study. Among them were the cell permeable peptides Ht-31 and PKI (14-22) amide. These peptides were found to both stabilize phospho-Nanog and produce ES cell colonies that uniformly express the Nanog protein. The development of a growth medium containing these peptides in order to maintain homogeneous pluripotent ES cells is currently in progress and received backing for a patent application by the University of Edinburgh on February 23, 2012.

Lau Kai-Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references. / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of Contents --- p.iv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The Gaussian´ؤ3 Method --- p.1 / Chapter 1.2 --- The G3 Method with Reduced MΦ ller-Plesset Order and Basis Set --- p.2 / Chapter 1.3 --- The Gaussian-3X Method --- p.2 / Chapter 1.4 --- The Modified G2 Method --- p.3 / Chapter 1.5 --- Calculation of Thermodynamical Data --- p.3 / Chapter 1.6 --- Remark on the Location of Transition Structures --- p.4 / Chapter 1.7 --- Scope of the Thesis --- p.4 / Chapter 1.8 --- References --- p.4 / Chapter Chapter 2 --- "A Gaussian-2 and Gaussian-3 Study of Alkoxide Anion Decompositions. I. H2 and CH4 Eliminations of the Methoxide, Ethoxide, i-Propoxide, and t-Butoxide Anions" --- p.6 / Chapter 2.1 --- Introduction --- p.6 / Chapter 2.2 --- Methods of Calcuations --- p.7 / Chapter 2.3 --- Results and Discussion --- p.8 / Chapter 2.3.1 --- Nature of ion-neutral complex --- p.8 / Chapter 2.3.2 --- Initial bond cleavage of alkoxide anions --- p.9 / Chapter 2.3.3 --- Dissociation of alkoxide anions --- p.10 / Chapter 2.4 --- Conclusions --- p.23 / Chapter 2.5 --- Publication Note --- p.25 / Chapter 2.6 --- References --- p.25 / Chapter Chapter 3 --- A Gaussian-2 and Gaussian-3 Study of Alkoxide Anion Decompositions. II. Alkane Eliminations of (CH3)2(C2H5)CO- and (i-Pr)(C2H5)2CO- --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Methods of Calculations --- p.29 / Chapter 3.3 --- Results and Discussion --- p.29 / Chapter 3.3.1 --- Initial bond cleavage of alkoxide anions --- p.30 / Chapter 3.3.2 --- Dissociation of alkoxde anions --- p.31 / Chapter 3.3.3 --- General dissociation mechanism of alkoxide anions --- p.35 / Chapter 3.4 --- Conclusions --- p.37 / Chapter 3.5 --- References --- p.37 / Chapter Chapter 4 --- A Gaussian-3 Study of the Photoionization and Dissociative Photoionization Channels of Dimethyl Disulfide --- p.40 / Chapter 4.1 --- Introduction --- p.40 / Chapter 4.2 --- Methods of Calculations --- p.41 / Chapter 4.3 --- Results and Discussion --- p.41 / Chapter 4.3.1 --- Bond cleavage reactions --- p.44 / Chapter 4.3.2 --- Dissociation channels involving transition structures --- p.45 / Chapter 4.4 --- Conclusions --- p.48 / Chapter 4.5 --- References --- p.48 / Chapter Chapter 5 --- A Gaussian´ؤ3 Study of the Photodissociation Channels of Propylene Sulfide --- p.50 / Chapter 5.1 --- Introduction --- p.50 / Chapter 5.2 --- Methods of Calculations --- p.51 / Chapter 5.3 --- Results and Discussion --- p.51 / Chapter 5.3.1 --- The dissociation channels involving transition structures --- p.53 / Chapter 5.3.2 --- The dissociations of sulfur atom --- p.56 / Chapter 5.4 --- Conclusions --- p.58 / Chapter 5.5 --- References --- p.59 / Chapter Chapter 6 --- Thermochemistry of Phosphorus Fluorides: A Gaussian´ؤ3 and Gaussian´ؤ3X Study --- p.60 / Chapter 6.1 --- Introduction --- p.60 / Chapter 6.2 --- Methods of Calculations --- p.62 / Chapter 6.3 --- Results and Discussion --- p.62 / Chapter 6.3.1 --- Comparison of the G3 and G3X methods --- p.62 / Chapter 6.3.2 --- Assessments of the experimental results --- p.65 / Chapter 6.4 --- Conclusions --- p.71 / Chapter 6.5 --- References --- p.71 / Chapter Chapter 7 --- Conclusions --- p.74 / Appendix A --- p.75 / Appendix B --- p.78

Melatonina, indolamina amplamente distribuída entre os seres vivos, é o hormônio da glândula pineal e também é produzida de forma parácrina por células imunocompetentes estimuladas. A produção de melatonina pela glândula pineal ocorre apenas no escuro e este hormônio serve para marcar a existência e a duração da noite. A monocamada de células endoteliais que reveste os vasos sanguíneos forma uma interface entre o sangue e os tecidos sendo, portanto, um sensor da presença de melatonina circulante. Essas células participam de diversos processos fisiológicos e fisiopatológicos, como na migração dos leucócitos durante a montagem de uma resposta inflamatória. Neste caso as células endoteliais são capazes de responder a padrões moleculares associados a patógenos tais como, lipopolissacarídeos (LPS) de bactérias Gram negativas. Melatonina circulante modula a interação leucócito endotélio em ratos e a expressão de moléculas de adesão em células endoteliais cultivadas. Este trabalho foi planejado com o objetivo de entender o efeito da melatonina sobre células endoteliais. Para tanto, todos os estudos foram feitos em células cultivadas ativadas ou não com LPS. Todos os ligantes foram administrados diretamente às culturas de células. Verificamos que LPS (1&um;g/mL, 2h) promove a expressão de moléculas de adesão (PECAM-1 e ICAM-1) e aumenta a adesão de neutrófilos. Melatonina (10-9 e 10-4M, 2h) inibe ambas as respostas. Um bloqueador dos receptores de melatonina, luzindol, não reverteu o efeito da melatonina. Um tratamento crônico com melatonina (20 dias) não dessensibiliza a resposta. Portanto, nossos dados mostram que o efeito da melatonina se dá diretamente sobre as células endoteliais, abrindo a perspectiva de um estudo direto dos mecanismos de ação envolvidos nos efeitos gerados pela melatonina / Melatonin, an indoleamine widely distributed among living beings, is the hormone of the pineal gland and is also produced by stimulated immunocompetent cells. Melatonin, which is konwn as the darkness hormone, is produced at night by the pineal gland. In blood vessels, a monolayer of endothelial cells forms the interface between blood and tissue. This monolayer, which participates in several physiological and pathophysiological processes, is a sensor of circulating substances, including melatonin. Regarding the migration of leukocytes during the initiation of an inflammatory response, endothelial cells are able to respond to molecular patterns associated with pathogens such as lipopolysaccharide (LPS) of Gram-negative bacteria. Circulating melatonin modulates the interaction between leukocytes and endotelial cells and the expression of adhesion molecules. This work was planned in order to understand the effect of melatonin on endothelial cells. All studies were performed in cultured cells activated or not with LPS. All ligands were administered directly to cell cultures. We found that LPS promotes the expression of adhesion molecules (PECAM-1 and ICAM-1) and increases the adhesion of neutrophils to endotelial cells. Melatonin (10-9 and 10-4M, 2 hours) inhibits both responses. A chronic treatment with melatonin (20 days) did not desensitize the response. Therefore, our data clearly show that the effect of melatonin occurs directly on endothelial cells, opening the prospect of a direct study of the mechanisms involved in the effects produced by melatonin

Orientador: Carmen Maria Andreazza / Banca: Edson Denis Leonel / Banca: José Williams dos Santos Vilas Boas / Resumo: De acordo com os resultados observacionais de condensações de nuvens moleculares escuras, grandes variações na razão 13CO/C18O são observadas quando se comparam os resultados obtidos nas condensações situadas dentro da mesma nuvem, bem como de nuvem para nuvem. O valor médio dessa razão na condensação principal de Ophiuchus é inferior a 5. Por outro lado, o valor encontrado nas condensações que estão situadas ao norte de Oph é maior que 10. Grandes diferenças também são encontradas quando se comparam os resultados observacionais de diferentes nuvens escuras, tais como Ophiuchus e Taurus, onde são observados também um decréscimo da razão C18O/C17O com o aumento da densidade. Os processos químicos e físicos que governam essas variações ainda não estão claros. Nesse sentido, o objetivo da presente proposta é analisar a influência do colapso gravitacional de condensações de nuvens moleculares escuras na síntese das moléculas CO, C17O, C18O, 13CO, 13C17O e 13C18O. Tal análise é feita com base em comparações entre modelos que consideram diferentes condições entre si, tais como, tamanho da cadeia química, velocidade de colapso, densidade inicial e processos de congelamento de espécies químicas na superfície de grãos de poeira. Os resultados obtidos mostram que o tamanho da cadeia química tem influência nas razões 13CO/C18O e C18O/C17O, mas não tanto quanto a densidade inicial e a velocidade do colapso. Além disso, o congelamento das espécies químicas nos grãos é mais significativo nos estágios mais avançados da evolução da condensação. Os modelos de condensações escuras que sofrem colapso gravitacional lento e em queda livre reproduzem satisfatoriamente as razões 13CO/C18O e C18O/C17O observadas, o que permite concluir que o colapso gravitacional pode ter um importante efeito nas referidas razões. / Abstract: According to the observational results of dark molecular clouds condensations, large variations in the ratio 13CO/C18O are observed when comparing the results obtained in the condensations located within the same cloud and cloud to cloud. The average value of this ratio in the main condensation of Ophiuchus is below 5. On the other hand, the value found in the condensations that are located north of Oph is larger than 10. Large differences are also found when comparing the observational results of different dark clouds such as Ophiuchus and Taurus, in which are also found a decrease of the C18O/C17O ratio with increasing density. The chemical and physical processes that govern these variations are still unclear. In this sense, the objective of this proposal is to analyze the influence of the gravitational collapse of centrally condensed clumps of dense molecular gas in the synthesis of the CO, C17O, C18O, 13CO, 13C17O and 13C18O molecules. This analysis is based on comparisons among models that consider different condition, such as, chemical chain, initial density, speed of collapse and freezing processes of the chemical species on the surface of dust grains. The results show that the size of the chemical chain has influence on the 13CO/C18O and C18O/C17O ratios, but they are not as important as the initial density and the speed of the collapse. Furthermore, the freezing of chemical species on the grains occurs at later times of the collapse. The models of a gravitational free-fall collapsing core and of slowly contracting core with higher initial density are consistent with observations. These results indicate that the gravitational collapse of molecular cores can have an important effect in the 13CO/C18O and C18O/C17O ratios. / Mestre

Melanoma primário da mucosa oral (MPMO) é um tumor raro e agressivo. Estudos recentes demonstraram uma correlação entre o aumento da invasão tumoral e o fenótipo metastático com uma alteração no padrão de expressão das moléculas de adesão. Neste estudo analisamos a expressão de integrinas e imunoglobulinas nos melanomas primários da mucosa oral e relacionamos os resultados com os parâmetros clínicos. As análises imunoistoquímicas dos padrões de expressão destas moléculas foram realizadas em 35 casos de melanomas primários da mucosa oral, e os resultados foram correlacionados com características clínicas e histológicas. Observou-se que a subunidade beta-4 de integrina foi negativa em casos com invasão vascular. A presença de integrina beta-3 e de CD166 (ALCAM) estavam estatisticamente associadas à extensa invasão vascular (p < 0,05). A menor expressão de CD54 (ICAM) foi marginalmente relacionada a casos com necrose extensa, enquanto a maioria dos casos com doença metastática foi negativa para CD66 (CEACAM). Conclusão: padrões alterados de expressão de moléculas de adesão, principalmente integrinas e imunoglobulinas, podem participar da patogênese e do desenvolvimento dos melanomas primários da mucosa oral / Primary oral mucosal melanoma is a rare and an aggressive tumor. Recent studies have demonstrated the correlation among increased tumor invasion, the metastatic phenotype and altered adhesion molecule expression profiles. The present study analyzed the expression of integrins and immunoglobulin-like adhesion molecules in oral mucosal melanomas and correlated results with clinical parameters. Immunohistochemical analyses of their expression patterns were performed on thirty-five cases of primary oral mucosal melanomas. The results were correlated with clinical and histological features of the cohort. The beta-4 subunit of integrin was negative and this was related with vascular invasion. Positivity of integrin beta-3 and CD166 (ALCAM) was statistically associated with extensive vascular invasion (p < 0.05). Lower expression of CD54 (ICAM) was associated with cases with extensive necrosis. Most cases with metastatic disease were negative for CD66 (CEACAM). Conclusion: Altered patterns of adhesion molecule expression, mainly integrins and immunoglobulin-like proteins, may participate in the pathogenesis and outcome of primary oral mucosal melanomas

Ácido clorogênico (ACG) é o termo utilizado para designar um grupo de compostos fenólicos oriundos da reação de esterificação entre ácidos hidroxicinâmicos (p-cumárico, caféico e ferúlico) e ácido quínico. São amplamente encontrados em produtos naturais e exercem ações antioxidantes, citotóxicas, antitumorais, antibacterianas, antifúngicas e antiinflamatórias. Apesar da descrição dos seus efeitos antiinflamatórios em diferentes modelos experimentais, a literatura é carente quanto suas ações específicas em funções inflamatórias de neutrófilos. Assim, o objetivo do presente trabalho foi investigar os efeitos do ACG sobre funções de neutrófilos in vitro. Neutrófilos foram obtidos do lavado peritoneal de ratos Wistar, machos, 4 horas após a injeção local de glicogênio de ostra a 1% e foram incubados, na presença ou ausência de LPS, com ACG nas concentrações de 25, 50, 100 ou 1000 &#181;M. A viabilidade celular (exclusão por trypan-blue), a secreção de citocinas e prostaglandina E2 (ensaio imunoenzimático); a produção de óxido nítrico (reação de Griess); a expressão de moléculas de adesão (citometria de fluxo); a aderência e a quimiotaxia foram avaliadas. Os resultados obtidos mostraram que o ACG não afetou as secreções do fator de necrose tumoral-&#945;,de interleucina 1&#946;, do óxido nítrico e de prostaglandina E2 em condições basais ou após estimulação pelo LPS. Diferentemente, a incubação com ACG inibiu a aderência de neutrófilos à cultura primária de célula endotelial de microcirculação de ratos e a quimiotaxia in vitro frente ao peptídeo formilado (N-formilmetionil- leucil-fenilalanina). Estes últimos efeitos podem estar associados à ação do ACG sobre a expressão de moléculas de adesão, já que o ACG elevou a expressão de L-selectina e reduziu as expressões de &#946;2 integrina e da molécula de adesão plaqueta e endotélio. Os dados obtidos não foram dependentes de alterações da viabilidade celular. Em conjunto, os resultados mostram que o ACG possui efeito direto sobre funções de neutrófilos responsáveis pela interação ao endotélio microvascular e pela migração orientada em resposta a estímulo inflamatório. Estes efeitos podem contribuir, pelo menos em parte, para redução da migração de neutrófilos para focos de inflamação na vigência de tratamento com ACG amplamente descrita na literatura. / Chlorogenic acid (CGA) is the term utilized to design a group of phenolic compounds from the sterification reaction between the hydroxycinnamic acids (p-coumaric, caffeic and ferulic acid) and the quinic acid. They are largely found in natural products and exert anti-oxidant, citoxic, anti-tumoral, anti-bactericidal, anti-fungicidal and anti-inflammatory activity. Besides the description of its anti-inflammatory effect in different experimental models, the literature is scarse regarding its specific actions in neutrophil inflammatory functions. Therefore, the aim of this present work was to investigate the CGA effects on neutrophils functions in vitro. Neutrophils were obtained from the peritoneal lavage of male Wistar rats four hours after a local injection of oyster glycogen 1% and were incubated, in the presence or absence of LPS, with CGA in the concentrations of 25, 50, 100 or 1000 &#181;M. The cellular viability (trypan-blue exclusion), the cytokines secretions (enzyme-linked immunosorbend assay), production of nitric oxide (Greiss reaction); adhesion molecules expression (flow citometry), adherence and chemotaxis in vitro were assessed. The results shows that the CGA did not affect the secretion of the tumor necrosis factor-&#945; , of nitric oxide and prostaglandin E2 and only the incubation with 50&#181;M of CGA inhibited the secretion of Interleukin 1&#946; after stimulation with LPS. Differently, the incubation with CGA inhibited the adherence of neutrophils on the primary culture of endothelial cell from the micro-circulation of rats and the chemotaxis in vitro against formylated peptide (fenyl-metyl-leucyl-alanin). This lasts effects might be associated with the action of CGA on the expression of adhesion molecules, hence this compound was capable of elevate the expression of L-selectin and reduce the expression of &#946;2 integrin and PECAM-1. The data here obtained are not dependent of cellular viability alterations. In conjunct, the data here obtained shows that the CGA has direct effect on neutrophils functions responsible of the interaction with the micro vascular endothelium and the oriented migration in response to an inflammatory stimuli. These effects can contribute, at least in part, to the decreased neutrophil migration in the inflammatory focus in the presence of CGA treatment.