INTRODUÇÃO: A pneumonectomia está associada à alta mortalidade e alto índice de complicações. Entre estas, o edema pulmonar pós-pneumonectomia é uma das mais graves, podendo chegar a 100% de mortalidade. Pouco se sabe acerca dos fatores etiológicos desta doença, bem como sua associação a um processo inflamatório ou estresse oxidativo. O objetivo deste estudo foi analisar os efeitos agudos da pneumonectomia esquerda sobre o pulmão remanescente de ratos quanto à avaliação funcional por gasometria e avaliação histológica por formação de edema, infiltrado inflamatório, estresse oxidativo e reatividade vascular. MÉTODOS: Trinta e um ratos Wistar foram submetidos ao estudo. Vinte e um foram submetidos à pneumonectomia esquerda, sendo sacrificados em 48 horas (11 animais) e 72 horas (10 animais). Como controle do tratamento, 10 ratos foram submetidos à operação sham, sendo 5 sacrificados em 48 horas e 5 em 72 horas. A avaliação funcional foi realizada por meio de coleta de sangue arterial, gasometria e análise da relação pO2/FiO2. A análise histológica consistiu da avaliação dos seguintes parâmetros: (1) grau de edema perivascular; (2) presença de infiltrado inflamatório obtido por meio da densidade de neutrófilos; (3) expressão tecidual imunoistoquímica da Óxido Nítrico Sintase (NOS) para a avaliação do estresse oxidativo e (4) do grau de reatividade vascular, medido por meio da relação luz parede (lumen/wall ratio). Na avaliação do estresse oxidativo, foram analisadas a isoformas induzida e endotelial da NOS (iNOS e eNOS). Além destes parâmetros, o edema pulmonar foi avaliado por meio do ganho de massa pulmonar proporcional, denominado de Índice Pulmonar (IP) e da relação do peso úmido e do peso seco (Razão U/S). A análise estatística foi realizada por meio do teste ANOVA. RESULTADOS: Não houve diferença entre os grupos quanto à relação pO2/FiO2. Quanto à análise histológica, houve diferença quanto ao edema perivascular, infiltrado inflamatório, imunoexpressão de iNOS e eNOS e reatividade vascular. Houve interação entre a pneumonectomia e o sacrifício mais tardio, com maior índice de edema perivascular neste grupo (p=0,0274). Houve menor densidade de neutrófilos nos animais submetidos à pneumonectomia tanto em 48 como 72 horas (p=0,0168). Não houve diferença na imunoexpressão tecidual de iNOS entre os animais submetidos à pneumonectomia e seus respectivos grupos controle, mas houve diminuição no grupos de 72 horas (p=0,0212). A análise imunoistoquímica da eNOS evidenciou maior expressão nos animais submetidos à pneumonectomia (p=0,0208). Quanto ao grau de reatividade vascular, houve menor razão L/P nos grupos sacrificados após 72 horas (p=0,0107), sugerindo maior vasoconstrição nestes grupos. Embora tenha havido maior ganho de massa pulmonar nos dois grupos de animais submetidos à pneumonectomia (p=0,0033), a Razão U/S não mostrou diferença entre os grupos. CONCLUSÕES: A pneumonectomia esquerda em ratos não causou alterações funcionais, mas causou alterações histológicas. Quanto a estas alterações, não foram de natureza inflamatória e nem relacionadas ao estresse oxidativo. Foram caracterizadas por edema perivascular e vasoconstrição, observados após 72 horas da operação. / INTRODUCTION: Pneumonectomy is associated with high mortality and complication rates. Of these complications, post-pneumonectomy pulmonary edema is one of the most severe with a mortality rate that can reach 100%. Little is known about the etiological factors involved in this process and its association with inflammatory process or oxidative stress. The objective of this study was to analyze the acute effects of left pneumonectomy on the remaining lung of rats based on functional assessment by blood gas analysis and on histological assessment by edema formation, inflammatory infiltrate, oxidative stress and vascular reactivity. METHODS: Thirty one Wistar rats were included in the study. Twenty one underwent left pneumonectomy and were sacrificed in 48 hours (11 animals) and 72 hours (10 animals). Ten rats underwent sham procedure for control and five were sacrificed in 48 hours and five in 72 hours. Functional assessment was conducted by arterial blood gas and pO2/FiO2 ratio analyses. Histological analysis consisted of the assessment of the following parameters: (1) degree of perivascular edema; (2) presence of inflammatory infiltrate suggested by neutrophil density; (3) immunohistochemical expression of Nitric Oxide Synthase (NOS) in tissues to assess oxidative stress and (4) the degree of vascular reactivity measured by lumen/wall ratio (L/W ratio). For the assessment of oxidative stress, induced and endothelial isoforms of NOS (iNOS and eNOS) were analyzed. In addition to these parameters, pulmonary edema was assessed by means of proportional pulmonary mass gain, called Pulmonary Ratio (PR) and of the wet/dry weight ratio (W/D Ratio). The statistical analysis was conducted using the ANOVA test. RESULTS: The histological analysis showed difference regarding perivascular edema, inflammatory infiltrate, immunoexpression of iNOS and eNOS and vascular reactivity. The rate of perivascular edema was higher in animals submitted to pneumonectomy and sacrificed after 72 hours (p=0.0274). Neutrophil density was lower in animals submitted to pneumonectomy for those sacrificed after 48 and 72 hours alike (p=0.0168). There was no difference in the immunoexpression of iNOS in tissues between animals submitted to pneumonectomy and control groups, but such immunoexpression was reduced in both 72-hour groups (p=0.0212). The immunohistochemical analysis of eNOS evidenced a higher expression in animals submitted to pneumonectomy (p=0.0208). As concerns the degree of vascular reactivity, there was a lower W/D ratio in the groups sacrificed after 72 hours (p=0.0107), suggesting greater vasoconstriction in these groups. There was no difference between the groups as to the pO2/FiO2 ratio. Although the two groups submitted to pneumonectomy had greater gain of mass (p=0.0033), there was no difference in the W/D ratio between the groups. CONCLUSIONS: Left pneumonectomy in rats did not cause functional alterations but caused histological alterations that were neither of inflammatory nature nor related to oxidative stress. The alterations included perivascular edema and vasoconstriction observed after 72 hours of the procedure.

Orientador: Elenice Aparecida de Moraes Ferrari / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T06:24:39Z (GMT). No. of bitstreams: 1 Faria_LarissaOliveiraMellonide_M.pdf: 1121073 bytes, checksum: 5a3108ca0447b5fae98988b4611d7616 (MD5) Previous issue date: 2013 / Resumo: O óxido nítrico (NO) é um neurotransmissor não convencional o qual tem papel importante em processos neurobiológicos de comportamento e de memória. Sua síntese é mediada por três isoformas de sintase do óxido nítrico (NOS): a neuronal (nNOS), a endotelial (eNOS) e a induzível (iNOS). Este trabalho investigou os efeitos da administração do 7- nitroindazol (7-NI), inibidor preferencial da nNOS, na consolidação e reconsolidação da memória do condicionamento clássico aversivo. Pombos adultos foram atribuídos a 5 grupos: Foram usados 5 grupos: grupo 7-nitroindazole (7-NI) (100nmol/0.5?/l; DMSO (20%), NaOH (50mM) e Tween-80 (16%) diluído em PBS; i.c.v.), grupo veículo (VEIC) (0,5?/l; DMSO (20%), NaOH (50mM) e Tween-80 (16%) diluído em PBS, i.c.v.), grupo condicionado/não tratado (COND), grupo contexto/não-tratado (CONT) e grupo não tratado/não condicionado (NÄIVE). Sete dias após implante de microcânula intracerebroventricular (i.c.v.), ocorreu o condicionamento com três associações contextochoque numa sessão de 20 min. O teste e o re-teste consistiram na re-exposição ao contexto do condicionamento por 5 min. O intervalo entre sessões foi de 24h. A administração de 7-NI ou do veículo ocorreu imediatamente após o treino (Experimento 1) ou após o re-teste (Experimento 2). A atividade enzimática da NOS dependente e independente de Ca2+ e da expressão protéica da nNOS foram realizadas no tecido hipocampal. No Experimento 1, a ocorrência de congelamento no teste do 7-NI foi menor do que no treino (p<0.01) e no teste do COND e VEIC (p < 0.001). A atividade da NOS dependente de Ca++ no 7-NI foi menor do que no COND e VEIC (p<0,01), mas não diferiu do CONT e do NÄIVE. A expressão protéica de nNOS não diferiu entre os grupos (p<0,05). No Experimento 2, houve diminuição dos comportamentos defensivos, incluindo o congelamento, no re-teste do 7-NI comparado com VEIC e COND (p<0.05), mas os grupos não diferiram quanto à atividade de NOS dependente de Ca2+ ou à expressão protéica da nNOS. Conclui-se que o 7-NI interferiu na consolidação e a reconsolidação da memória, indicando a ativação da via de sinalização do óxido nítrico no hipocampo em processos da memória de medo condicionado ao contexto em pombos / Abstract: Nitric oxide (NO) is an unconventional neurotransmitter which plays an important role in neurobiological processes of behavior and memory. Its synthesis is mediated by three isoforms of nitric oxide synthase (NOS): the neuronal (nNOS), the endothelial (eNOS) and the inducible (iNOS). This study investigated the effects of the administration of 7- nitroindazole (7-NI), a preferential nNOS inhibitor, in the consolidation and reconsolidation of aversive classical conditioning memory. Adult male pigeons were assigned to 5 groups: 7-nitroindazole, 7-NI (100nmol/0.5?/l; DMSO (20%), NaOH (50 mM) and Tween-80 (16%) diluted in PBS; i.c.v.) Vehicle group; VEH (0.5 ? / L; DMSO (20%), NaOH (50 mM) and Tween-80 (16%) diluted in PBS; i.c.v.), conditioning/non-treated group (COND), context/non-treated group (CONT) and non-conditioning/non-treated group (NÄIVE). Seven days after implantation of intracerebral ventricular (i.c.v.) microcannula the conditioning occurred with three context-shock associations in a session of 20 min. During the testing and retesting sessions pigeons were reexposed to the conditioning context for 5 min. The between sessions interval was 24h. Administration of 7-NI or vehicle occurred immediately after training (Experiment 1) or after testing (Experiment 2). The enzymatic activity of Ca2+ dependent and independent NOS and protein expression of nNOS in the hippocampus tissue were carried out following the behavioral test or retest. In Experiment 1, the occurrence of freezing in the testing session of 7-NI group was lower than in the training (p <0.01) and the testing sessions of COND and VEH groups (p <0.001). The activity of Ca2+ dependent NOS in the 7-NI group was lower than in COND and VEH groups (p <0.01) but did not differ from CONT and NÄIVE groups. The nNOS protein expression in the hippocampus did not differ among the different groups (p<0.05). In Experiment 2, there was a decrease of defensive behaviors, which include freezing, in the retest of the 7-NI compared with VEH and COND groups (p <0.05), but the groups did not differ in the activity of Ca2+ dependent NOS or the protein expression of nNOS. We conclude that 7-NI interfered on the consolidation and reconsolidation of memory, indicating activation of the nitric oxide signaling pathway in the hippocampus and in memory processes of conditioned fear context in pigeons / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Interferons are known cytokines that display antiviral, anti-proliferative and immuno-modulatory functions in the host. Interferon-gamma (Ifnγ) is the only type II family interferon that binds to the heterodimeric receptor consisting of two subunits, IfnγR1 and IfnγR2. This specific interaction between Ifnγ and its receptor triggers the activation of Janus Kinase (Jak) – Signal Transducer and Activator of Transcription (Stat) pathway. This triggers a cascade of events leading to modulation of a wide variety of genes resulting in a plethora of responses including antimicrobial activities, induction of Major Histocompatibility Complex encoded molecules etc. The impact of Ifnγ in regulating host defense is observed in patients who lack functional IFNγ or its receptor as they succumb to less virulent strains of intracellular bacteria such as Mycobacterium and Salmonella. Also, mice lacking important downstream signaling components such as Stat1 and Interferon Regulated Factor 1 (Irf1) are known to be highly susceptible to a variety of bacterial and viral infections. Consequently, studies on uncharacterized signaling and regulatory molecules downstream to Ifnγ are of great interest. The modulatory functions of Ifnγ have been attributed to its ability to regulate the expression of a vast number of genes in a Stat1 and Irf1 dependent manner. Also, gene regulation in response to Ifnγ in a target cell such as mouse hepatoma cell line, H6, can be categorized broadly into two subsets: Reactive Oxygen Species (ROS) - Reactive Nitrogen Intermediates (RNI) dependent (e.g. Nos2, Catalase, Id2 etc.) as well as ROS – RNI independent (e.g. Tap1, Lmp2 etc.). However, the effect of Ifnγ induced ROS and RNI in the regulation of the expression of genes at the level of transcriptome and how these could impact cellular and host responses are not well characterized. To investigate these questions, we standardized an in vitro Ifnγ responsive primary cell culture system using mouse adherent peritoneal macrophages (PMs). It needs to be highlighted that this study has, primarily, utilized unstimulated resident PMs. The adherent cells from the peritoneal cavity were positive for macrophage markers such as F4/80 and CD11b, but negative for granulocyte marker Gr1. Also, PMs were resistant to Ifnγ induced cell death, unlike cell lines such as the mouse fibroblast cell line L929, for the time points studied. There are three distinct parts to this study involving the system of PMs: I. To understand the contribution of Nitric Oxide (NO) in regulating the expression of novel Ifnγ responsive genes, PMs from C57BL/6 mice and mice lacking nitric oxide synthase 2 (Nos2-/-), the enzyme isoform responsible for the generation of NO in PMs, were stimulated with Ifnγ for 8 h and microarray analysis was performed. Detailed analysis led to identification of several annotated genes that were uniquely regulated in C57BL/6 and Nos2-/- PMs. Further analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several differentially regulated pathways. Interestingly, a large number of metabolism related pathways, Butirosin and neomycin, Galactose, Phenylalanine and glyoxylate and dicarboxylate, were specifically up-regulated in the C57BL/6 PMs treated with Ifnγ. Similarly, other metabolism related pathways were differentially regulated by Ifnγ in PMs from C57BL/6 and Nos2-/- mice. One of the pathways that was up regulated in a Nos2 dependent manner in the C57BL/6 PMs upon Ifnγ treatment was that of circadian rhythm, which consisted of genes Per1, Bhlhb2 and Bhlhb3. All three are known circadian rhythm regulators, with Bhlhb2 and Bhlhb3 being transcriptional repressors that bind to E-box consensus sequence (CANNTG) as heterodimers, using the basic helix-loop-helix (bHLH) domains, along with other transcriptional regulators. Both Bhlhb2 and Bhlhb3 were up regulated at RNA and protein levels in a kinetic manner upon Ifnγ treatment in L929 cells. Studies with inhibitors to ROS and RNI revealed that up regulation of Bhlhb2 and Bhlhb3 was RNI, but not ROS, dependent in response to Ifnγ. Interestingly, the RNI inhibitor, NG-Methyl-L-Arginine (LNMA) rescued Ifnγ induced ROS. On the other hand, ROS inhibitors, e.g. Apocyanin and polyethylene glycol Catalase (PEG-Catalase), did not affect the nitrite amounts in the supernatant. These experiments suggested that RNI was upstream to ROS in L929 cells and both contributed to Ifnγ induced cell death. The knockdown of Bhlhb3 using specific siRNAs in the untreated L929 cells increased Bhlhb2 amounts, but not vice versa. This observation is consistent with the fact that Bhlhb3 is a known repressor of Bhlhb2. However, this repression of Bhlhb2 by Bhlhb3 was not detected upon Ifnγ treatment in L929 cells possibly because of heterodimerization of Bhlhb3 with other Ifnγ induced transcriptional modulators. Finally, knockdown of either of the proteins did not affect induced nitrite but decreased ROS amounts resulting in significant rescue of Ifnγ induced cell death of L929 cells. Thus, Bhlhb2 and Bhlhb3 are novel Ifnγ induced proteins that are NO dependent and contribute to Ifnγ induced cell death. Ifnγ and Nos2 are known to elicit antibacterial defense in the host. Interestingly, recent studies have implicated circadian rhythm to regulate bacterial infection in mice. Therefore, regulation of both Bhlhb2 and Bhlhb3 upon Ifnγ treatment and during Salmonella enterica Serovar Typhimurium (S. typhimurium) infection in the bone marrow derived macrophages (BMDMs) was performed. Both Bhlhb2 and Bhlhb3 were induced in a Nos2 dependent manner upon Ifnγ addition in BMDMs. Similar to L929 cells, Bhlhb3 repressed Bhlhb2 in the untreated BMDMs. Also, infection of BMDMs with S. typhimurium increased the protein amounts of Bhlhb2, while repressing Bhlhb3. Importantly, knockdown of Bhlhb2 resulted in higher colony forming units (CFU), whereas knockdown of Bhlhb3 reduced CFU in BMDMs 18 h post infection with S. typhimurium. Thus, Bhlhb2 induced whereas Bhlhb3 repressed antibacterial defense in BMDMs. The exact mechanism downstream to these two proteins and their inter-relationship in regulating S. typhimurium infection is of considerable interest and will be studied in future. II. Macrophages are known to produce a large number of different cytokines and chemokines upon activation. To identify novel cytokines and chemokines that may be differentially regulated in response to Ifnγ, a protein array was performed using the supernatants of C57BL/6 PMs treated with and without Ifnγ. Chemokine Ccl3 was found to be repressed by Ifnγ in the supernatant of PMs. Further analysis using Enzyme Linked Immuno-Sorbent Assay (ELISA) revealed that both Ccl3 and Ccl4, highly homologous proteins that chemo-attract almost all types of leukocytes, were repressed upon Ifnγ treatment. This response was ligand and cell specific as Lip polysaccharide (LPS) stimulation of PMs and Ifnγ stimulation of thioglycollate elicited PMs did not result in repression of Ccl3 and Ccl4. Also, studies with fludarabine, an inhibitor to Stat1, revealed that the repression of Ccl3 and Ccl4 as well as induction of Cxcl10 in response to Ifnγ was Stat1 dependent. Importantly, the use of LNMA as well as PMs from Nos2-/- mice established the role of Nos2 in the repression of Ccl3 and Ccl4, but not Cxcl10 induction, in response to Ifnγ. Furthermore, activation of p38 Mapk, but not Jnk, was downstream to Nos2 activation and contributed functionally to the repression of Ccl3 and Ccl4 in response to Ifnγ. Finally, the transcriptional repressor, Activating Transcription Factor 3 (Atf3), was induced in a Stat1-Nos2-p38 Mapk dependent manner and knockdown of Atf3 using siRNAs established the functional role of the same in the repression of Ccl3 and Ccl4 in response to Ifnγ. Further, to understand the regulation of Ccl3 and Ccl4, their modulation upon S. typhimuirum infection of BMDMs was performed. Apart from regulating the CFU in BMDMs, Ifnγ and Nos2 functionally repressed Ccl3 and Ccl4 upon S. typhimurium infection. Oral infection of mice with S. typhimurium was performed and mice lacking Ifnγ and Nos2 were found to have greater CFU in their organs as well as more leukocytes in the infected liver sections in comparison to the infected C57BL/6 mice. Importanly, absence of Ifnγ as well as Nos2 increased the amounts of Ccl3 and Ccl4 in the sera upon S. typhimurium infection in comparison to the C57BL/6 infected mice. Overall, this part of the study identified Ifnγ and Nos2 to repress chemokines Ccl3 and Ccl4 in macrophages and in mice upon S. typhimurium infection. III. While working on the above mentioned studies, it was noticed that addition of Ifnγ to PMs induced in a dose and time dependent manner aggregation of cells. Experiments with LPS, TG PECs and BMDMs established that Ifnγ induced aggregation of PMs was ligand and cell type specific. A panel of cell surface integrins and selectins were screened for regulation upon Ifnγ addition, namely Icam1, Lfa1, CD11b, P-selectin and E-selectin. Most of the cell surface integrins were repressed by Ifnγ in a kinetic manner. Interestingly, CD11b as well as E-Selectin co-localized to the sites of interactions between the PMs upon Ifnγ treatment. Studies with Reopro, a purified F(ab’)2 to glycoprotein GPIIb that is also known to functionally block CD11b, revealed the functional contribution of CD11b during Ifn induced aggregation of PMs. Further, studies with specific inhibitors identified RNI, but not ROS, to contribute to Ifnγ induced PM aggregation. Also, lack of Nos2 in PMs upon Ifnγ treatment resulted in minimal aggregation together with morphological changes, e.g. flattening of cells. Since differences in the morphology of PMs was observed in the absence of Nos2 upon Ifnγ treatment, the regulation and roles of cytoskeleton proteins, Actin and tubulin, during Ifnγ induced aggregation of PMs was studied. Upon Ifnγ stimuli, actin and tubulin get stabilized. On the other hand, the absence of Nos2 leads to depolymerization of actin, while tubulin failed to stabilize to the membrane, in response to Ifnγ. Further, addition of actin and tubulin depolymerizing agents, Cytochalasin D and Colcemid respectively, decreased Ifnγ induced aggregation of PMs. Live cell imaging studies revealed that PMs needed actin, but not tubulin or CD11b, for mobility. Upon Ifnγ treatment, PMs from C57BL/6 mice exhibited reduced mobility and aggregated with each other. The Nos2-/- PMs exhibited lower mobility compared to C57BL/6 PMs and, upon Ifnγ treatment, underwent morphological changes with time, e.g. flattening. On the other hand, Nos2 is important for endogenous mobility and maintaining the cellular morphology in response to Ifnγ. To understand the physiological relevance of our observations, oral infection of C57BL/6 and Nos2-/- mice with S. typhimurium was performed. Four days post infection, no significant differences in the number of peritoneal cells were found. Importantly, PMs from infected Nos2-/- mice had higher CFU in comparison to C57BL/6 mice. However, the amounts of cytokines such as Ifnγ, Tnfα, Il6 and Il1β in the peritoneal lavage were not significantly different between the two infected strains. Interestingly, PMs isolated from infected Nos2-/- mice displayed distinct morphology, e.g. flattening. In comparison, infected C57BL/6 PMs aggregated when cultured for 24 h in vitro. In the future, it will be interesting to address the functional roles of aggregates of macrophages during physiologically relevant responses such as combating intracellular bacterial infection. This part of the study adds a new dimension to the ability of Ifnγ in the regulation of macrophage-macrophage interactions and their roles during intracellular bacterial infections. Overall, the present study has elucidated hitherto uncharacterized roles of Nos2 and mechanisms involved in regulation of novel functional responses of PMs to Ifnγ and during S. typhimurium infection.

Orientador: Maria Alice da Cruz-Höfling / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T16:36:47Z (GMT). No. of bitstreams: 1 Soares_EdileneSiqueira_M.pdf: 11448561 bytes, checksum: 00aace7be2d8fec579aea2fd4166f813 (MD5) Previous issue date: 2015 / Resumo: Neste trabalho investigamos a permeabilização da barreira hematoencefálica (BHE) pela peçonha da aranha Phoneutria nigriventer (PNV) através da via transcelular no cerebelo de ratos. As cavéolas foram analisadas nas células endoteliais pela expressão de proteínas associadas à sua formação (caveolina-1, Cav-1 e dinamina-2, Din2) e internalização (caveolina-1 fosforilada, pCav-1 e quinase da família Src, SKF), e nos astrócitos com avaliação da caveolina-3 (Cav-3) e da conexina-43 (Cx43) (formadora de junções comunicantes). A ação do PNV sobre o endotélio também foi avaliada pela ativação (acoplamento) ou inativação (desacoplamento) da enzima eNOS, produtora de óxido nítrico (NO). As estruturas que compõe a BHE foram avaliadas através de microscopia eletrônica de transmissão. Inicialmente, o estudo de Cav-1 contemplou sua localização, expressão gênica e proteica após o envenenamento em diferentes idades, ratos neonatos eram mais suceptíveis ao envenenamento do que ratos adultos. Após PNV, a imunomarcação para Cav-1 foi mais evidente na camada granular e molecular e em neurônios de Purkinje. A expressão Cav-1 e Din2 (foramdoras das vesículas e seu gargalo, respectivamente) aumentou em períodos de envenenamento agudo (1 h), de recuperação (5 h) e na ausência de sinais clínicos (24 h); em contrapartida SKF e pCav-1 envolvidas na internalização caveolar foram superexpressas em períodos opostos (às 2 h e 72 h). O PNV induziu aumento da metaloproteinase-9 da matriz (MMP9), importante mediadora de quebra da BHE e aumentou a formação e o tráfego de vesículas no endótelio após envenenamento. A análise de eNOS revelou desacoplamento (aumento de monômeros) nos períodos de envenenamento agudo (1-2 h) com progressivo retorno e super-expressão de dímeros (re-acoplamento) às 72 h; essas alterações foram relacionadas à ação do PNV sobre os níveis intracelulares de cálcio investigado pelo aumento na expressão de calmodulina e confimado pela localização de calbindina-D28. Os dados revelam a interferência do PNV sobre a homeostase endotelial e função vascular ao afetar o sistema eNOS/NO, importante controlador do tônus vascular. Nos astrócitos, as cavéolas são formadas por Cav-3 e sua superexpressão é associada a doenças neurológicas. O PNV aumentou significativamente os níveis basais de Cav-3 em astrócitos GFAP positivos (astrogliose reativa) em períodos de aumento de Cx43 (às 1, 5 e 24 h), e na vigência de edema citotóxico nos pés astrocitários e alterações nos contatos sinápticos axo-dendríticos e axo-somáticos. Em conjunto os resultados revelam que: (a) a quebra da via transcelular da BHE pelo PNV tem aumento da endocitose via cavéolas; (b) componentes da unidade neurovascular, como endotélio, astrócitos e neurônios estão intimamente envolvidos; (c) no endotélio, os efeitos são mediados pelo sistema eNOS/NO; (d) a SKF ativa o sistema endocítico e de transporte vesicular; (e) nos astrócitos, a dinamica expressão de Cx43 e Cav-3 e o retorno aos níveis basais em paralelo com a ausência de sinais de intoxicação nos animais (72 h) dá evidências de que ambas as proteínas interagem na resposta astrocitária. Os dados permitem sugerir que a presença de peptídeos neurotóxicos no veneno de Phoneutria nigriventer estão no centro dos efeitos aqui relatados / Abstract: In this work, we investigated the blood-brain barrier (BBB) permeabilization induced by Phoneutria nigriventer venom (PNV) in the transcellular route of rats¿ cerebellum. Caveolae was analyzed in endothelial cells accessing the expression of proteins involved in caveolae formation (caveolin-1, Cav-1 and dynamin-2, Dyn2) and internalization (phosphorylated Caveolin-1, pCav-1 and Src kinase family, SKF), in astrocytes caveolae role were evaluated with caveolin-3 (Cav-3) and connexin-43 (Cx43) (gap-junction main protein). PNV action on the endothelium was also investigated through activation (coupling) or inactivation (uncoupling) of eNOS enzyme, responsible for nitric oxide (NO) production. BBB components were evaluated using transmission electron microcopy. Initially, Cav-1 study addressed its localization along with Cav-1 protein and gene expression after envenoming in different age animals, neonate rats were more susceptible to envenoming than adult rats. After PNV, Cav-1 labeling was intense in granular and molecular layers and in Purkinje neurons. Cav-1 and Dyn2 (responsible for caveolae vesicles formation and scission, respectively) expression increased in periods of acute envenomation (1 h), recovery (5 h) and in the absence of clinical signals (24 h); in opposition SKF and pCav-1 involved in caveolae internalization were overexpress in opposite periods (at 2 h and 72 h). PNV induced increases in matrix metaloproteinases-9 (MMP9) an important BBB breakdown mediator, and increases in vesicles formation and traffic in the endothelium after envenoming. The study of eNOS activity revealed uncoupling (increasing in eNOS monomers) in acute periods after envenomation (1 h and 2 h) and progressive return followed by overexpression of dimers (re-coupling) at 72 h; those alterations were related to PNV action on calcium intracellular levels confirmed by Calmodulin increased expression and confirmed using Calbindin-D28 localization. Data revealed PNV interference on endothelial homeostasis and vascular function once affects the eNOS/NO system, an important vascular tonus controller. In astrocytes, caveolae are formed by Cav-3 and its overexpression is related to neurological disorders. PNV increased the basal levels of Cav-3 in GFAP-positive astrocytes (reactive astrogliosis) in the same periods as increased Cx43 (at 1, 5 e 24 h), during cytotoxic edema in astrocytes end-feet and alterations in axo-dendrites and axo-somatic synaptic contacts. Together, the results revealed that: (a) the BBB breakdown in transcellular route by PNV involves upregulation of caveolae endocytosis (b) the neurovascular unit components such as the endothelium, astrocytes and neurons are intimal involved (c) in the endothelium the effects are mediated by the eNOS/NO system and (d) SKF activates endocytic system and vesicular transport; (e) in the astrocytes, Cx43 and Cav-3 dynamic expression and their return to basal level in parallel with the absence of toxic signals in the animals (72 h) provides evidence that both protein interacts in astrocytes response. The data allows us to suggest that the neurotoxic peptides presented in Phoneutria nigriventer venom are in the center of the effects reported here / Mestrado / Biologia Tecidual / Mestra em Biologia Celular e Estrutural

1 Univerzita Karlova v Praze 1. lékařská fakulta Některé aspekty patofyziologie plicní arteriální hypertenze a její výskyt v České republice Some aspects of pathophysiology of pulmonary arterial hypertension and its epidemiology in the Czech Republic MUDr. Pavel Jansa Praha 2011 2 Abstract Pulmonary arterial hypertension (PAH) is a group of diseases characterized by a progressive increase of resistance and pressure in pulmonary vascular bed. In all types of PAH the same four pathological processes are reported: vasoconstriction, inflammation, thrombosis and remodelling. The genetic background is essential for the development of PAH. We aimed to investigate the role of polymorphisms of endothelial nitric oxide synthase (eNOS) genes in PAH. We studied 142 PAH patients and 189 healthy subjects. We examined 3 polymorphisms of the eNOS gene, including the Glu298Asp polymorphism, 27-base pair (bp) variable numbers of tandem repeats (VNTR) and -786 T/C promoter gene polymorphism. Prevalence of 27-bp VNTR allele A was higher in patients with PAH compared with healthy controls. Patients with PAH associated with connective tissue diseases had higher prevalence of AA genotype compared with other PAH subgroups. The Glu298Asp polymorphism and -786 T/C polymorphism are not associated with PAH. Thrombotic arteriopathy is...

Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...

Indiana University-Purdue University Indianapolis (IUPUI) / Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The epidemic of obesity and associated cardiovascular complications continues to grow at an alarming rate. Currently, obesity is thought to initiate a state of chronic inflammation, which if unresolved potentially causes cardiovascular dysfunction and disease. Although poorly understood, release of inflammatory mediators and other cytokines from adipose tissue (adipocytokines) has been proposed to be the molecular link between obesity and coronary artery disease. Furthermore, the anatomic location of adipose has been increasingly recognized as a potential contributor to vascular disease. Importantly, the development of coronary atherosclerosis, a key component of heart disease, is typically found in segments of coronary arteries surrounded by perivascular adipose tissue. Accordingly, the goal of this project was to determine how perivascular adipose tissue affects coronary artery function and elucidate the critical mechanisms involved. Initial studies assessing arterial function were conducted with and without perivascular adipose tissue. Preliminary results demonstrated that factors released by perivascular adipose tissue effectively impaired coronary endothelial function both in vitro and in vivo. This observation was determined to be caused by direct inhibition of nitric oxide synthase (NOS), a critical enzyme for the production nitric oxide. Attenuation of endothelium-dependent vasodilation was independent of changes in superoxide production, smooth muscle response, or peroxide-mediated vasodilation. Additional studies revealed that perivascular adipose-induced impairment of NOS was due to increased inhibitory regulation by the β isoform of protein kinase C (PKC-β). Specifically, perivascular adipose-derived factors caused site specific phosphorylation of nitric oxide synthase at Thr-495. Additional experiments investigated how perivascular adipose-derived factors contributed to coronary artery disease in an animal model of obesity. Results from these studies indicated that perivascular adipose-derived leptin markedly exacerbated underlying endothelial dysfunction, and significantly contributed to coronary endothelial dysfunction through a PKC-β dependent mechanism. Findings from this project confirm epicardial perivascular adipose tissue as a local source of harmful adipocytokines. In addition, perivascular adipose-derived leptin was demonstrated to be a critical mediator of coronary vascular dysfunction in obesity. Together, the results strongly suggest that perivascular adipose tissue is a key contributor to coronary artery disease in obesity.

No description available.