O glioblastoma multiforme (GBM) é o tumor mais agressivo do Sistema Nervoso Central (SNC) e caracteriza-se por um alto poder angiogênico que está diretamente relacionado com a capacidade invasiva e inversamente com o prognóstico. A angiogênese contribui para a malignidade por prover a oxigenação e o suprimento nutricional necessários para o crescimento e invasão do tumor. O GBM prolifera sob um ambiente de hipóxia e o fator induzido por hipóxia (HIF-1) apresenta um papel fundamental na ativação da transcrição de genes alvo que favorecem a angiogênese e evitam a morte celular. O fator de crescimento do endotélio vascular (VEGF) e o fator de crescimento derivado da plaqueta C (PDGF-C) são agentes de grande interesse na angiogênese tumoral e que podem ser modulados pelo HIF-1. Foi realizado tissue microarray(TMA) de 208 casos de GBM e estudados pelo método de imunoistoquímica os marcadores HIF-1, VEGF e PDGFC. Os resultados foram correlacionados com a angiogênese avaliada através da densidade microvascular (DMV) pelo CD34, CD105, VEGF e PDGF-C, com a proliferação celular endotelial através da marcação nuclear pelo KI-67 e também com a sobrevida. A expressão tumoral do HIF-1 foi observada em 184 casos (88,5%), a do VEGF em 131 (63%) e a do PDGF-C em 160 (77%). As DMVs medianas pelo CD34, PDGF-C, VEGF e CD105 foram, respectivamente, 20, 16, 5 e 6. Os GBMs com marcação positiva pelo HIF-1 tiveram uma DMV mediana pelo CD34 de 30, enquanto que nos negativos a DMV mediana foi 14 (p<0,001). A expressão tumoral positiva pelo HIF-1 teve correlação com a marcação tumoral pelo VEGF e PDGF-C (p<0,001). Houve também uma correlação entre a marcação do VEGF e do PDGF-C tanto no tumor (p=0,001) como na célula endotelial (p<0,001). A expressão do VEGF no tumor teve correlação com a sua expressão no vaso (p<0,001). Células endoteliais marcadas pelo PDGF-C e pelo VEGF também foram marcadas pelo CD105 e seus núcleos pelo KI-67 confirmando seu padrão neoangiogênico e proliferativo. A marcação nuclear das células tumorais pelo VEGF teve impacto na sobrevida (p=0,002), bem como as marcações nucleares pelo HIF-1 e VEGF (p=0,005). Em conclusão, este estudo mostrou que a expressão do HIF-1 está associada com as expressões do VEGF e do PDGF-C e que houve uma correlação desses marcadores com a angiogênese no GBM. / Glioblastoma multiforme (GBM) is the most aggressive tumour of the central nervous system (CNS) and is characterized by a high angiogenic power that is directly related to the invasiveness and inversely to the prognosis. Angiogenesis contributes to malignancy by providing the necessary oxygenation and nutritional supplement for the tumour\'s growth and invasion. GBM proliferates under a hypoxia environment and the hypoxia-inducible factor (HIF-1) plays a key role on the transcription of essential genes which promote angiogenesis and prevent cellular death. The vascular endothelial growth factor (VEGF) and the platelet-derived growth factor C (PDGF-C) are agents of great interest on tumour angiogenesis and may be modulated by the HIF-1. Tissue micro-array (TMA) was conducted on 208 cases of GBM and the HIF-1, VEGF and PDGF-C markers were studied under immunohistochemistry method. The results were correlated with angiogenesis assessed by micro-vessel density (MVD) for CD34, CD105, VEGF and PDGF-C, with endothelial cell proliferation by nuclear staining for KI-67, and also with survival. The tumour expression by HIF-1 was observed in 184 cases (88.5%), by VEGF in 131 (63%) and by PDGF-C in 160 (77%). The median MVD for CD34, PDGF-C, VEGF and CD105 was respectively 20, 16, 5 and 6.The GBMs which stained positive for HIF-1 showed a median MVD of 30 for CD34, whereas in negative ones, median MVD was 14 (p <0.001). The positive tumour expression for HIF-1 was correlated with tumour markings for VEGF and PDGF-C (p <0.001). There was also a correlation between the markings for VEGF and PDGF-C in both the tumour (p = 0.001) and endothelial cell (p <0.001). VEGF expression in the tumour was correlated with its expression in the blood vessel (p <0.001). Endothelial cells marked for the PDGF-C and VEGF were also marked for CD105 and their nuclei for KI-67 confirming its neoangiogenic and proliferative pattern. The nuclear staining on tumour cells for VEGF had an impact on survival (p = 0.002), as well as the cases with nuclear staining for HIF-1 and VEGF (p = 0.005). In conclusion, this study showed that HIF-1 expression is associated with VEGF and PDGF-C and correlated with angiogenesis on GBM.

A heterogeneidade intratumoral é um fenômeno extremamente importante para entender a progressão tumoral e a resposta à intervenção terapêutica. A galectina-3 pertence à família das lectinas, possuem a função de reconhecimento e ligação à ?-galactosídeos ramificados de glicolipídeos e glicoproteínas, e está envolvida em processos fisiológicos e patológicos como o câncer. Nesse trabalho, a heterogeneidade intratumoral em relação à expressão de galectina-3 foi observada em amostras de diferentes lesões melanocíticas de pacientes. Além disso, o inóculo de células de melanoma murino negativas para galectina-3 em animais gal3-/- gerou tumores constituídos por uma fração de células tumorais que passaram a expressar de novo galectina-3, sugerindo que pressões do microambiente tumoral modulam a expressão dessa lectina em melanomas. A acidose extracelular atuou como regulador negativo de galectina-3 in vitro, diminuindo a expressão dessa lectina tanto em células de melanoma murino e humano quanto em melanócito murino. Entretanto, a hipóxia, seja pela exposição aguda ou intermitente, não alterou a expressão in vitro de galectina-3 em células de melanoma humano. Por fim, tumores originados pelo inóculo de células tumorais positivas e negativas para galectina-3 (mimetizando tumores heterogêneos) obtiveram a maior taxa de crescimento tumoral comparados aos tumores constituídos por uma única população de células, seja positiva ou negativa para galectina-3. Portanto, foram apresentadas evidências de que a heterogeneidade intratumoral em relação à galectina-3 parece estar envolvida com o sucesso evolutivo do melanoma e que a acidose é indicada como uma das pressões microambientais que contribuem para o estabelecimento e manutenção da fração de células tumorais negativas para galectina-3 dentro da massa tumoral / The intratumoral heterogeneity observed in human tumors is extremely important to understand tumor progression and its therapeutic response. Galectin-3 belongs to animal lectin family and it is a ?-galactosidase binding protein which is involved in physiological and pathological processes, including cancer. In this work, an intratumor heterogeneous galectin-3 expression was observed in tissue sessions containing different human melanocytic lesions. Moreover, negative galectin-3 murine cells injected into gal3-/- mice were able to generate tumors composed of a positive galectin-3 cell fraction, suggesting that selective forces in tumor microenvironment modulate galectin-3 expression in melanoma. Extracellular acidosis acts as a negative regulator to galectin-3 in vitro, decreasing its expression in murine and human melanoma cells and even in murine melanocytes. However, intermittent or acute hypoxia exposure did not alter galectin-3 expression in human melanoma cells in vitro. In addition, tumors originated from a mixture of positive and negative galectin-3 cells (mimicking heterogeneous tumors) showed higher growth rate compared to those derived from only galectin-3 positive or negative cells. Therefore, we showed evidences that galectin-3 intratumoral heterogeneity seems to be involved with the evolutionary success of melanoma and that acidosis may be the microenvironmental pressure responsible for the establishment and maintenance of galectin-3 negative cell fraction into the tumor bulk

A hipóxia mantida de curta duração (HM) está associada a alterações cardiorrespiratórias e ao desencadeamento de processo inflamatório em humanos e modelos experimentais. Ademais, há evidências de que a HM pode alterar a transmissão sináptica na região do Núcleo do Trato Solitários (NTS). No presente estudo, utilizamos a minociclina, um inibidor da ativação microglial e antiinflamatório, para avaliar a influência da inflamação desencadeada pela HM sobre a neurotransmissão glutamatérgica nos neurônios do NTS que enviam projeções para a região ventrolateral da medula (NTS-VLM). A hipótese geral do nosso estudo foi a seguinte: a HM induz processo inflamatório no tronco encefálico, o qual contribui para o aumento da neurotransmissão glutamatérgica em neurônios NTS-VLM, colaborando para a elevação da pressão arterial média (PAM) observada nestes ratos. Embora tenhamos observado aumento da pressão arterial média em ambos os grupos de ratos tratados com veículo (solução salina + água destilada, ip) ou minociclina [(30mg/Kg ip por 3 dias) submetidos a 24h de HM (FiO2 0.1) em relação aos seus respectivos grupos controle (FiO2 0,28), o aumento da MAP foi menor nos ratos previamente tratados com minociclina. Os registros eletrofisiológicos utilizando a técnica de whole cell patch-clamp mostraram que a HM não produziu alterações nas propriedades ativas e passivas dos neurônios NTS-VLM. No entanto, os neurônios de ratos submetidos a HM apresentaram aumento nas correntes glutamatérgicas espontâneas e evocadas pelo estímulo do trato solitário. Esse grupo de animais também apresentou aumento no número de microgliais na região do NTS. As alterações mencionadas foram atenuadas pelo tratamento prévio com minociclina. Concluímos que a inflamação induzida pela HM contribui para o aumento da neurotransmissão glutamatérgica nos neurônios NTS-VLM o qual poderia estar relacionado com a hipertensão arterial observada nestes ratos. / Short-term Sustained hypoxia (SH) is associated with cardiorespiratory changes and inflammatory process in humans and experimental models. There is also evidence that SH can change the synaptic transmission in the nucleus tractus solitarius (NTS) region. Here we use the minocycline, an anti-inflammatory and microglial inhibitor, to evaluate the role of inflammation triggered by SH on the excitatory neurotransmission in the NTS neurons sending projections to the ventrolateral medulla (NTS-VLM). We hypothesized that SH induces brainstem inflammatory process, which may contribute to increase in excitatory neurotransmission and excitability of the NTS-VLM neurons, collaborating to the high blood pressure observed on these rats. Although we have observed increased MAP in both groups of rats treated with vehicle (saline + distilled water, i.p) or minociclina [(30mg/Kg i.p for 3 days) submitted to 24h of SH (FiO2 0.1) in relation to their respective control groups (FiO2 0.28), the MAP increase was lower in rats treated with minociclina. The whole cell patch-clamp recordings showed that SH produced no changes in active properties of NTS neurons. However, neurons of rats submitted SH presented an increase in the glutamatergic neurotransmission and the number of microglial at the NTS region. These increases were prevented in the groups previously treated with minociclina. We conclude that inflammation induced by SH contributes to the increased excitatory neurotransmission in NTS-VLM neurons that could be associated to high blood pressure observed in these rats.

The pulmonary vein (PV) constricts to hypoxia however little is known about the underlying mechanisms. Hypoxic PV constriction is proposed to recruit upstream capillary beds and optimise gas exchange in healthy humans and may play a role in high altitude pulmonary oedema. The PV is also intrinsic to disease states including pulmonary hypertension and pulmonary veno-occlusive disease. Blood vessel culture can be a powerful tool to enable assessment of the impact of environmental factors on vessel function and as a disease model. However culture conditions alone affect vessel contractility; the effect of culture conditions on PV function remained to be established. The aim of this project was to investigate hypoxic responses of porcine PVs including the impact of maintenance in culture. Maintenance of PVs in culture conditions for 24 hours increased contraction to hypoxia and inhibited hypoxic relaxation post-contraction. These changes to PV hypoxic responses were thought to result from endothelial dysfunction. However, the endothelial nitric oxide synthase inhibitor L-NAME inhibited PV hypoxic contraction and enhanced relaxation. The impact of K+ channel inhibitors on hypoxic contraction was also investigated. Penitrem A, 4AP, DPO-1, ZnCl2 and glyburide had no significant effect however TEA and BDM inhibited the hypoxic contraction. This suggested that TASK, KV1.5, BKCa and KATP do not play a role in the mechanism of hypoxic pulmonary venoconstriction however KV channels containing KV2.1 α subunits may modulate the response. Results with L-NAME suggested endothelial dysfunction may not fully account for the change in PV function after exposure to culture. Therefore the impact of PV maintenance in culture was further explored using an isolated PV smooth muscle cell (PVSMC) model. Maintenance of PVs in culture conditions had minimal impact on morphology and electrical properties of PVSMCs. Notably, resting membrane potential and hypoxia-induced depolarisation were not significantly different. Based on the findings of this study, the endothelium in PVs appears to a) play a major role in modulation of the hypoxic response b) be sensitive to short-term exposure to culture conditions. K+ channels appear to play a minor role in PV hypoxic contraction and SMCs isolated from PVs maintained in culture conditions have similar morphological and electrophysiological characteristics to freshly isolated PVSMCs. Taking all this into account, endothelial regulation of contractility should be a key focus for future PV research.

A qualidade de sementes é fundamental para as decisões para a produção agrícola e as formas para determinar a qualidade de sementes requerem inúmeros estudos visando à semeadura. Adicionalmente é essencial que haja condições ideais de armazenamento até que as sementes sejam utilizadas estabelecidas em função da espécie e da temperatura e a umidade relativa do ar, e que são fundamentais para a manutenção do parâmetro fisiológico das sementes. Alguns pesquisadores têm afirmado que outros fatores podem contribuir significativamente para a deterioração das sementes durante o armazenamento, tal como o oxigênio. Dessa forma, nessa pesquisa foi avaliada a ação do oxigênio na deterioração das sementes de arroz durante o armazenamento. Num primeiro momento, a qualidade das sementes de arroz foi avaliada com a aplicação de técnicas multivariadas em contraste com a análise univariada, para classificar as sementes quanto ao parâmetro fisiológico. Em seguida, as sementes foram testadas quanto à qualidade ao serem submetidas ao aumento da pressão da atmosfera com o intuito de aumentar a interferência do oxigênio na qualidade das sementes. Complementarmente, foi avaliada a utilização de embalagem com aplicação de vácuo, para verificar a interferência na conservação dessas sementes. Há possibilidade de utilização da análise estatística multivariada para classificar as sementes de arroz quanto à qualidade. O oxigênio reduz a qualidade das sementes de arroz durante o armazenamento em condições em que há a redução da temperatura e da umidade relativa do ar. A embalagem com uso de vácuo é uma alternativa viável para reduzir a deterioração das sementes de arroz. / Seed quality is fundamental for decision for agricultural production. Ways to determine seed quality are targets of numerous studies to identify the quality of the lots for sowing. Once identified the quality of seeds, secured storage ideal conditions should be proceeded until the seeds are ready for use. Ideal conditions for seed storage are depending on the species, as temperature and air relative humidity, important for the maintenance of the physiological quality of seeds. Some researchers have raised the hypothesis that more factors can significantly contribute to the deterioration of seeds, such as oxygen. Through the chapters of this thesis sought to evaluate the oxygen action in the deterioration of rice seeds during storage. At first it evaluated the quality of the seeds with the use of multivariate techniques in contrast to the univariate analysis to classify seeds as the physiological. Then seeds were tested for quality being subjected to high-pressure atmosphere in order to enhance the effects of oxygen on the seed deterioration. Finally, an additional chapter discusses the use of application of vacuum packaging to verify the effect on conservation. It is concluded that the multivariate statistical analysis is a potential tool to classify rice seeds as quality. Oxygen reduces the quality of rice seeds during storage under conditions of lower temperature and relative humidity and vacuum packing is a good alternative to decrease seed deterioration.

Objetivos: Avaliar as alterações retinianas em modelo experimental de hipóxia em coelhos aclimatizados em ambiente hipóxico-normobárico e investigar os efeitos do tratamento com bevacizumabe intravítreo (IV). Métodos: Vinte e dois coelhos New Zealand, com pesos entre 2,4 a 3,8 kg, foram divididos em quatro grupos. Os grupos S12% (n=5) e B12% (n=5) foram aclimatizados durante três dias consecutivos em concentrações de oxigênio (O2) a 12%. Os grupos S8% (n=5) e B8% (n=7) foram aclimatizados durante três dias consecutivos, com reduções graduais da concentração de O2, até atingir o nadir de 8%. Os olhos direitos (OD) foram mantidos como controle e os olhos esquerdos (OE) dos animais dos grupos S12% e S8% receberam injeção IV de 0,05 ml de solução salina balanceada (SSB), enquanto os OE dos grupos B12% e B8% receberam 0,05 ml (1,25 mg) de bevacizumabe IV. Foram realizados exames de tomografia de coerência óptica (OCT) para avaliação da espessura dos segmentos retinianos (SR) e coroidianos (SC), angiografia por fluoresceína sódica (AF) para observação da presença ou ausência de vasodilatação e tortuosidade da vasculatura retiniana e quantificação do Vascular Endothelial Growth Factor (VEGF) do humor aquoso e soro no primeiro dia (D0), antes do tratamento, no terceiro dia de hipóxia (D7) e no décimo primeiro dia (D11), utilizando-se a técnica do Luminex®. Após, os animais foram sacrificados e amostras do tecido retiniano foram avaliadas por histologia e imuno-histoquímica (IHQ). Resultados: Comparando-se os cortes horizontais dos OD (controle) nos períodos D0 e D7, notou-se redução de 8% (p<0,0001) e 10% (p<0,0001) da espessura do SR nas concentrações de O2 a 12% e 8%, respectivamente. Comparando-se os cortes verticais nos mesmos períodos, verificou-se redução da espessura do SR de 7%, tanto nas concentrações a 12% (p<0,0001) como a 8% (p<0,0001). Nos olhos tratados com bevacizumabe, a redução das médias das espessuras do SR para os cortes horizontais entre os períodos D0 e D7 foi de 6% (<0,0001) e 9% (<0,0001), para as concentrações de O2 a 12 e 8%, respectivamente. Enquanto que nos olhos tratados com SSB no mesmo período, observou-se redução de 8% (<0,0001) e 6% (<0,0001) para as concentrações de O2 a 12 e 8%, respectivamente, nos cortes horizontais. Nos cortes verticais, para os olhos tratados com bevacizumabe, houve redução de 5% (p=0,0005) e 8% (<0,0001) para concentrações de O2 a 12% e 8%, respectivamente; e para os olhos tratados com SSB foi encontrada redução de 7% (<0,0001) e 8% (<0,0001) nas concentrações de O2 a 12% e 8%, respectivamente. As espessuras dos SC não apresentaram alterações. O grupo B8 apresentou diferença estatisticamente significativa na análise da proporção dos olhos que não evidenciaram vasodilatação e tortuosidade dos vasos retinianos durante o período hipoxêmico, e não foram observados neovasos retinianos. A histologia e IHQ dos olhos tratados com SSB e bevacizumabe não demonstraram alterações quando comparados com os controles. Conclusões: A aclimatização de coelhos em ambiente hipóxico-normobárico resultou na redução da espessura do SR no terceiro dia de hipóxia. Notou-se, ainda, aumento da tortuosidade e vasodilatação. O bevacizumabe IV não inibiu a redução da espessura retiniana, mas sim a vasodilatação e tortuosidade vascular. / Objectives: Evaluate retinal changes in experimental model of hypoxia in rabbits acclimatized in normobaric-hypoxic environment and to investigate the effects of the treatment by intravitreal (IV) bevacizumab drug. Methods: Twenty two New Zealand rabbits weighing between 2,4 to 3,8 kg were divided into 4 groups. The groups S12 (n=5) and B12 (n=5) were acclimatized for 3 consecutive days in oxygen concentration (O2) to 12%. The groups S8 (n=5) and B8 (n=7) were acclimatized for 3 consecutive days with gradual reductions in O2 concentration until the nadir of 8%. The right eye (RE) were kept as controls and the left eye (LE) of the animals belonging to S12 and S8 groups received IV injection of 0,05 ml of balanced salt solution (BSS), while the LE belonging to groups B12 and B8 received 0,05 ml (1,25 mg) of bevacizumab IV. Optical coherence tomography (OCT) to evaluate the thickness of the retinal segments (RS) and choroidalsegments (CS), sodium fluorescein angiography (FA) for evaluation of the presence or absence of vasodilation and tortuosity of the retinal vasculature and quantification of VEGF in the aqueous fluid and peripheral blood sample were conducted at the first day (D0) before treatment, on the third day of hypoxia (D7) and day 11 (D11) using the Luminex® technique. After the animals were sacrificed, the retinal tissue samples were evaluated by histology and immunohistochemistry (IHC). Results: Comparing the horizontal sections of the RE (control) in D0 and D7 periods, a reduction of 8% (p<0,0001) and 10% (p<0,0001) the thickness of the RS in O2 concentration at 12% and 8%, respectively. Comparing the vertical cuts in the same period, there was reduced RS thickness of 7% in both concentrations to 12% (p<0,0001) and 8% (p<0,0001). In the eyes treated with bevacizumab, to reduce the average thickness of the retinal segment for horizontal cuts between D0 and D7 periods were 6% (<0,0001) and 9% (<0,0001) for O2 concentrations to 12 and 8%, respectively. While in the eyes treated with BSS in the same period, there was an 8% reduction (<0,0001) and 6% (<0,0001) for the O2 concentration at the 12% and 8%, respectively, in the horizontal cuts. In the vertical sections is observed for the eyes treated with bevacizumab, 5% reduction (p=0.0005) and 8% (<0,0001) O2 concentration at 12% and 8%, respectively; and BSS treated eyes was reduced by 7% (<0,0001) and 8% (<0,0001) in the O2 concentrations of 12% and 8%, respectively. The thickness of the CS did not show changes. The B8 group showed statistical difference in the analysis of the eyes that did not have vasodilation and tortuosity of the retinal vessels during the hypoxic period. Retinal neovascularization were not observed. Histology and IHC of the eyes treated with BSS and bevacizumab showed no changes compared to the control eyes. Conclusions: The acclimatization of the rabbits in normobaric-hypoxic environment has the effect of reducing the thickness RS on the third day of hypoxia. It is observed also increased tortuosity and vasodilation. The intravitreal bevacizumab does not inhibit retinal thickness decrease, but inhibits vasodilation and vascular tortuosity.

Foram avaliadas as características hematológicas, mais especificamente a resposta dos elementos de proteção do sangue, de juvenis de pacu, espécie endêmica e neotropical, mantidos em condições adversas e alimentados com dietas contendo colostro bovino liofilizado. Esta primeira secreção láctea, rica fonte de proteínas, moléculas biologicamente ativas e elementos antioxidantes, pode atuar positivamente no sistema de proteção dos animais. No experimento de estresse crônico, juvenis de pacu foram alocados em elevada densidade (50 kg peixe/m3) por 30 dias. No experimento de estresse agudo, os juvenis permaneceram por 15 dias em gaiolas de alimentação, sendo submetidos à baixa concentração de oxigênio (0,75 mg/L). Durante o período experimental, quatro dietas contendo 0, 10, 20 e 30% de colostro bovino liofilizado (CBL) (com 32% de proteína bruta), foram fornecidas duas vezes diariamente (considerando-se quadruplicatas para cada dieta). Antes do início e ao final de cada experimento, analisou-se para cada lote os parâmetros biométricos de biomassa e comprimento para obtenção de análises de desempenho. Oito juvenis para a situação experimental de adensamento, e dez juvenis para a de hipóxia, de cada tratamento, foram anestesiados com benzocaína e amostras de sangue foram coletadas do vaso caudal para análises hematológicas. Foram realizadas análises de eritrograma, leucograma (contagem total e diferencial de leucócitos), hematócrito e calculados os índices hematimétricos. As imunoglobulinas séricas dos juvenis de pacu foram quantificadas através do método de Turvação por Sulfato de Zinco (ZST), que foi padronizado para peixes no presente trabalho. Os juvenis de pacu foram distribuídos em um delineamento experimental inteiramente casualizado no experimento de estresse crônico, e para o de estresse agudo, em blocos, ambos com quatro tratamentos. Os dados foram submetidos à análise de variância, através do procedimento \"General Linear Model\" (PROC GLM) do programa estatístico SAS (1989). Para a avaliação de diferenças entre médias foram efetuados contrastes entre pares de médias utilizando-se o método de Tukey, onde foi considerada a probabilidade de 5% (P<0,05). Não foi observada influência do colostro bovino no desempenho e nas variáveis hematológicas analisadas em ambas situações experimentais, estresse crônico e agudo. Entretanto, para o experimento de estresse agudo, a inclusão de CBL na dieta dos pacus influenciou o número de células granulocíticas especiais, sendo que o grupo que recebeu 0% de CBL apresentou menor número de células que o grupo que recebeu 10% de CBL, assim como o número de monócitos, sendo que o grupo que recebeu 20% de CBL apresentou menor número de células que o grupo que recebeu 30% de CBL na dieta (P<0,05). Embora o colostro bovino não tenha influenciado as variáveis hematológicas analisadas, não houve efeito negativo à esta fonte de proteína heteróloga fornecida aos peixes, uma vez que o desempenho dos animais que receberam o composto na dieta foi semelhante aos animais que receberam dieta sem esta secreção láctea. Considerando os presentes resultados, o colostro bovino liofilizado, uma fonte rica de moléculas bioativas, não contribuiu para a proteção de juvenis de pacu sujeitos à alta densidade de estocagem e baixa oxigenação. / Hematological characteristics were evaluated, specifically the response of blood protection elements of juvenile pacu, endemic and neotropical species, kept under adverse conditions and fed diets containing lyophilized bovine colostrum. This first milk secretion, a rich protein source, biologically active molecules and antioxidant elements, can act positively in the animal protection system. In the chronic stress experiment, juvenile pacu were allocated at high density (50 kg fish/m3) for 30 days. In the acute stress experiment, the juveniles remained for 15 days in feed cages and were submitted to low oxygen concentration (0.75 mg/L). During the experimental period, four diets containing 0, 10, 20 and 30% of lyophilized bovine colostrum (CBL) (with 32% crude protein) were given twice daily (considering quadruplicates for each diet). Before the beginning and the end of each experiment, the biometric parameters of biomass and length were analyzed in each batch for performance analyses. Eight juveniles for the experimental situation of densification and ten juveniles for hypoxia of each treatment were anesthetized with benzocaine and blood samples were collected from the caudal vein for the hematological analysis. Erythrogram, leukogram (total and differential counts of leukocytes), hematocrit and hematimetric indexes were performed. The serum immunoglobulins of the pacu juveniles were quantified by the Zinc Sulfate Turbid (ZST) method, which was standardized for fish in this study. Juveniles pacu were distributed in a completely randomized experimental design for cronic stress experiment, and for acute stress in randomized blocks, both with four treatments. Data were submitted to the \"General Linear Model\"procedure (PROC GLM) of the statistical program SAS (1989). For a mean-to-average assessment, contrasts were performed between pairs of means using the Tukey method, where a probability of 5% (P <0.05) was considered No influence of bovine colostrum was observed on performance and hematological variables analyzed in both experimental situations, chronic and acute stress. However, for the experimental situation of acute stress, the addition of CBL to the diet of pacu influenced the number of special granulocytic cells, and the group that received 0% CBL presented lower number of cells than the group that received 10% CBL, as well as the number of monocytes. The group that received 20% of CBL presented lower number of monocytes than the group that received 30% of CBL in the diet (P <0.05). Although bovine colostrum did not influence the hematological variables analyzed, there was no negative effect on this source of heterologous protein supplied to the fish, since the performance of the animals that received the compound in the diet was similar to the animals that received diet without this milk secretion. Considering theresults, lyophilized bovine colostrum, a rich source of bioactive molecules, does not contribute to the protection of juvenile pacu subjected to high stocking density and low oxygenation.

Obstructive sleep apnea (OSA) is a common breathing and sleeping disorder, characterized by repeated episodes of airway obstruction during sleep resulting in intermittent hypoxia (IH). From clinical reports, patients with OSA are associated with behavioral and neuropsychological deficits, including impaired spatial learning memory and cognitive deficiencies. Previous studies proposed that reactive oxygen species (ROS) and apoptosis caused by intermittent hypoxia (IH) contributed to this cognitive deficits. However, the exact mechanism is still poorly understood and not settled. / The endoplasmic reticulum (ER) is a cellular organelle in which all secretory and integral membrane proteins are folded and is also the site where proteins are post-translationally modified in ATP-dependent chaperone-mediated processes. In this study, we hypothesized that ER stress in the hippocampus is initiated in the OSA via elevated levels of ROS. Four groups of adult male mice were used, with two of them exposed to normoxia as control, and the other two exposed to IH treatment, each receiving either vehicle or tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. Eight-armed radial maze was used to investigate the performance of reference memory during the whole IH/normoxia treatment. After behavior test, long-term potentiation (LTP) was measured to investigate synaptic plasticity in hippocampus. Furthermore, ER stress-associated pro-apoptotic effectors were detected by Western blotting, and ultra-structure of rough ER and the morphology of hippocampal dendritic spines and synapses in the hippocampal CA1 area were observed. / LTP was impaired in the hippocampus after IH treatment, which was rescued by TUDCA. Furthermore, ER stress-associated pro-apoptotic effectors, CHOP and caspase-12, were up-regulated after chronic IH treatment and was abolished by co-infusion of TUDCA. Meanwhile, increased cleaved-caspase-3 after chronic IH treatment was reduced by TUDCA via increased expression of Bcl-2. On the other hand, ultrastructural analysis of rough ER in the hippocampal CA1 revealed IH-induced ER luminal swelling, and was attenuated by TUDCA. In addition, the length of synaptic active zone was significantly reduced after chronic IH treatment and was partially rescued by the application of TUDCA. Golgi staining also showed a decrease in mature dendritic spines in IH group, and reversed by TUDCA. In behavioral analysis, the number of reference memory errors significantly increased after IH treatment and rescued by TUDCA injection. Overall, the data suggest a critical role of ER stress underlying the impairment of long-term synaptic plasticity and neurocognitive deficits in chronic IH. Targeting ER stress could be a potential therapeutic strategy for neural dysfunction in OSA. / On the other hand, neuronal firing, especially robust persistent activity of neuron in hippocampus, is critical role in memory formation. Increased ROS induced by IH has been implicated in long-term potentiation of neural activity. IH could be involved in a variety of K⁺ channels which eventually leads to excitotoxicity by increased Ca2⁺-dependent glutamate release. Although the results were just shown in acute IH treatment, the chronic effect of IH on the firing frequency of hippocampus is still unknown. / Therefore, to investigate the effect of chronic IH treatment on firing activities and local field potentials of hippocampal neurons, implantation of multi-channel micro-wires electrode array into hippocampus of OSA model rat was performed to monitor spontaneous discharge. The results were shown the firing frequency of pyramidal neurons (PNs) was significantly elevated after 8 hours IH in second and third days, on the other hand, interneurons (INs) seem to be more sensitive to intermittent hypoxia since the higher firing frequency was sustained from third day to seventh day after 8 hours IH, however, at the end of 14 days IH treatment, the firing frequencies of PNs and INs are all both dramatically reduced. Meanwhile, the results in this part will enable us to understand the exact change of firing pattern and local field potential during intermittent hypoxia. The percentage of complex burst spikes was decreased after 14 days IH in PNs and the power of theta rhythms was also impaired. It suggests that the disorder of neuronal pattern and the change of local field potential are associated with cognitive impairment in OSA model. After 1 week recovery, the firing frequency of PNs was rescued again, but not for that of INs. We also found that the power of theta rhythms which had an important role in memory formation was weaker after 2 weeks IH treatment, however, the precise mechanism was still unknown. From the effect of intermittent hypoxia on spontaneous discharges and LFP of hippocampal neurons in free moving rat, it may reveal some roles of IH in cognitive impairment via disorder neuronal function in CA1 region. / 阻塞性睡眠呼吸暫停(OSA) 是一種常見的睡眠障礙疾病，這種疾病的主要特徵是在睡眠過程中反復發作的氣道阻塞，從而導致间歇性缺氧(IH)。從臨床報導中發現，OSA患者表現出行為和神經心理缺陷，包括空間學習記憶的受損和認知缺陷。通過之前的研究表明，活性氧(ROS)的增多和細胞凋亡是間歇性缺氧所引起認知功能障礙的主要機制之一，然而，其具體的機制仍不清楚。 / 作為細胞重要的細胞器，內質網是分泌蛋白和膜蛋白折疊組裝的主要場所，同時，由ATP依賴的分子伴侶所介導的蛋白質翻譯後修飾這一過程也主要在內置網中完成。在本課題中，我們假設在OSA模型的海馬組織中，內質網應激的啟動是由於缺氧引起的與活性氧(ROS)的升高。在本課題中，我們使用了四組成年雄性小鼠，其中兩組作為正常對照組，分別接受生理鹽水和牛磺去氧膽酸（一種常用的內質網抑制劑）的腹腔注射，另外兩組接受缺氧處理，同時也分別接受照生理鹽水和牛磺去氧膽酸注射。八臂放射迷宮被用來研究參考記憶的表現。行為學結束之後，長時程增強(LTP)用來測定海馬的突觸可塑性。用免疫印跡的方法檢測內質網應激的相關凋亡蛋白的表達情況，並且觀察海馬CA1區域中，內質網超微結構和海馬樹突棘數目及突觸形態的變化。 / 從實驗結果中，LTP在缺氧後減弱，而TUDCA能夠部分恢復由於缺氧所導致的LTP的降低。除此之外，內質網應激相關的促凋亡蛋白(CHOP和caspase-12)在缺氧組中表達升高，但是在TUDCA組中有所減低，同時，我們還發現，TUDCA也能夠減低缺氧組中cleaved-caspase-3的表達，而這一作用，可能與提高Bcl-2蛋白的表達（一個可標記的抗凋亡蛋白）有關。在間歇性缺氧組的海馬CA1區域中，粗面內質網出現管腔的腫脹，這一超微結構的變化表明在內質網出現官腔中有的許多未折疊蛋白聚集，並通過TUDCA的注射能夠降解未折疊蛋白來緩解這一現象的發生。同時，在IH處理後，突觸超微結構也發生了形態上的變化。突觸活性區的長度在IH處理組中顯著減少，但是在TUDCA組中有一定程度的恢復。高爾基染色顯示，成熟樹突棘（海馬突觸可塑性的結構基礎）的數目在間歇性缺氧組中有所下降，而在TUDCA治療後，成熟樹突棘的數目有所上升。我們發現參考記憶錯誤次數在缺氧後都有明顯的升高，而在注射TUDCA後，參考記憶錯誤次數都有所降低。總之，這些結果證明，內質網應激在間歇性缺氧的所引起的長時程突觸可塑性減弱和神經認知功能的損傷起到關鍵的作用，而抑制內質網應激對OSA中的出現神經功能紊亂起到一定的預防和治療效果。 / 而另一方面，神經元的放電，特別是海馬中神經元穩定持久的放電形式，對記憶的形成起到關鍵的作用。間歇性缺氧所引起的ROS的升高對於長時程增強的神經活動存在一定的關係，因為，通過以往的研究發現，間歇性缺氧可以通過多種鉀離子通道的啟動，最終由於鈣離子依賴的谷氨酸釋放的增多从而導致興奮性毒性的神經遞質的釋放。而這些結果只在急性缺氧模型中發現，慢性的間歇性缺氧對海馬的放電頻率的影響仍是未知之數。 / 因此，為了探討長時程的間歇性缺氧對海馬神經元的放電頻率和局部場電位的影響，多管道微絲電極陣列植入OSA大鼠的海馬中來監控自發放電的影響。結果表明，錐體細胞的放電頻率在第二天和第三天的8小時的間歇性低氧後明顯的升高了。另一方面，我們觀察到中間神經元似乎對間歇性缺氧更敏感，因為，從第三天到第七天缺氧8小時後，神經元的放電頻率都明顯的增高。但是在間歇性缺氧14天后，錐體細胞和中間神經元的放電頻率都所有顯著性的減少。同時，這一部分結果準確表明了海馬神經元的放電模式和局部場電位在間歇性缺氧的模型的是如何變化的。我們發現錐體細胞所具有的複合簇狀放電的比例減少，同時，theta波(與記憶的形成有關)的能量也有所減低。而這種神經元活動和局部的場電位的異常變化可能與OSA模型中出現的總認知功能障礙有關。在恢復一周後，錐體細胞的放電頻率有所增加，基本上可以恢復到缺氧前的狀態，但是中間神經元的頻率卻沒有多大的改變, 但是，其具體機制仍不清楚。從間歇性缺氧對大鼠海馬神經元自發放電和場電位影響的結果，它揭示了間歇性缺氧通過擾亂海馬CA1區域神經元的功能從而導致認知功能損傷。 / Xu, Linhao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 167-199). / Abstracts also in Chinese. / Title from PDF title page (viewed on 03, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

L’obtention d’un nombre élevé de cellules souches hématopoïétiques (CSH) représente un enjeu majeur pour le développement de protocoles de thérapies cellulaires d’hémopathies ou de tumeurs solides. L’expansion ex vivo de ces cellules met en jeu différents acteurs (cytokiniques, environnementaux) et notamment les basses concentrations d’oxygène (O2), qui reflètent des conditions physiologiques retrouvées au sein de structures spécifiques de la moelle osseuse où résident les CSH et auxquelles notre équipe s’intéresse depuis plusieurs années. Les effets bénéfiques de ces basses concentrations d’O2 sur le maintien des CSH sont actuellement bien établis lors de courtes cultures de cellules de moelle osseuse, de sang placentaire ou mobilisées dans le sang. Nous avons cherché à confirmer et à étendre ces résultats à des cellules peu étudiées, les cellules souches de sang périphérique en situation d’homéostasie (CSSP-H). Ces cellules représentent en effet une source possible de CSH à usage thérapeutique, du fait de leur disponibilité et de leur facilité d’accès. Nos travaux ont permis d’établir et d’optimiser un protocole, rapide et simple, de purification de cellules CD34+ à partir de filtres de leucoréduction (LRF). La quantité et la pureté de ces cellules adaptées à la poursuite de nos travaux, ainsi que leur validation fonctionnelle, nous ont permis de les utiliser comme modèle pour l’étude des effets de cultures de 7 jours très faiblement oxygénées (0,1% d’O2). La détermination de combinaisons cytokiniques assurant le maintien et l’expansion des cellules primitives a révélé un rôle bénéfique de l’IL-3 et du SCF couplé à la TPO. Ces conditions de culture ont permis de révéler, comparativement à des cultures réalisées à 20% d’O2, le rôle majeur des faibles concentrations d’O2 dans le maintien de cellules quiescentes, indifférenciées, ne se divisant pas ou très peu et capables de reconstituer une hématopoïèse, suite à leur injection dans des souris NOG. Les mécanismes moléculaires et métaboliques intervenant dans ces processus restent, cependant, encore à établir. / Obtaining a high number of hematopoietic stem cells (HSCs) is a major challenge for developing cell therapies for blood diseases. Ex vivo expansion of HSCs involves various factors (cytokines, environment), including low oxygen (O2) concentrations, that reflect the physiological conditions found in specific structures of the bone marrow where HSCs reside. Our team is interested with the study of these low O2 levels for several years and their beneficial effects are currently well established during short-term cultures of cells from bone marrow, cord blood or mobilized in the blood. We sought to confirm and extend these results to poorly studied cells: stem cells from steady state peripheral blood (SSPB). Indeed, these cells represent a possible HSCs source devoted to the therapeutic use, because of their availability and their easy access. Our work has led to the establishment and the optimisation of a procedure, rapid and easy to set up, for CD34+ cells purification from leukoreduction filters (LRFs). The cell quantities and purities, adapted to our further work, together with their functional validation, led us to use these cells as a model for 7-days in vitro cultures performed at very low O2 concentration (0.1%). Cytokine combination assays, allowing the maintenance and the expansion of primitive cells, have revealed a beneficial influence of IL-3 or SCF + TPO additions. These cultures have revealed, comparatively to those performed at 20% O2, a major role of the very low O2 concentrations in the maintenance of quiescent and undifferentiated cells, showing an un- or low-cycling status and able to reconstitute hematopoiesis, consecutively to their injection into NOG mice. However, the molecular and metabolic mechanisms involved in these processes remain unknown.

A hipóxia-isquemia neonatal (HI) faz parte da etiologia de diversas patologias neurológicas e é causa de graves sequelas. Os mecanismos patofisiológicos dessa lesão começam com o insulto imediato após a HI e se estendem por dias ou semanas, pelo aumento da liberação de espécies reativas de oxigênio associada a redução da defesas anti-oxidantes e reação glial, sendo a lesão secundária parte crucial no processo que culmina no dano final. A acetilcolina (ACh) é um neurotransmissor do sistema nervoso central (SNC) que parece ter uma importante ação neuroprotetora após a HI. A acetilcolinaesterase (AChE) é responsável pela degradação da ACh, inibidores dessa enzima vêm sendo utilizados para o tratamento de danos neurológicos. Sua ação positiva sobre a HI foi demonstrada em estudos realizados em nosso laboratório, onde a administração do extrato de Huperzia quadrifariata (inibidor de AChE) reduziu os déficits cognitivos e histológicos causados por essa lesão Para avaliar os efeitos das administrações pré e pós-hipóxia de galantamina, inibidor da AChE, no modelo de HI perinatal, ratos Wistar no 7º dia de vida pós-natal (DPN7) foram submetidos à combinação da oclusão unilateral da artéria carótida direita e exposição a uma atmosfera hipóxica (8% de O2) durante 60 minutos. Foram aplicadas injeções intraperitoniais de salina para os grupos Sham e HI+Salina (HIS) e de galantamina nos grupos HI+Galantamina 5 mg/kg pré-hipóxia (HIG5-Pré), HI+Galantamina 10 mg/kg pré-hipóxia (HIG10-Pré), HI+Galantamina 5 mg/kg pós-hipóxia (HIG5-Pós) e HI+Galantamina 10 mg/kg pós-hipóxia (HIG10-Pós). Os grupos Pré receberam galantamina imediatamente antes da hipóxia e os grupos Pós nos intervalos de 1, 24, 48 e 72 horas após a cirurgia. No DPN45 foi feita a análise do volume das estruturas encefálicas que demonstrou a redução do volume do hipocampo do grupo HIS em relação ao Sham e uma prevenção desse efeito no grupo HIG10-Pré, mas não nos demais grupos. Análises bioquímicas foram feitas no hipocampo ipsilesional 24 horas após a lesão e revelaram: através da citometria de fluxo uma redução na sobrevivência de neurônios no grupo HIS em relação ao Sham que foi prevenida no grupo HIG10-Pré; através de ELISA uma hipertrofia dos astrócitos no grupo HIS que foi revertida no grupo HIG10-Pré e um aumento na atividade da enzima anti-oxidante catalase. O tratamento pré-hipóxia com galantamina foi capaz de prevenir os déficits histológicos, aumentar a sobrevivência celular, reduzir a reação astrocitária e aumentar a atividade anti-oxidante em ratos submetidos à HI. / Neonatal hypoxia ischemia (HI) has a role in etiology of several neurological pathologies and causes severe sequelae. The pathophysiological mechanisms of this lesion start immediately after HI and last for days or weeks, with the secondary injury being a crucial part the process that culminates in the final damage. Acetylcholine (ACh) is a neurotransmitter of the central nervous system that seems to have an important neuroprotective action after HI. Acetylcholinesterase (AChE) degradates ACh and inhibitors of this enzyme have been used to treat neurological damage. Its positive action on HI has been demonstrated in studies performed in our laboratory, where the administration of the alkaloid extract of Huperzia quadrifariata (An inhibitor of AChE) reduced the cognitive and histological deficits caused by this lesion. To evaluate the effects of the pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in the model oh perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right charotid artery and of exposure to a hypoxic exposure (8% O2) for 60 minutes. Intraperitoneal injections of saline in the groups Sham anf HI+Saline (HIS) and of galantamine in the groups HI+Galantamine 5 mg/kg pre-hypoxia (HIG5-Pre), HI+Galantamine 10 mg/kg pre-hypoxia (HIG10-Pre), HI+Galantamine 5 mg/kg post-hypoxia (HIG5-Post) and HI+Galantamine 10 mg/kg post-hypoxia (HIG5-Post). The Pre groups received galantamine immediately before hypoxia and the Post groups in the intervals of 1, 24, 48 and 72 hours after HI. On PND45 the analysis of the volume of brain structures showed a reduction of the volume of the ipsilesional hippocampus in the HIS group when compared to the sham and a prevention of this effect in the HIG10-Pre, but not in any other group. Biochemical analysis was performed in the ipsilesional hippocampus 24 hours after the lesion and revealed: a reduction of the number of surviving neurons in the HIS group when compared to the Sham that was prevented in the HIG10-Pre; a hypertrophy of the astrocytes in the HIS group that was prevented in the HIG10-Pre group and an increase in the activity of the anti-oxidant enzyme catalase in the HIG10-Pre group. The treatment with galantamine was able to prevent the histological deficits, increase the survival of neurons, reduce astrocytic reaction and increase the anti-oxidant activity in rats submitted to HI.