Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100 mu g/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4,6 or 24 h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24 h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24 h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6 h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24 h after surgery, and in both hypothalamic nuclei only at 6 h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood. (C) 2011 Elsevier B.V. All rights reserved.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Vascular dysfunction and heart failure: Epiphenomenon or etiologic agent?

Endothelial function plays a key role in the local regulation of vascular tone. Alterations in endothelial function may result in impaired release of endothelium-derived relaxing factors or increased release of endothelium-derived contracting factors. Heart failure may impair endothelial function by means of reduced synthesis and release of nitric oxide (NO) or by increased degradation of NO and increased production of endothelin-1. Endothelial dysfunction may worsen heart function by means of peripheral effects, causing increased afterload and central effects such as myocardial ischemia and inducible nitric oxide synthase (iNOS)-induced detrimental effects. Evidence from clinical studies has suggested that there is a correlation between decreased endothelial function and increasing severity of congestive heart failure (CHF). Treatments that improve heart function may also improve endothelial dysfunction. The relationship between endothelial dysfunction and heart failure may be masked by the stage of endothelial dysfunction, the location of vessels being tested, and the state of endothelial-dependent vasodilatation response.

Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum

Background The ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such cell-mediated immunity was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density. Methods We enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite. Findings After challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon gamma but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells. Interpretation People can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasite-specific antibodies, suggesting an additional strategy for development of a malaria vaccine.

Caveolae — from ultrastructure to molecular mechanisms

Almost 50 years after the first sighting of small pits that covered the surface of mammalian cells, investigators are now getting to grips with the detailed workings of these enigmatic structures that we now know as caveolae.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.

Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

Iron Overload Favors the Elimination of Leishmania

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host’s iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host’s oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Basement membrane alterarions in kernicteric brain microvasculature and pericyte response to bilirubin

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

The pattern of immune cell infiltration in chromoblastomycosis: involvement of macrophage inflammatory protein-1 alpha/CCL3 and fungi persistence

Chromoblastomycosis (CR) is a subcutaneous chronic mycosis characterized by a granulomatous inflammatory response. However, little is known regarding the pattern of leukocyte subsets in CR and the pathways involved in their recruitment. The objective of this study was to assess the cellular subsets, chemokine, chemokine receptors and enzymes in CR. The inflammatory infiltrate was characterized by immunohistochemistry using antibodies against macrophages (CD68), Langerhans'cells (S100), lymphocytes (CD3, CD4, CD8, CD45RO, CD20 and CD56) and neutrophils (CD15). The expression of MIP-1alpha (Macrophage inflammatory protein-1alpha), chemokine receptors (CXCR3 and CCR1) and enzymes (superoxide dismutase-SOD and nitric oxide synthase-iNOS) was also evaluated by the same method. We observed an increase in all populations evaluated when compared with the controls. Numbers of CD15+ and CD56+ were significantly lower than CD3+, CD4+, CD20+ and CD68+ cells. Statistical analysis revealed an association of fungi numbers with CD3, CD45RO and iNOS-positive cells. Furthermore, MIP-1alpha expression was associated with CD45RO, CD68, iNOS and CXCR3. Our results suggest a possible role of MIP-1alpha and fungi persistence in the cell infiltration in CR sites.

Role of oxidative stress-induced systemic and cavernosal molecular alterations in the progression of diabetic erectile dysfunction

Background Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. Methods Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and α-smooth muscle actin (α-SMA) and eNOS–α-SMA. Results There was a significant increase in urinary H2O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. Conclusions The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.

Immunoactivation and immunopathogeny during active visceral leishmaniasis

Visceral leishmaniasis is caused by protozoan parasites of the Leishmania donovani complex. During active disease in humans, high levels of IFN-γ and TNF-α detected in blood serum, and high expression of IFN-γ mRNA in samples of the lymphoid organs suggest that the immune system is highly activated. However, studies using peripheral blood mononuclear cells have found immunosuppression specific to Leishmania antigens; this poor immune response probably results from Leishmania antigen-engaged lymphocytes being trapped in the lymphoid organs. To allow the parasites to multiply, deactivating cytokines IL-10 and TGF-β may be acting on macrophages as well as anti-Leishmania antibodies that opsonize amastigotes and induce IL-10 production in macrophages. These high activation and deactivation processes are likely to occur mainly in the spleen and liver and can be confirmed through the examination of organ samples. However, an analysis of sequential data from studies of visceral leishmaniasis in hamsters suggests that factors outside of the immune system are responsible for the early inactivation of inducible nitric oxide synthase, which occurs before the expression of deactivating cytokines. In active visceral leishmaniasis, the immune system actively participates in non-lymphoid organ lesioning. While current views only consider immunocomplex deposition, macrophages, T cells, cytokines, and immunoglobulins by diverse mechanism also play important roles in the pathogenesis.

Regulação integrada do metabolismo da glucose no estado pós-prandial : fisiologia e reversão de processos patológicos

Resumo: Os mecanismos que regulam a homeostase da glucose no pós-prandial são distintos dos mecanismos desencadeados em situações de jejum. Desta forma o fígado parece desempenhar um papel fundamental na acção periférica da insulina após a refeição através de um mecanismo que envolve os nervos parassimpáticos hepáticos e o óxido nítrico (NO). Esta dissertação procura evidenciar a importância de ambos na fi siologia de manutenção da glicémia pós-prandial e na fi siopatologia da resistência à insulina. Dos resultados obtidos observou-se que após a administração de uma refeição mista o perfi l glicémico foi distinto em animais com ou sem ablação dos nervos parassimpáticos hepáticos. A desnervação parassimpática hepática aumentou as excursões de glucose imediatamente após a refeição. Estas diferenças nas excursões de glucose dependentes do parassimpático ocorreram devido a uma diminuição da clearance de glucose, sem que fosse afectada a taxa de aparecimento de glucose no sangue, a produção endógena de glucose e secreção de insulina ou péptido-C. Este aumento das excursões de glucose revelou-se ser devida à diminuição da clearance de glucose pós-prandial exclusivamente no músculo-esquelético, coração e o rim. Concluiu-se que o fígado teria uma função endócrina nestes três órgãos. Surgiu assim a hipótese dos S-nitrosotiois (RSNOs) poderem mimetizar essa resposta endócrina. Testou-se o seu efeito in vivo na sensibilidade à insulina. Para níveis baixos de sensibilidade à insulina, como jejum, desnervação no estado pós-prandial e resistência à insulina os RSNOs potenciaram a sensibilidade à insulina para valores semelhantes ao pós-prandial indicando-os como potenciais fármacos no tratamento da resistência à insulina. O NO e seus derivados ganharam assim uma evidência cada vez maior na acção periférica da insulina e portanto fez-se uma caracterização dos seus níveis desde a fi siologia à fi siopatologia. Os resultados obtidos nesta dissertação permitiram correlacionar a sintetase de óxido nítrico (NOS), enzima responsável pela síntese de NO como um possível marcador da resistência à insulina. Os resultados obtidos contribuíram substancialmente para compreender os mecanismos fi siológicos e fi siopatológicos de manutenção da glicémia após a refeição, colocando o fígado como órgão primordial na regulação periférica (extra-hepática) da captação de glucose.-------- ABSTRACT: The mechanisms responsible for the postprandial response are different from the ones in the fasted state. Therefore the liver seems to play a fundamental role in postprandial insulin action through a mechanism that evolves the hepatic parasympathetic nerves (HPN) and nitric oxide (NO). This work focused on the importance of both, HPN and NO, on postprandial glycemic control and on the pathophysiology of insulin resistance. We observed that after administration of a mixed meal the glycemic profi les with or without the parasympathetic nerves were distinct, increasing glucose excursions after ablation of HPN.This increase in glucose excursions was due to a decrease on the rate of glucose disappearance in extra-hepatic tissues. Glucose appearance rate, endogenous glucose production and insulin secretion were not related to this mechanism. The increase on glucose excursions after the ablation of hepatic parasympathetic system was due to a decrease on glucose clearance on extra-hepatic tissues, namely skeletal-muscle, heart and kidney. We concluded that the liver has an endocrine function on those tissues increasing their glucose uptake.This mechanism led to propose the hypothesis that S-nitrosothiols (RSNOs) could mimic this mechanism. Therefore RSNOs effects on insulin sensitivity were tested. For low insulin sensitivity levels, i.e. fasted state, ablation of the HPN or insulin resistance state induced by a high sucrose diet RSNOs increased insulin sensitivity to levels normally observed in the postprandial state. These results indicated these drugs as potential pharmacological tools in the treatment of insulin resistance. NO and their derivates emerged as fundamental parts of insulin action. A characterization of nitric oxide and nitric oxide synthase (NOS), the enzyme responsible for NO synthesis was part of the work performed. We concluded that NO could be used as a biomarker for insulin resistance states. This work contributed for understanding the mechanism underlying postprandial glycemic control indicating the liver as a key organ in the regulation of peripheral (extra-hepatic) insulin action.

Exploring female mice interstrain differences relevant for models of depression

Depression is an extremely heterogeneous disorder. Diverse molecular mechanisms have been suggested to underlie its etiology. To understand the molecular mechanisms responsible for this complex disorder, researchers have been using animal models extensively, namely mice from various genetic backgrounds and harboring distinct genetic modifications. The use of numerous mouse models has contributed to enrich our knowledge on depression. However, accumulating data also revealed that the intrinsic characteristics of each mouse strain might influence the experimental outcomes, which may justify some conflicting evidence reported in the literature. To further understand the impact of the genetic background, we performed a multimodal comparative study encompassing the most relevant parameters commonly addressed in depression, in three of the most widely used mouse strains: Balb/c, C57BL/6, and CD-1. Moreover, female mice were selected for this study taken into account the higher prevalence of depression in women and the fewer animal studies using this gender. Our results show that Balb/c mice have a more pronounced anxious-like behavior than CD-1 and C57BL/6 mice, whereas C57BL/6 animals present the strongest depressive-like trait. Furthermore, C57BL/6 mice display the highest rate of proliferating cells and brain-derived neurotrophic factor (Bdnf) expression levels in the hippocampus, while hippocampal dentate granular neurons of Balb/c mice show smaller dendritic lengths and fewer ramifications. Of notice, the expression levels of inducible nitric oxide synthase (iNos) predict 39.5% of the depressive-like behavior index, which suggests a key role of hippocampal iNOS in depression. Overall, this study reveals important interstrain differences in several behavioral dimensions and molecular and cellular parameters that should be considered when preparing and analyzing experiments addressing depression using mouse models. It further contributes to the literature by revealing the predictive value of hippocampal iNos expression levels in depressive-like behavior, irrespectively of the mouse strain.