Chemokines impact in Alzheimer’s disease

Alzheimer’s Disease (AD) is a neurodegenerative disorder neuropathologically characterized by the presence of extracellular senile plaques, intracellular neurofibrillary tangles and synaptic loss. Neuroinflammation has been associated with some neurodegenerative diseases, such as AD. In AD, increased Aβ production and aggregation, have a fundamental role in the activation of the inflammatory process. In turn, this could be fundamental in the early stages of this pathology, regarding the Aβ clearance and brain protection. However, chronic inflammation leads to an increase of the inflammatory mediators, such as cytokines, released by activated microglia, astrocytes, and neurons. The excessive production of these inflammatory components promotes alterations in both amyloid precursor protein (APP) expression and processing, stimulating the increase of Aβ accumulation and abnormal tau phosphorylation. This results in neurotoxic effects, irreversible damage and neuronal loss. Chronic inflammation is a feature of AD however, little is known about the effects of some chemokines on its pathogenesis. Thus, the main aim of this thesis was to study the impact of the interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) on apoptosis, APP and tau. The both studied chemokines resulted in small alterations regarding the cytotoxicity on SH-SY5Y differentiated cells, being a significant increase in apoptosis observed only for the MCP-1 at the highest concentration. For the APP processing no significant differences were obtained, although a tendency to increase at different concentrations and periods was registered for both IL-8 and MCP-1. With respect to tau and other cytoskeleton-associated proteins, it was possible to observe a tendency to increase in the phosphorylated residue (Ser396) at the higher concentrations, as well as alterations on actin and tubulin with an increase on acetylated-α tubulin. This effect can be translated by neuronal architectural and survival alterations. Therefore additional studies could contribute to a better understanding of the way that these chemokines act on AD pathogenesis.

Avaliação do extrato do fungo caripia montagnei e de agonistas de PPAR - α no processo inflamatório

The mushrooms have been object of intense research in view of its potential raising of application in different sectors of the pharmacology and alimentary industry. Among diverse bioactive composites of polyssacharides nature that exist in the fungus the glucans are much searched. These are polymers of glucose and classified as the type of glicosidic linking [α, β]. Peroxisome proliferator-activated receptors (PPARs), ranscription factors belonging to the family of nuclear receptors that bind themselves o specific agonists, have shown their importance in controlling the inflammatory process. The aim of this study was to perform a chemical characterization of extract rom the mushroom Caripia montagnei, assess its antiinflammatory and antibacterial effect and determine if this effect occurs via PPAR. This mushroom is composed of carbohydrates (63.3±4.1%), lipids (21.4l±0.9%) and proteins (2.2± 0.3%). The aqueous solution resulting from the fractionation contained carbohydrates (98.7±3.3%) and protein (1.3±0.25%). Analyses of infrared spectrophotometry and of nuclear magnetic esonance demonstrated that the extract of mushroom C. montagnei is rich in β-glucans. In hioglycolate-induced peritonitis, the C. montagnei glucans (50 mg/kg) educed the inflammatory process in 65.5±5.2% and agonists, pharmacological igands, for PPAR: Wy-14643 (49.3±6.1%), PFOA (48.9±3.8%) and clofibrate in 45.2±3.2%. Sodium diclofenac showed a reduction of 81.65±0.6%. In the plantar edema, the glucans from C. montagnei (50 mg/kg) and L-NAME reduced the edema to a similar degree 91.4±0.3% and 92.8±0,5 %, respectively. In all the groups tested, nitric oxide (NO), an inflammation mediator, showed a significant reduction in the nitrate/nitrite levels when compared to the positive control (P<0.001). The C. montagnei glucans did not show cytotoxicity in the concentrations tested (2.5, 5.0, 10.0, 20.0 and 40.0 µg/100 µL). Antibacterial activity demonstrated that, unlike total extract, there was no inhibition of bacterial growth. The C. montagnei glucans show great potential for antiinflammatory applications. This effect suggests that it is mediated by PPAR activation and by COX and iNOS inhibition

Effect of vitamin D supplementation on bone status, glucose homeostasis and immune function in children with vitamin D deficiency

Background: Between 1961-1971 vitamin D deficiency was recognized as a public health issue in the UK, because of the lack of effective sunlight and the population mix [1, 2]. In recent years, health care professionals have cited evidence suggesting a re-emergence of the vitamin D deficiency linked to a number of health consequences as a concern [3-6]. Evidence from observational studies has linked low vitamin D status with impairment in glucose homeostasis and immune dysfunction [7-9]. However, interventional studies, particularly those focused on paediatric populations, have been limited and inconsistent. There is a need for detailed studies, to clarify the therapeutic benefits of vitamin D in these important clinical areas. Objective: The aims of this PhD thesis were two-fold. Firstly, to perform preliminary work assessing the association between vitamin D deficiency and bone status, glucose homeostasis and immune function, and to explore any changes in these parameters following short term vitamin D3 replacement therapy. Secondly, to assess the effectiveness of an electronic surveillance system (ScotPSU) as a tool to determine the current incidence of hospital-based presentation of childhood vitamin D deficiency in Scotland. Methods: Active surveillance was performed for a period of two years as a part of an electronic web-based surveillance programme performed by the Scottish Paediatric Surveillance Unit (ScotPSU). The validity of the system was assessed by identifying cases with profound vitamin D deficiency (in Glasgow and Edinburgh) from the regional laboratory. All clinical details were checked against those identified using the surveillance system. Thirty-seven children aged 3 months to 10 years, who had been diagnosed with vitamin D deficiency, were recruited for the bone, glucose and immunity studies over a period of 24 months. Twenty-five samples were analysed for the glucose and bone studies; of these, 18 samples were further analysed for immune study. Treatment consisted of six weeks taking 5000 IU units cholecalciferol orally once a day. At baseline and after completion of treatment, 25 hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), alkaline phosphatase (ALP), collagen type 1 cross-linked C-telopeptide (CTX), osteocalcin (OCN), calcium, phosphate, insulin, glucose, homeostasis model assessment index, estimated insulin resistance (HOMA IR), glycated hemoglobin (HbA1c), sex hormone binding globulin (SHBG), lipids profiles, T helper 1 (Th1) cytokines (interleukin-2 ( IL-2), tumor necrosis factors-alpha (TNF-α), interferon-gamma (INF-γ)), T helper 2 (Th2) cytokines (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6)), T helper 17 (Th17) cytokine (interleukin-17 (IL-17)), Regulatory T (Treg) cytokine (interleukin-10 (IL-10)) and chemokines/cytokines, linked with Th1/Th2 subset balance and/or differentiation (interleukin-8 (IL-8), interleukin-12 (IL-12), eosinophil chemotactic protein ( EOTAXIN), macrophage inflammatory proteins-1beta (MIP-1β), interferon-gamma-induced protein-10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein-1(MCP-1)) were measured. Leukoocyte subset analysis was performed for T cells, B cells and T regulatory cells and a luminex assay was used to measure the cytokiens. Results: Between September 2009 and August 2011, 163 cases of vitamin D deficiency were brought to the attention of the ScotPSU, and the majority of cases (n = 82) were reported in Glasgow. The cross-validation checking in Glasgow and Edinburgh over a one-year period revealed only 3 (11%) cases of clearly symptomatic vitamin D deficiency, which had been missed by the ScotPSU survey in Glasgow. While 16 (67%) symptomatic cases had failed to be reported through the ScotPSU survey in Edinburgh. For the 23 children who are included in bone and glucose studies, 22 (96%) children had basal serum 25(OH)D in the deficiency range (< 50 nmol/l) and one (4%) child had serum 25(OH)D in the insufficiency range (51-75 nmol/l). Following vitamin D3 treatment, 2 (9%) children had final serum 25(OH)D lower than 50 nmol/l, 6 (26%) children had final serum 25(OH)D between >50-75 nmol/l, 12 (52%) children reached a final serum 25(OH)D >75-150 nmol/l and finally 3 (13%) exceeded the normal reference range with a final 25(OH)D >150 nmol/l. Markers for remodelling ALP and PTH had significantly decreased (p = 0.001 and <0.0001 for ALP and PTH respectively). In 17 patients for whom insulin and HOMA IR data were available and enrolled in glucose study, significant improvements in insulin resistance (p = 0.04) with a trend toward a reduction in serum insulin (p = 0.05) was observed. Of those 14 children who had their cytokines profile data analysed and enrolled in the immunity study, insulin and HOMA IR data were missed in one child. A significant increase in the main Th2 secreted cytokine IL-4 (p = 0.001) and a tendency for significant increases in other Th2 secreted cytokines IL-5 (p = 0.05) and IL-6 (p = 0.05) was observed following vitamin D3 supplementation. Conclusion: An electronic surveillance system can provide data for studying the epidemiology of vitamin D deficiency. However, it may underestimate the number of positive cases. Improving vitamin D status in vitamin D deficient otherwise healthy children significantly improved their vitamin D deficient status, and was associated with an improvement in bone profile, improvements in insulin resistance and an alteration in main Th2 secreting cytokines.

Silencing the gip gene of Phytophthora cinnamomi by iRNA and studying the subcellular localization of GIP and NPP1 proteins

Ink Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is caused by two species: Phytophthora cinnamomi and Phytophthora cambivora, being the first one the foremost pathogen of ink disease in Portugal. P. cinnamomi is one of the most aggressive and widespread plant pathogen with nearly 1,000 host species. This oomycete causes enormous economic losses and it is responsible for the decline of many plant species in Europe and worldwide. Up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels, especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP (glucanase inhibitor protein) and NPP1 (necrosis-inducing Phytophthora protein 1) produced by P. cinnamomi during the infection. The technique of RNA interference was used to knockdown the gip gene of P. cinnamomi. Transformants obtained with the silenced gene have been used to infect C. sativa, in order to determine the effect of gene silencing on the plant phenotype. To know more about the function of GIP and NPP1 involved in the mechanism of infection, the ORF’s of gip and npp1 genes have been cloned to the pTOR-eGFP vector for a future observation of P. cinnamomi transformants with fluorescent microscopy and determination of the subcellular localization. Moreover the prediction by bioinformatics tools indicates that both GIP and NPP1 proteins are secreted. The results allow to predict the secretory destination of both GIP and NPP1 proteins and confirm RNAi as a potential alternative biological tool in the control and management of P. cinnamomi. Keywords:

Transcription of TIR1-Controlled Genes Can be Regulated within 10 Min by an Auxin-Induced Process. Can TIR1 be the Receptor?

ABP1 and TIR1/AFBs are known as auxin receptors. ABP1 is linked to auxin responses several of which are faster than 10 min. TIR1 regulates auxin-induced transcription of early auxin genes also within minutes. We use transcription of such TIR1-dependent genes as indicator of TIR1 activity to show the rapid regulation of TIR1 by exogenous auxin. To this end, we used quantification of transcription of a set of fifteen early auxin-induced reporter genes at t = 10 and t = 30 min to measure this as a TIR1-dependent auxin response. We conducted this study in 22 mutants of auxin transporters (pin5, abcb1, abcb19, and aux1/lax3), protein kinases and phosphatases (ibr5, npr1, cpk3, CPK3-OX, d6pk1, d6pkl1-1, d6pkl3-2, d6pkl1-1/d6pkl2-2, and d6pkl1-1/d6pkl3-2), of fatty acid metabolism (fad2-1, fad6-1, ssi2, lacs4, lacs9, and lacs4/lacs9) and receptors (tir1, tir1/afb2, and tir1/afb3) and compared them to the wild type. After 10 min auxin application, in 18 out of 22 mutants mis-regulated expression of at least one reporter was found, and in 15 mutants transcription of two-to-three out of five selected auxin reporter genes was mis-regulated. After 30 min of auxin application to mutant plants, mis-regulation of reporter genes ranged from one to 13 out of 15 tested reporter genes. Those genes chosen as mutants were themselves not regulated in their expression by auxin for at least 1 h, excluding an influence of TIR1/AFBs on their transcription. The expression of TIR1/AFB genes was also not modulated by auxin for up to 3 h. Together, this excludes a feedback or feedforward of these mutant genes/proteins on TIR1/AFBs output of transcription in this auxin-induced response. However, an auxin-induced response needed an as yet unknown auxin receptor. We suggest that the auxin receptor necessary for the fast auxin-induced transcription modulation could be, instead, ABP1. The alternative hypothesis would be that auxin-induced expression of a protein, initiated by TIR1/AFBs receptors, could initiate these responses and that this unknown protein regulated TIR1/AFB activities within 10 min.

Estudo da proteína humana centrossomal TBCCD1 em condições de stress oxidativo causado por H2O2 em estado estacionário

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

A infecção por Leishmania infantum chagasi altera o metabolismo lipídico do hospedeiro

American visceral leishmaniasis (AVL), caused by Leishmania infantum chagasi (L.i.chagasi), stands as a public health problem in Brazil, with human and canine cases related in all states..Lipid metabolism can be modified in several status of infection. For example, experimental studies show that the cholesterol is necessary to internalization and replication of L.i.chagasi in macrophages through caveolar domains. Patients with AVL present low levels of cholesterol and a visible triglycerides increase. This work aimed to evaluate the lipid metabolism in several post-infection status by L.i.chagasi, including individuals with symptomatic infection (AVL), and asymptomatic. The levels of cholesterol, triglycerides, HDL and reactive C protein, were measured. Individuals with AVL were compared with individuals with assymptomatic infection and presented low levels of total cholesterol (128 ± 6.180 mg/dL vs. 158 ±5.733 mg/dL, p=0.0001), HDL (29 ± 1.746 mg/dL vs. 37 ± 1.647 mg/dL, p=0.0001), increased levels of triglycerides (149.5 mg/dL ± 12.72 vs. 78.00 ± 10.43 mg/dL, p=0.0095) and higher levels of reactive C protein (1.750± 0.4939 mg/dL vs. 0.40 ± 0.1707 mg/dL; p=0.0001). The expression of genes related to lipid metabolism, such as LXR-a, LXR-b, PPAR-a, PPAR-d, PPAR-g and APOE was evaluated by real time PCR. A reduction in the expression of those genes was found in the group of AVL patients corroborating the serum levels of the metabolites earlier quantified. Our findings suggest a modulation of metabolism of lipids, in the chronic phase of AVL, this could facilitate the survival of leishmania, due to the known reduction on the ability of macrophages in presenting antigens efficiently to the T cells due to the reduction in the cholesterol available, it results in a subversion of the host immunity.

Anticorpos bloqueadores dos checkpoints imunes

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Efeito do GQ-02, derivado de tiazolidinadionas, sobre a aquisição de fenótipo termogênico pelo adipócito in vivo e em cultura de células

Dissertação (mestrado)—Universidade de Brasília, Faculdade em Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016.

Avaliação do extrato do fungo caripia montagnei e de agonistas de PPAR - α no processo inflamatório

The mushrooms have been object of intense research in view of its potential raising of application in different sectors of the pharmacology and alimentary industry. Among diverse bioactive composites of polyssacharides nature that exist in the fungus the glucans are much searched. These are polymers of glucose and classified as the type of glicosidic linking [α, β]. Peroxisome proliferator-activated receptors (PPARs), ranscription factors belonging to the family of nuclear receptors that bind themselves o specific agonists, have shown their importance in controlling the inflammatory process. The aim of this study was to perform a chemical characterization of extract rom the mushroom Caripia montagnei, assess its antiinflammatory and antibacterial effect and determine if this effect occurs via PPAR. This mushroom is composed of carbohydrates (63.3±4.1%), lipids (21.4l±0.9%) and proteins (2.2± 0.3%). The aqueous solution resulting from the fractionation contained carbohydrates (98.7±3.3%) and protein (1.3±0.25%). Analyses of infrared spectrophotometry and of nuclear magnetic esonance demonstrated that the extract of mushroom C. montagnei is rich in β-glucans. In hioglycolate-induced peritonitis, the C. montagnei glucans (50 mg/kg) educed the inflammatory process in 65.5±5.2% and agonists, pharmacological igands, for PPAR: Wy-14643 (49.3±6.1%), PFOA (48.9±3.8%) and clofibrate in 45.2±3.2%. Sodium diclofenac showed a reduction of 81.65±0.6%. In the plantar edema, the glucans from C. montagnei (50 mg/kg) and L-NAME reduced the edema to a similar degree 91.4±0.3% and 92.8±0,5 %, respectively. In all the groups tested, nitric oxide (NO), an inflammation mediator, showed a significant reduction in the nitrate/nitrite levels when compared to the positive control (P<0.001). The C. montagnei glucans did not show cytotoxicity in the concentrations tested (2.5, 5.0, 10.0, 20.0 and 40.0 µg/100 µL). Antibacterial activity demonstrated that, unlike total extract, there was no inhibition of bacterial growth. The C. montagnei glucans show great potential for antiinflammatory applications. This effect suggests that it is mediated by PPAR activation and by COX and iNOS inhibition

Immune modulation by vitamin D and its relevance to food allergy

Apart from its classical function in bone and calcium metabolism, vitamin D is also involved in immune regulation and has been linked to various cancers, immune disorders and allergic diseases. Within the innate and adaptive immune systems, the vitamin D receptor and enzymes in monocytes, dendritic cells, epithelial cells, T lymphocytes and B lymphocytes mediate the immune modulatory actions of vitamin D. Vitamin D insufficiency/deficiency early in life has been identified as one of the risk factors for food allergy. Several studies have observed an association between increasing latitude and food allergy prevalence, plausibly linked to lower ultraviolet radiation (UVR) exposure and vitamin D synthesis in the skin. Along with mounting epidemiological evidence of a link between vitamin D status and food allergy, mice and human studies have shed light on the modulatory properties of vitamin D on the innate and adaptive immune systems. This review will summarize the literature on the metabolism and immune modulatory properties of vitamin D, with particular reference to food allergy.

Salmonella Enterica serovar Typhimurium Infection in Mouse Pregnancy

Salmonella are Gram-negative, intracellular food-borne pathogens that cause pregnancy complications. In pregnant mice, Salmonella enterica serovar Typhimurium (S.Tm) infection results in placental bacterial replication, inflammation, necrosis, and fetal loss by unknown mechanisms. Necroptosis, or programmed necrosis mediated by RIPK3 (receptor-interacting protein kinase 3), an inflammatory cell death pathway, is implicated in the pathogenesis of S.Tm in non-pregnant mice. This goal of this thesis was to investigate the role of necroptosis in the pathogenesis of S.Tm infection during mouse pregnancy. I hypothesized that elimination of the key necroptotic cell death protein RIPK3 would decrease placental inflammation and trophoblast cell death, and increase conceptus survival compared to controls. Mice expressing a functional Slc11a1 (encodes the natural resistance-associated macrophage protein 1, NRAMP1) gene with or without RIPK3 function (Ripk3-/-Slc11a1+/+ compared to Slc11a1+/+) were infected with 103 S.Tm by tail vein injection on gestational day (GD) 12. Mice were euthanized on GD 14 (48h post-infection) or GD 15 (72h post-infection) and implantation sites (IS) and maternal serum were harvested for analyses. In nearly all challenged mice (except one outlier), S.Tm were detected in most IS within a litter but there was limited immune cell infiltration, placental damage or cell death in Slc11a1 competent mice regardless of Ripk3 gene deletion. Maternal serum cytokine analyses confirmed lack of maternal immune responses to S.Tm infection. IS amongst the litter of a single dam (Ripk3-/-Slc11a1+/+ at 72h postinfection) displayed heavy but not universal placental S.Tm infection of decidual tissues and spongiotrophoblast, associated with elevated maternal serum pro-inflammatory cytokines. S.Tm infection of the fetal yolk sac (YS) was observed in 54.5% of IS from this dam. YS infection was confirmed in archival samples in mice expressing Ripk3 with intact Slc11a1 and in mice lacking functional Slc11a1. In Slc11a1 incompetent mice, S.Tm were detected in placental labyrinthine trophoblast. Based on the available data, this thesis suggests that Ripk3 and necroptosis have no significant roles in either promotion or prevention of progressive Salmonella infection during mouse pregnancy. It also provides pilot data that NRAMP1 controls placental localization and lethality due to YS infection.

Nouveaux inducteurs covalents de la voie de signalisation Keap1/Nrf2/ARE

Le stress électrophile et oxydant est un souci grandissant pour la santé avec l'évolution de nos modes de vie. L'exposition aux ultraviolets, à la pollution, aux substances carcinogènes, à la fumée de cigarette et la pratique intensive d'activités sportives sont autant de causes de stress oxydant pour l'organisme. Ces dommages sont associés à plusieurs maladies et conditions pathologiques telles que cancers, diabètes, infections pulmonaires et maladies neurodégénératives. L'élément de réponse antioxydant (ARE) est un des composants principaux des défenses de la cellule contre ce phénomène. Ce promoteur agit sous le contrôle de Nrf2 (Nuclear factor erythroid 2-related factor 2). Une stratégie populaire pour l'activation de ce mécanisme est l'utilisation d'inducteurs covalents. Ces molécules agissent par la formation de liens covalents avec les nombreux résidus cystéine de Keap1 (Kelch-like ECH-associated protein 1), une protéine chaperonne qui contrôle l'activité de Nrf2. Cette thèse présente la synthèse, les propriétés biologiques et l'étude des relations structure-activité d'une librairie d'électrophiles capables d'induire la transcription des gènes cibles de la voie de signalisation Keap1/Nrf2/ARE. Le premier volet fait état de la comparaison d'une variété d'électrophiles simples pour étudier les préférences de la cible. Le deuxième volet montre que la présence d'une seconde fonction capable de piéger un résidu cystéine fournit des analogues très puissants.