34 resultados para Constitutive metabolites


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All life forms need to monitor carbon and energy availability to survive and this is especially true for plants which must integrate unavoidable environmental conditions with metabolism for cellular homeostasis maintenance. Sugars, in the heart of metabolism, are now recognized as crucial signaling molecules that translate those conditions. One such signal is trehalose 6- phosphate (T6P), a phosphorylated dimer of glucose molecules which levels correlate well with those of sucrose (Suc). Central integrators of stress and energy regulation include the conserved plant Snf1-related kinase1 (SnRK1) which respond to low cellular energy levels by up-regulating energy conserving and catabolic metabolism and down-regulating energy consuming processes. In 2009 T6P was shown to inhibit SnRK1. The in vitro inhibition of SnRK1 by T6P was confirmed in vivo through the observation that genes normally induced by SnRK1 were repressed by T6P and vice-versa, promoting growth processes. These observations provided a model for the regulation of growth by sugar.(...)

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The industrialization of traditional processes relies on the scientific ability to understand the empirical evidence associated with traditional knowledge. Cork manufacturing includes one operation known as stabilization, where humid cork slabs are extensively colonized by fungi. The implications of fungal growth on the chemical quality of cork through the analysis of putative fungal metabolites have already been investigated. However, the effect of fungal growth on the mechanical properties of cork remains unexplored. This study investigated the effect of cork colonization on the integrity of the cork cell walls and their mechanical performance. Fungal colonization of cork by Chrysonilia sitophila, Mucor plumbeus Penicillium glabrum, P. olsonii, and Trichoderma longibrachiatum was investigated by microscopy. Growth occurred primarily on the surface of the cork pieces, but mycelium extended deeper into the cork layers, mostly via lenticular channels and by hyphal penetration of the cork cell wall. In this first report on cork decay in which specific correlation between fungal colonization and mechanical proprieties of the cork has been investigated, all colonizing fungi except C. sitophila, reduced cork strength, markedly altering its viscoelastic behaviour and reducing its Young’s modulus.

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33rd IAHR Congress: Water Engineering for a Sustainable Environment

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River Flow 2010

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Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Doutor em Engenharia Civil

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Dissertation presented to obtain the Ph.D. degree in “Biology” at the Institute of Chemical and Biological Technology of the New University of Lisbon

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FEBS journal, Volume 278, Issue 14, pages 2511-2524, July 2011

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Dissertation presented to obtain a Doctoral Degree in Biology by Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

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Dissertation presented to obtain a Ph. D. degree in Biochemistry by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica.

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Dissertation presented to obtain the Ph.D degree in Biochemistry

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Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

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RESUMO: O uso de ratinhos transgénicos em neurociências aumentou consideravelmente nos últimos anos devido ao crescente interesse em compreender o cérebro e a necessidade de solucionar situações clínicas do foro neurológico e psiquiátrico. Para esse efeito, diferentes métodos de produção de animais transgénicos têm sido testados. O objectivo desta tese foi comparar métodos de integração aleatória de um transgene no genoma de ratinhos em termos de eficiência, estabilidade da integração do transgene, número de animais e de horas de trabalho necessárias para cada método. Assim, foi comparado o método mais utilizado - microinjecção pronuclear (PNMI) - com duas outras técnicas cujo desempenho foi considerado promissor – a transferência génica através dos testículos por electroporação e transfecção por lentivírus in vivo. As três técnicas foram realizadas usando um gene repórter sob o controlo de um promotor constitutivo, e depois reproduzidas usando um gene de interesse de modo a permitir obtenção de um animal capaz de ser usado em experimentação laboratorial. O transgene de interesse utilizado codifica uma proteína de fusão correspondendo a uma variante da rodopsina (channelrhodopsin) fundida à proteína enhanced yellow fluorescente protein ((EYFP) resultando num produto designado ChR2-EYFP. Este animal transgénico apresentaria expressão deste canal iónico apenas em células dopaminergicas, o que, com manipulação optogenética, tornaria possivel a activação especifica deste grupo de neurónios e, simultaneamente, a observação do impacto desta manipulação no comportamento num animal em livre movimento. Estas ferramentas são importantes na investigação básica em neurociências pois ajudam a esclarecer o papel de grupos específicos de neurónios e compreender doenças como a doença de Parkinson ou a esquizofrenia onde a função de certos tipos de neurónios de encontra alterada. Quando comparados os três métodos realizados verifica-se que usando um gene repórter PMNI resulta em 31,3% de, a de animais transgénicos obtidos, a electroporação de testículos em 0% e a injecção de lentivírus em 0%. Quando usado o gene de interesse, os resultados obtidos são, respectivamente, 18,8%, 63,9% e 0%.--------------ABSTRACT: The use of transgenic mice is increasing in all fields of research, particularly in neuroscience, due to the widespread need of animal models to solve neurological and psychiatric medical conditions. Different methodologies have been tested in the last decades in order to produce such transgenic animals. The ultimate goal of this thesis is to compare different methods of random integration of a transgene in the genome of mice in terms of efficiency, stability of the transgene integration, number of animals required and the labour intensity of each technique. We compared the most used method – pronuclear microinjection (PNMI) – with two other promising techniques – Testis Mediated Gene Transfer (TMGT) by electroporation and in vivo lentiviral transfection. The three techniques were performed using a reporter gene – green fluorescent protein (GFP), whose transcription was driven by the constitutive cytomegalovirus (CMV) promoter. These three techniques were later reproduced using the tyrosine hydroxylase promoter (TH) and the neuronal manipulator, channelrhodopsin-2 fused to the enhanced yellow fluorescent reporter protein (ChR2-EYFP). The transgenic animal we sough to produce would express the light driven channel only in dopaminergic cells, making possible to specifically activate this group of neurons, while simultaneously observe the behaviour in a freely moving animal. This is a very important tool in basic neuroscience research since it helps to clarify the role of specific groups of neurons, map circuits in the brain, and consequently understand neurological diseases such as Parkinson’s disease or schizophrenia, where the function of certain types of neurons is affected. When comparing the three methods, it was verified that using a reporter gene PNMI resulted in 31.3% of transgenic mice obtained, testis electroporation in 0% and lentiviral injection in 0%. When using the gene of interest, the results obtained were, respectively, 18.8%, 63.9% and 0%.

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Dissertação apresentada para obtenção do Grau de Doutor em Biologia, na especialidade de Genética Molecular, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

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Dissertação para obtenção do Grau de Doutor em Engenharia Civil

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ABSTRACT: In the late seventies the term “Haematological Stress Syndrome” defined some haematological abnormalities appearing in the course of acute and chronic disorders, such as raised plasma levels of fibrinogen (FNG) and factor VIII, reduced fibrinolytic activity and hyperviscosity. In the early nineties the “Membrane stress syndrome hypothesis” proposed the unification of the concepts of haematological stress syndrome with those of oxidation, inflammation and immune activation to explain the pathogenesis of the antiphospholipid syndrome (APS) Antiphospholipid antibodies, coagulation, fibrinolysis and thrombosis. This chapter investigated the occurrence of the “Haematological Stress Syndrome” and thrombosis in 144 participants positive for aPL detected by clotting and immune tests. Among the clotting assays for the detection of lupus anticoagulant, dilute Russell's viper venom time better correlated with a history of venous thrombosis than activated partial thromboplastin time (p<0.0002 vs p<0.009) and was the only test correlated with a history of arterial thrombosis (p<0.01). By regression analysis, serum levels of IgG anticardiolipin antibodies (aCL) associated with the number of venous occlusions (p<0.001). With regards to FNG and von Willebrand factor (vWF), the former rose by 36% (95% CI; 21%, 53%) and the latter by 50% (95% CI; 29%, 75%) at the first venous occlusion and remained unchanged after subsequent occlusions. At variance FNG rose by 45% (95% CI; 31%, 60%) per arterial occlusion and vWF by 27% (95% CI; 10%, 47%) per arterial occlusion throughout. The coagulation/fibrinolytic balance was cross-sectionally evaluated on 18 thrombotic PAPS patients, 18 subjects with persistence of idiopathic aPL and in healthy controls. Markers of thrombin generation prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and of fibrin turnover D-Dimer (D-D) were higher in thrombotic (p=0.006)and non-thrombotic subjects (p=0.0001) than in controls as were those of D-D (p<0.0001 and p=0.003 respectively). TAT levels did not differ. Gender analysed data revealed blunted tPA release (hence a negative venous occlusion test) in thrombotic females but neither in thrombotic males (p=0.01) nor in asymptomatic subjects of either sex. Also, in both patient groups females had higher mean PAI than males (p<0.0002) and control females (p<0.02). The activity of factor XIII (FXIIIa) was evaluated was evaluated in 29 patients with PAPS, 14 persistent carriers of aPL without thrombosis, 24 thrombotic patients with inherited thrombophilia, 28 healthy controls and 32 patients with mitral and aortic valve prosthesis as controls for FXIII only. FXIIIa was highest in PAPS (p=0.001), particularly in patients with multiple (n=12) than single occlusion (p=0.02) and in correlation with PAI (p=0.003) and FNG (p=0.005). Moreover FXIIIa was strongly associated with IgG aCL and IgG anti-2GPI (p=0.005 for both) in the PAPS group and to a lesser degree in the aPL group (FXIIIa with IgG aCL, p=0.02, with IgG anti-2GPI, p=0.04). Altogether these results indicate: 1) a differential relationship of aPL, vWF and FNG with venous and arterial thrombosis; 2) heightened thrombin generation, accelerated fibrin turnover and fibrinolysis abnormalities also in asymptomatic carriers of aPLs; 3) enhanced FXIIIa that may contribute to atherothrombosis via increased fibrin/fibrinogen cross-linking. Lipid profile, lipid peroxidation and anti-lipoprotein antibodies in thrombotic primary antiphospholipid syndrome. Given the atherogenic lipid profile of SLE, the same possibility was explored in PAPS by comparing high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (CHO), apolipoprotein AI (ApoAI), apolipoprotein B (ApoB), triglycerides (TG), anti-lipoprotein antibodies, beta-2-glycoprotein I complexed to oxidized low-density lipoprotein (oxLDL-2GPI) and C-reactive protein (CRP) in 34 thrombotic PAPS patients compared to 36 thrombotic patients with inherited thrombophilia (IT), to 18 subjects persistently positive for antiphospholipid antibodies (aPL) with no underlying autoimmune or non-autoimmune disorders and to 28 healthy controls. Average concentrations of HDL (p<0.0001), LDL (p<0.0001), CHO (p=0.0002), ApoAI (p=0.002) were lower in PAPS whereas average TRY was higher (p=0.01) than other groups. Moreover PAPS showed higher IgG anti-HDL (p=0.01) and IgG anti-ApoAI (p<0.0001) as well as greater average oxLDL-2GPI (p=0.001) and CRP (p=0.003). Within PAPS, IgG anti-HDL correlated negatively to HDL (p=0.004) and was an independent predictor of oxLDL-2GPI (p=0.009). HDL and ApoAI correlated negatively with CRP (p=0.001 and p=0.007, respectively). IgG anti-HDL may hamper the antioxidant and anti-inflammatory effect of HDL favouring low-grade inflammation and enhanced oxidation in thrombotic PAPS. Indeed plasma 8-epi-prostaglandin F2α (a very specific marker of lipid peroxidation) was significantly higher in 10 patients with PAPS than 10 age and sex matched healthy subjects (p=0.0002) and strongly related to the titre of plasma IgG aCL (r=0.89, p=0.0004). Hence oxidative stress, a major player in atherogenesis, also characterises PAPS. Nitric oxide and nitrative stress in thrombotic primary antiphosholipid syndrome. Oxidative stress goes hand in hand with nitrative stress and to address the latter plasma nitrotyrosine (NT, marker of nitrative stress), nitrite (NO2-) and nitrate (NO3-) were measured in 46 thrombotic PAPS patients, 21 asymptomatic but persistent carriers of antiphospholipid antibodies (PCaPL), 38 patients with inherited thrombophilia (IT), 33 patients with systemic lupus erythematosus (SLE) and 29 healthy controls (CTR). Average crude NT was higher in PAPS and SLE (p=0.01) whereas average plasma NO2- was lower in PAPS and average NO3- highest in SLE (p<0.0001). In PAPS, IgG aCL titer and number of vascular occlusions negatively predicted NO2-, (p=0.03 and p=0.001, respectively) whereas arterial occlusions and smoking positively predicted NO3- (p=0.05 and p=0.005). Moreover CRP (an inflammatory marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade inflammation and both phenomena may have implications for thrombosis and atherosclerosis in PAPS Inflammation and immune activation in thrombotic primary antiphospholipid syndrome. To investigate inflammation and immune activation in thrombotic PAPS high-sensitivity CRP (hs-CRP), serum amyloid A (SAA), oxLDL-2GPI, CRP bound to oxLDL-2GPI (CRP-oxLDL-2GPI) (as inflammatory markers) neopterin (NPT) and soluble CD14 (sCD14) (as immune activation markers) were measured by ELISA in 41 PAPS patients, in 44 patients with inherited thrombophilia (IT) and 39 controls (CTR). Compared to other groups, PAPS presented with higher plasma concentrations of inflammatory, hs-CRP (p=0.0004), SAA (p<0.01), CRP-oxLDL-2GPI (p=0.0004) and immune activation markers, NPT (p<0.0001) and sCD14 (p=0.007). By regression analysis SAA independently predicted thrombosis number (p=0.003) and NPT independently predicted thrombosis type (arterial, p=0.03) and number (p=0.04). These data confirm that low-grade inflammation and immune activation occur and relate to vascular features of PAPS. Antiphosholipid antibodies, haemostatic variables and atherosclerosis in thrombotic primary antiphospholipid syndrome To evaluate whether IgG aCL titre, haemostatic variables and the lipid profile bore any relationship to the intima media thickness (IMT) of carotid arteries high-resolution sonography was applied to the common carotid (CC), carotid bifurcation (CB) and internal carotid (IC) of 42 aPL subjects, 29 with primary thrombotic antiphospholipid syndrome and 13 with persistence of aPL in the absence of any underlying disorder. The following were measured: plasma FNG, vWF, PAI, homocysteine (HC), CHO, TG, HDL, LDL, platelet numbers and aCL of IgG and IgM isotype. By multiple regression analysis, IgG aCL titre independently predicted IMT at all carotid segments examined (p always <0.005). Plasma FNG and HC independently predicted IMT at the CB (p=0.001 and p<0.0001, respectively) and IC (p=0.03 and p<0.0001, respectively). These data strongly support an atherogenic role for IgG aCL in patients with aPL in addition to traditional risk factors. The atherosclerosis hypothesis was investigated in an age and sex-matched case-double-control study including 49 thrombotic PAPS patients (18 M, 31 F, mean age 37 ± 11), 49 thrombotic patients for IT and 49 healthy subjects. Average IMT was always greater in PAPS than control patients (CC: p=0.004, CB: p=0.013, IC: p=0.001). By dividing participants into age tertiles the IMT was greater in the second (CC: p=0.003, CB: p=0.023, IC: p=0.003) and third tertiles (CC: p=0.03, CB: p=0.004, IC: p=0.007). Conclusion: Coagulation activation, fibrinolysis depression, hightened fibrin turnover, oxidative and nitrative stress in parallel with low grade inflammation and immune activation characterise thrombotic PAPS: all these are early atherogenic processes and contribute to the demonstrated premature atherosclerosis that should be considered a clinical feature of PAPS.