18 resultados para SENSITIVE K CHANNELS
Resumo:
Glauconite K-Ar ages (6.880.4; 7.030.4 MY) confirm earlier reports to Upper Tortonian of silt beds near Morgadinho, Luz de Tavira and Tavira. Taking stratigraphical position and age into account it is possible now to correlate these beds with similar ones at Quelfes and Cacela (Formao de Cacela, lower member, ascribed to the upper part of N16 or to NI7 Blow's zone, Globorotalia humerosa - G. dutertrei; Tortonian to Messinian, according to the ostracod fauna). Limit between the above quoted zones is thus placed at about 7 MY. New K-Ar ages greatly improve the knowledge about Upper Miocene in eastern Algarve, and on regional tectonic evolution. This is particulary so in what concerns an intra-Tortonian phase.
Resumo:
In the southern part of Tagus basin, North of paleozoic rocks of Valverde-Senhor das Chagas (near Alccer do Sal) horst, a marine transgression has been recognized, Upper Serravallian, and maybe Lower Tortonian in age. There are no earlier marine deposits, and no younger ones are known either. Paleozoic behaved as a barrier separating two basins, distinct at least since Middle Miocene until Upper Pleistocene. Until now, both were regarded as a single entity, the so-called Sado basin Southwards (Alvalade basin) there has been a single transgression. It was assummed that it was the same one as the former. Indeed it is not definitely so. Later transgression accounts for Esbarrondadoiro Formation, whose deposits have been ascribed to Tortonian or even to Middle Miocene. However they are Upper Messinian to Lower Zanclean. Esbarrondadoiro Formation is younger than Lower Member of Cacela Formation in Algarve and, with even stronger reason, than the upper-most well dated marine levels in Tagus basin. Age of Miocene units dealt with here has been based on small mammals found in marine sands.
Resumo:
Dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Science in Geospatial Technologies
Resumo:
RESMO: Introduo: A anemia de clulas falciformes doena hereditria, com repercusso multi-orgnica, tem grande variabilidade na sua expresso clnica. Da o interesse do estudo de indicadores de prognstico. A investigao realizada foi precedida de um resumo histrico incidindo sobre a compreenso de aspectos fundamentais da doena ao longo dos tempos. Na primeira parte do estudo e aps reviso bibliogrfica, foram referidos dados da fisiopatologia como base para os estudos que integram a presente dissertao. Abordou-se o estado da arte relativamente s complicaes, aos indicadores de prognstico e teraputica utilizada. Objectivos: Constituram objectivos deste estudo realizado numa amostra populacional representativa: identificar as leses a nvel dos sistemas cardio-respiratrio e nervoso central, avaliando-se as respectivas repercusses; avaliar a presena de indicadores de prognstico entre as variveis seleccionadas; estudar a eficcia e toxicidade da HU nos doentes com as formas graves da ACF. Para a prossecuo destes objectivos foram delineados para alm do estudo global trs estudos especficos: Estudo 1- repercusso no sistema cardio-respiratrio; Estudo 2- repercusso no sistema nervoso central; Estudo 3- teraputica com hidroxiureia. Doentes e mtodos: Procedeu-se a um estudo prospectivo e multi-institucional durante um perodo de trs anos tendo-se seleccionado para a amostra, e de acordo com critrios pr-definidos, 30 doentes com ACF na fase estvel da doena, com idades compreendidas entre os sete e os 18 anos, todos de origem africana excepo de um caucasiano. O diagnstico baseou-se em tcnicas de electroforese e estudo molecular que definiu o genotipo da doena e a presena da deleco da -talassmia assim como os haplotipos da amostra populacional. Foram utilizadas diferentes metodologias para avaliar a existncia de leso pulmonar e cerebral. Atravs do estudo estatstico foram seleccionadas diversas variveis como hipotticos indicadores de prognstico. Estudo 1. Para determinar a existncia de leso a nvel pulmonar usaram-se duas metodologias diferentes, a avaliao da funo pulmonar com estudo da saturao da Hb em O2 no sangue arterial e a tomografia computadorizada de alta resoluo. Estudou-se tambm a possvel disfuno cardaca como repercusso da leso pulmonar, atravs do ecocardiograma, e os indicadores de prognstico com significado estatstico para a leso encontrada. Estudo 2. O desenho deste estudo foi sobreponvel ao anterior, mas com metodologia adequada para o SNC. Procedeu-se ao estudo das leses cerebrais por meio de exames imagiolgicos, (RMN-CE e DTC) e de testes psicolgicos. Correlacionaram-se as trs metodologias utilizadas e a importncia de cada uma para a deciso de atitudes teraputicas preventivas. Estudo 3. Consistiu num estudo aberto prospectivo no controlado com nove crianas e adolescentes com formas graves de ACF, com o objectivo de avaliar a eficcia da teraputica com hidroxiureia, durante um perodo de 24 meses. Todos os doentes completaram no mnimo 15 meses de teraputica, com uma dose final mdia de 194 mg/K/dia. Resultados globais: Durante o perodo anterior investigao caracterizou-se a amostra populacional estudada quanto ao fenotipo gentico, clnico e hematolgico de acordo com os critrios utilizados por outros investigadores. Verificou-se: predomnio do haplotipo Bantu na forma homozigtica em 53% dos doentes; nmero total de EVO 3/ano em 87,5% dos doentes; crises de sequestrao em 18,75%; dactilites no primeiro ano de vida em 31,2%; quadro de spsis grave apenas num doente; crises de hiper-hemlise em 50%; e STA em 59,38% dos doentes. Quanto ao fenotipo hematolgico evidenciaram-se como factores de risco reticulocitose (13,1x103/l) e hiperbilirrubinmia (2,5 mg/dl) e como factores de bom prognstico a presena de deleco de um gene da -talassmia em 46,9% dos doentes e valor mdio de Hb 8,1 g/dl. Resultados dos estudos parcelares: Estudo 1. Deste estudo infere-se que a DPR ligeira foi diagnosticada em 70% dos doentes, uma vez que as alteraes da difuso no foram estatisticamente significativas, o estudo dos gases no sangue no evidenciaram resultados anormais e a TCAR evidenciou alteraes em 43,3% dos doentes. Apenas num doente se verificou doena pulmonar obstrutiva relacionada com maior nmero da STA.O estudo da disfuno cardaca encontrada em 86,7% dos doentes no reflecte a repercusso da DPR a nvel cardaco, podendo estar associada s alteraes fisiopatolgicas da prpria anemia crnica. Encontraram-se indicadores de prognstico hematolgicos e clnicos. Entre os primeiros, valores de Hb 8,5 g/dl e de HbF 13% foram considerados indicadores de bom prognstico para a leso pulmonar. Em relao aos parmetros clnicos, as STA no foram consideradas indicadoras de prognstico para a DPR ao contrrio do que se verificou com o nmero de EVO. Pela anlise dos parmetros genticos e socio-econmicos provou-se a ausncia de relao estatisticamente significativa com leso pulmonar. Estudo 2. Pela RMN-CE foram diagnosticados ES em 33,3% com uma localizao preferencial na substncia branca profunda em 26,6% dos doentes. Relativamente aos parmetros hematolgicos seleccionados, o valor mdio da HbF 8,6% constituu um indicador de bom prognstico para o aparecimento de ES, enquanto o valor mdio de leuccitos 12.39x103/l foi considerado um indicador de mau prognstico. No estudo do DTC apenas um doente apresentou aumento da velocidade do fluxo cerebral na ACM igual a 196 cm/segundos, associado a vasculopatia grave. Os testes psicolgicos alterados em 80% dos doentes mostraram ser o mtodo mais sensvel para detectar alteraes do neurodesenvolvimento, mas sem correlao com os ES em 10% dos doentes. Reala-se a baixa percentagem de DTC patolgicos encontrados neste estudo em relao ao nmero elevado de ES e de testes psicolgicos alterados, no se verificando concordncia entre os trs exames. Dos indicadores de prognstico estudados a -talassmia foi considerada um factor de proteco para o coeficiente de inteligncia da escala de Wechsler. Em relao a parmetros clnicos estudados os doentes com maior nmero de EVO, tem em mdia valores inferiores nos testes psicolgicos. Estudo 3. Neste estudo verificou-se que o valor mdio da HbF aumentou significativamente de 7,04% para 13,75,3% (p=0,028) ao fim de 15 meses de teraputica com hidroxiureia. Clinicamente todos os doentes responderam significativamente com uma reduo de 80% no nmero de EVO, 69% no nmero de internamentos, 76% no nmero de dias de hospitalizao e 67% no nmero de transfuses. Deste modo comprovou-se no s a eficcia desta teraputica neste grupo peditrico como tambm a falta de efeitos secundrios significativos. Considera-se a necessidade de estudos mais prolongados e em grande sries, para com segurana se usar a HU antes que a leso orgnica se estabelea, portanto logo nos primeiros anos de vida. Concluso: Na amostra populacional estudada foram evidenciadas leses pulmonares e cerebrais na grande maioria dos doentes que condicionaram a sua qualidade de vida. Foram identificados indicadores de prognstico que podero eventualmente ditar medidas teraputicas precoces com o objectivo de diminuir a morbilidade e a mortalidade neste grupo etrio. Demonstrou-se que a teraputica com a HU foi eficaz e bem tolerada----------ABSTRACT: Background: Sickle cell anemia (SCA), a hereditary disease characterized by pain and lifetime multi-organic lesion, is a challenge for all that work with carriers of this disease. The clinical expression variability of SCA is a constant reality and a problem to be solved in the current world of investigation, for which the knowledge of prognostic indicators responsible for the different aspects of clinical evolution diversity wiil be an added value. The study is preceded by a historical summary of the most important factors in the evolution of SCA, which are in themselves, an incentive for future research. In the first part of the study, after an extensive bibliographical revision, physiopathology data is referred to in general and specifically regarding the target organs, that constituted the base for the studies presented in the dissertation. The state of the art for the complications to be studied, the choice of prognostic indicators and the therapeutics application, were approached for the renewed interest in the theme. Aims: In regard to the investigation, the objective was to study the lesions in the most affected organs of a chosen pediatric group, to investigate prognostic indicators for lung and cerebral lesions and to evaluate the protective effect of hydroxyurea in children with severe outcomes. Patients and methods: A prospective and multi-institutional study was carried out during a three-year period, February 1998 to March 2001, with children and adolescents followed up at a Immunohematology Outpatient Clinic of Dona Estefnia's Hospital, Lisbon. Based in predefined criteria, 30 children with SCA were selected in a stable phase of the disease, aged from seven to 18 years old, all of whom were of African origin with exception of one who was Caucasian. The diagnosis was based on electrophoresis techniques and molecular study that allowed to define the genotype, the presence of deletional alpha-thalassemia as well as haplotypes in the population. Different methodologies were used to evaluate the existence of lung and cerebral lesion. Statistical study of the different variables selected the prognostic indicators. In Study 1, to determine the existence of lung lesion two different methodologies were used: pulmonar function study with arterial blood gases determination; and high resolution computerized tomography. Heart dysfunction as a repercussion of lung lesion was also studied through echocardiography, and prognostic indicators were statistically significant for lesions found. The design of Study 2 was similar to Study 1, but with the appropriate methodology for CNS. After neurological examination, which was normal in all patients (control group), cerebral lesions were studied with imagiologic exams (MRN-CE and TCD) and psychological tests. These three methodologies were correlated and the importance of each one in the decision of the therapeutic profilactic attitudes. Study 3 consisted of a controlled prospective open study in children with severe forms of SCA, with the aim of the evaluating therapeutic effectiveness of hydroxyurea, during a period of 24 months. Results: In the global overall study preceding the Studies 1,2 and 3, there were a prevalence of haplotype Bantu (53%) and other risk factors, namely the number of VOC (87,5%), sequestration crisis (18,75%), dactilytis in first year of life(31,2%), hyperhemolysis crisis (50%) and ATC in more than half of the patients (59,38%). This group of bad prognostic indicators, associated with the population of the lower class according to the Graffar scale, demonstrates the importance of primary health care services, information provided to the children and their relatives, as well as the interest in prophylactic therapeutics, specific screening and prenatal diagnosis. Study 1. It was evident from this study that slight RPD was diagnosed in 70% of the patients, because alterations of the diffusion had no statistical significance and arterial blood gases determinations were normal. Only one patient had restrictive lung disease related with numerous ACS. However ACS was not considered a prognostic indicator for RPD, contrary to the number of EVO. HRTC revealed discreet fibrotic lines that could be related with slight RPD, but the lack of correlation of these two exams (33%) supports the value of lung function tests for precocious diagnosis of RPD. Heart dysfunction was found in 86,7% of patients, does not reflect the repercussion of RPD, but with the physiopathology of chronic anemia. Hematologic and clinical prognostic indicators were found. Good prognostic indicators for the non-evolution of RPD with average Hb values of 8,5 g/dl and average HbF values of 13%, respectively. The genetic and social-economic factors had no statistical significance; nevertheless, they were more prevalent among Bantu haplotype (53,3%) in patients with RPD. Study 2. RMN-CE detected SI in 33,3% of the patients, with preferential location in deep white substance in 26,6% and in front lobe in 20%. This distribution can be related to structural aspects of the brain and with the high sensibility of this organ to hypoxia. From the hematological parameters selected, average HbF value 8,6% and average leucocyte count 12.39x103/l were prognostic indicators with different meaning to SI. The increase in the total bilirubin related to hyperhemolysis clinically explains the genesis of SI In the TCD study, only one patient had increased cerebral flow speed >196 cm/sec in CMA, which corresponded to serious vasculopathy in AngioMR. This patient never present previously neurological symptoms and had several hyperhemolysis crisis and VOC as risk factors. Low percentage of pathological TCD in this study, in relation to the high number of SI and altered tests, although without correlation among the three exams, is probably attributed to factors related to the methodology, aspects of cerebral physiopathology or perhaps a sign of good prognostic if the duration of study had not been so short. TCD should be used as a screening method in the age groups with higher risk of AVC and should never be considered separately in prophylactic therapeutics indication. Psychological tests were the most sensitive method to detect neurodevelopment impairment; in 80% of patients the neuropsychologics tests were altered, but without correlation with SI (10%). Since SI can become evident during the first two years of life and develop with time, the first psychological tests should be carried out between 3 and 5 years of age to timely be referred to special education and stimulation programs. Prognostic indicators to psychological tests were also found: alpha-thalassemia was found to be a protection factor of the IQ, just as other hematologic factors (hematocrit, MGCV and erythrocytes count). In relation to clinical parameters, although without statistical significance, patients with larger number of VOC had average lower scores versus the average in tests, except in TP. Results from different studies were conclusive as to the type of lesion found and the importance of prognostic indicators. Study 3. All the patients completed a minimum of 15 months therapeutic treatment with the final average daily dose of 194 mg/kg/day. The average value of the fetal hemoglobin increased significantly from 7,03,9% to 13,75,3% (p=0.028). The HbF average values increased from 6% to 15% after 15 months of therapeutic treatment. Clinically there was a reduction of 80% in the number of VOE , 69% in the number of hospitalization, 76% in the number of days of hospitalization and 67% in the number of transfusions. Once again the effectiveness of this treatment in this pediatric group, as well as the lack of any significant secondary effects, was evident. The study confirms the need for further detailed research in order to safely effect the appropriate treatment prior to the development of organic lesions, which ideally should be in the first year of life. Conclusions: These results allow us to clarify the importance of either pulmonary lesions or either nervous central system impairment among patients, children and adolescents, with sickle cell anemia. These lesions were demonstrated in most of the patients studied compromising their quality of life and the mortality. The treatment with HU is proved to be effective and having low toxicity.
Resumo:
Dissertao para obteno do grau de Doutor em Cincias da Comunicao Especialidade de Comunicao e Cultura
Resumo:
RESUMO: A esporulao em Bacillus subtilis controlada por uma cascata de factores sigma da polimerase do RNA. F e E controlam os estgios precoces do desenvolvimento no pr-esporo e na clula me, respectivamente. Numa fase intermdia da diferenciao, quando a clula me acaba por envolver o pr-esporo, F substitudo por G e E substitudo por K. Vrios mecanismos asseguram que a actividade dos diferentes factores sigma seja confinada a uma janela temporal precisa na clula adequada. Neste estudo, investigmos a funo de um factor anti-G, designado por CsfB. Mostramos que para alm da sua funo de inibio da actividade do factor G em clulas pr-divisionais, CsfB tambm necessrio na clula me num estgio tardio do desenvolvimento. Mostramos que a expresso de csfB activada na clula me a partir de um promotor dependente de K. Contudo, demonstramos que CsfB interage directamente com E e no com K, e que CsfB suficiente para inibir a actividade transcricional dependente de E em clulas vegetativas de B. subtilis. Propomos que CsfB contribui para reduzir o perodo dependente de E, na linha de expresso gentica da clula me, desse modo reduzindo a sobreposio entre os regules E e K e aumentado a fidelidade do processo de desenvolvimento. Uma segunda protena, YabK, partilha semelhana estrutural com CsfB. YabK produzida no pr-esporo sob o comando de F, e necessria para a esporulao. YabK contribui para a transio F/G no programa gentico do pr-esporo, porque uma mutao que torna F sensvel a CsfB ultrapassa parcialmente a funo de YabK na esporulao. No entanto, YabK e CsfB funcionam por mecanismos diferentes, uma vez que YabK no liga directamente a F.---------ABSTRACT: Gene expression during spore development in Bacillus subtilis is governed by a cascade of RNA polymerase sigma factors. F and E control the early stages of development in the forespore and in the mother cell, respectively. At an intermediate stage in the differentiation process, when the larger mother cell finishes engulfment of the smaller forespore, F is replaced by G and E is replaced by K. Several mechanisms ensure the proper timing of activation of the cell type-specific sigma factors. Here, we have investigated the funtion of an anti-sigma G factor, called CsfB. We show here that in addition to its role in inhibiting G in pre-divisional cells, CsfB is also required in the mother cell at a late stage in development. We show that the expression of csfB is activated in the mother cell from a K-specific promoter. However, we demonstrate that CsfB binds directly to E but not to K in a yeast two-hybrid assay, and that CsfB is sufficient to inhibit E-dependent transcriptional activity in vegetative cells of B. subtilis. We posit that CsfB contributes to shutting off the early, E-controlled period in the mother cell line of gene expression, thus reducing the overlap between deployment of the E and K regulons and increasing the fidelity of the developmental process. A second protein, YabK, shares structural similarity with CsfB. YabK is produced in the forespore under F control, and is required for efficient sporulation. YabK contributes to the transition from the F- to the G-dependent period of gene expression, because a mutation that renders F sensitive to CsfB partially bypasses the need for YabK. Yet, YabK and CsfB must function in the control of sigma factor activity by different mechanisms because YabK does not bind directly to F.
Resumo:
Dissertao para obteno do Grau de Mestre em Engenharia Geolgica (Georrecursos)
Resumo:
Ligand K-edge XAS of an [Fe3S4]0 model complex is reported. The pre-edge can be resolved into contributions from the 2Ssulfide, 3Ssulfide, and Sthiolate ligands. The average ligand-metal bond covalencies obtained from these pre-edges are further distributed between Fe3+ and Fe2.5+ components using DFT calculations. The bridging ligand covalency in the [Fe2S2]+ subsite of the [Fe3S4]0 cluster is found to be significantly lower than its value in a reduced [Fe2S2] cluster (38% vs 61%, respectively). This lowered bridging ligand covalency reduces the superexchange coupling parameter J relative to its value in a reduced [Fe2S2]+ site (-146 cm-1 vs -360 cm-1, respectively). This decrease in J, along with estimates of the double exchange parameter B and vibronic coupling parameter 2/k-, leads to an S ) 2 delocalized ground state in the [Fe3S4]0 cluster. The S K-edge XAS of the protein ferredoxin II (Fd II) from the D. gigas active site shows a decrease in covalency compared to the model complex, in the same oxidation state, which correlates with the number of H-bonding interactions to specific sulfur ligands present in the active site. The changes in ligand-metal bond covalencies upon redox compared with DFT calculations indicate that the redox reaction involves a two-electron change (one-electron ionization plus a spin change of a second electron) with significant electronic relaxation. The presence of the redox inactive Fe3+ center is found to decrease the barrier of the redox process in the [Fe3S4] cluster due to its strong antiferromagnetic coupling with the redox active Fe2S2 subsite.
Resumo:
Dissertao para obteno do Grau de Mestre em Engenharia Civil (Perfil de Estruturas e Geotecnia)
Resumo:
Inorg. Chem., 2003, 42 (4), pp 938940 DOI: 10.1021/ic0262886
Resumo:
RESUMO: O corpo carotdeo (CB) um pequeno rgo sensvel a variaes na PaO2, PaCO2 e pH. As clulas tipo I (clulas glmicas) do corpo carotdeo, as unidades sensoriais deste rgo, libertam neurotransmissores em resposta s variaes dos gases arteriais. Estes neurotransmissores atuam quer em recetores pr-sinpticos, localizados nas clulas tipo I, quer em recetores pssinpticos, localizados nas terminaes do nervo do seio carotdeo, ou em ambos. A activao dos recetores pr-sinpticos modula a atividade do corpo carotdeo, enquanto que, a activao dos recetores ps-sinpticos, de carater excitatrio, desencadeia um aumento da frequncia de descarga das fibras do CSN, com subsequente despolarizao dos neurnios do gnglio petroso, e posterior despolarizao de um grupo especfico de neurnios do centro respiratrio central, desencadeando, como resposta final, hiperventilao. Estes recetores pr- e ps-sinpticos podem ser classificados em ionotrpicos ou metabotrpicos, estando os ltimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variaes dos gases arteriais so detetadas pelo CB no se encontra ainda completamente elucidado, mas tem sido sugerido que alteraes nos nveis de cAMP estejam associadas ao mecanismo de deteo de variaes de O2 e CO2. Os nveis de cAMP podem ser regulados atravs da sua via de sntese, mediada por dois tipos de adenilatos ciclases: tmAC sensvel aos eurotransmissores e adenilato ciclase solvel (sAC)sensvel a variaes de HCO3/CO2, e pela sua via de degradao mediada por fosfodiesterases. A via de degradao do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo teraputico para o tratamento de patologias do foro respiratrio (e.g. asma, hipertenso pulmonar, doena pulmonar obstructiva crnica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertao partiu da hiptese de que a actividade do CB manipulada por frmacos, que interferem com a via de sinalizao do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransduo do CB de rato, e determinar se a actividade dos enzimas responsveis pela via de sinalizao do cAMP ou no regulada por variaes de O2/CO2. Assim, a relevncia deste trabalho a de estudar e identificar possveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratrio. A primeira parte do presente trabalho, centrou-se na caracterizao farmacolgica da PDE4 no CB e em tecidos no quimiorecetores (e.g. gnglio cervical superior e artrias cartidas), e na observao do efeito de hipxia aguda na acumulao dos nveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificao de cAMP foi efectuada por tcnica imunoenzimtica (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, no especficos (IBMX) e especficos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos nveis de cAMP acumulados, em situaes de normxia (20%O2/5%CO2) e hipxia (5%O2/5%CO2). A caracterizao das PDE no gnglio cervical superior foi aprofundada, utilizando-se a tcnica de transferncia de energia de ressonncia por fluorescncia (FRET) em culturas primrias de neurnios, na presena de inibidores no especficos (IBMX) e especficos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, atravs de RT-qPCR, as alteraes na expresso de PDE3A-B e PDE4A-D, no gnglio cervical superior, em resposta a diferentes percentagens de oxignio. Na segunda parte do trabalho investigou-se a via de sntese do cAMP no CB em resposta a variaes na concentrao de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterizao da sAC, dado que a sua actividade regulada por variaes de HCO3/CO2. A caracterizao da expresso e regulao da sAC, em resposta a variaes de HCO3/CO2 ,foi efectuada no CB e em tecidos no quimioreceptores perifricos (e.g. gnglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente atravs da quantificao dos nveis de cAMP (quantificao por EIA), induzidos por diferentes concentraes de HCO3/CO2, na presena de MDL-12,33-A, um inibidore da tmAC. A expresso das isoformas da tmAC no CB e gnglio petroso foi determinada por RT-qPCR. Adicionalmente, estudmos a contribuio relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alteraes: 1) nos nveis de cAMP (quantificado por EIA) na presena de diferentes concentraes de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativao da protena cinase A (PKA, FRET baseado em sensores) em clulas tipo I do CB; e 3) na frequncia de descarga do CSN (registos) na presena e ausncia de ativadores e inibidores da sAC,tmAC e PKA. Por ltimo, foi caracterizada a expresso e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) atravs de tcnicas de RT-qPCR e EIA. A expresso das isoformas da tmAC foi aprofundada no locus coeruleus atravs de RT-qPCR. Por fim, comparmos a contribuio da tmAC e sAC nos nveis de cAMP no locus coeruleus em condies de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 est funcional no corpo carotdeo, artrias cartidas e gnglio cervical superior de rato, embora a PDE2 s se encontre funcional neste ltimo; 2) Os efeitos dos inibidores de PDE nos nveis de acumulao de cAMP foram exacerbados em situaes de hipxia aguda no CB e artrias cartidas, mas foram atenuados no gnglio cervical superior; 3) No gnglio cervical superior, diferentes tipos de clulas apresentaram uma caracterizao especfica de PDEs, sugerindo uma subpopulao de clulas neste gnglio com funes fisiolgicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gnglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubaes de gnglio cervical superior, em diferentes percentagens de oxignio, no alteraram (no regularam) significativamente a expresso das diferentes isoformas de PDE neste rgo; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expresso no CB comparativamente aos restantes orgos estudados, exceptuando os testculos, orgo controlo. Variaes de HCO3/CO2 de 0/0 para 24/5 aumentaram os nveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentraes acima dos 24mM HCO3/5%CO2 no induziram alteraes nos nveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condies fisiolgicas (normocapnia) e que este enzima no desempenha qualquer papel na deteo de situaes de hipercapnia; 6) No CB, a expresso das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 mais elevada comparativamente expresso da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500M, 25-ddADO, 30-300M, SQ 22536, 200M) e da sAC (KH7, 10-100M) para estudar a contribuio relativa destes enzimas na acumulao do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulao dos nveis de cAMP em condies de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condies de 12 mM HCO3/2.5%CO2 do que em condies de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situaes de hipocapnia; 8) No se observaram variaes nos nveis de cAMP em resposta a diferentes concentraes de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitrio induzido por ddADO e KH7 foi sobreponvel aps 5 ou 30 minutos de incubao em todas as concentraes de HCO3/CO2 estudadas; 9) Por ltimo, verificou-se um aumento na frequncia da descarga do nervo do seio carotdeo entre as condies de normocapnia e hipercapnia acdica. Ao contrrio do KH7 (10M), o 25-ddADO reduziu significativamente a frequncia de descarga do nervo, quer em condies de normocapnia quer de hipercapnia acdica. Contudo, no se verificou aumento na frequncia de descarga do nervo entre normocapnia e hipercapnia isohdrica, sugerindo que a sensibilidade hipercapnia no CB mediada por variaes de pH. Em concluso, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalizao do cAMP possam ser bons alvos teraputicos em condies particulares, a sua actividade no especfica para o CB. Os resultados sugerem ainda que o cAMP no um mediador especfico da transduo hipercapnia neste orgo. Contudo, os nossos resultados demonstraram que os nveis de cAMP so mais elevados em condies fisiolgicas, o que sugere que o cAMP possa ter uma funo homeosttica neste orgo. Por ltimo, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condies de hipercapnia, no so observveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ.
Resumo:
A thermal Energy Storage Unit (ESU) could be used to attenuate inherent temperature fluctuations of a cold finger, either from a cryocooler working or due to sudden income heat bursts. An ESU directly coupled to the cold source acts as a thermal buffer temporarily increasing its cooling capacity and providing a better thermal stability of the cold finger (Power Booster mode). The energy storage units presented here use an enthalpy reservoir based on the high latent heat of the liquid-vapour transition of neon in the temperature range 38 - 44 K to store up to 900 J, and that uses a 6 liters expansion volume at RT in order to work as a closed system. Experimental results in the power booster mode will be described: in this case, the liquid neon cell was directly coupled to the cold finger of the working cryocooler, its volume (12 cm3) allowing it to store 450 J at around 40 K. 10 W heat bursts were applied, leading to liquid evaporation, with quite reduced temperature changes. The liquid neon reservoir can also work as a temporary cold source to be used after stopping the cryocooler, allowing for a vibration-free environment. In this case the enthalpy reservoir implemented (24 cm3) was linked to the cryocooler cold finger through a gas gap heat switch for thermal coupling/decoupling of the cold finger. We will show that, by controlling the enthalpy reservoirs pressure, 900 J can be stored at a constant temperature of 40 K as in a triple-point ESU.
Resumo:
Cryocoolers have been progressively replacing the use of the stored cryogens in cryogenic chains used for detector cooling, thanks to their higher and higher reliability. However, the mechanical vibrations, the electromagnetic interferences and the temperature fluctuations inherent to their functioning could reduce the sensors sensitivity. In order to minimize this problem, compact thermal energy storage units (ESU) are studied, devices able to store thermal energy without significant temperature increase. These devices can be used as a temporary cold source making it possible to turn the cryocooler OFF providing a proper environment for the sensor. A heat switch is responsible for the thermal decoupling of the ESU from the cryocoolers temperature that increases when turned OFF. In this work, several prototypes working around 40 K were designed, built and characterized. They consist in a low temperature cell that contains the liquid neon connected to an expansion volume at room temperature for gas storage during the liquid evaporation phase. To turn this system insensitive to the gravity direction, the liquid is retained in the low temperature cell by capillary effect in a porous material. Thanks to pressure regulation of the liquid neon bath, 900 J were stored at 40K. The higher latent heat of the liquid and the inexistence of triple point transitions at 40 K turn the pressure control during the evaporation a versatile and compact alternative to an ESU working at the triple point transitions. A quite compact second prototype ESU directly connected to the cryocooler cold finger was tested as a temperature stabilizer. This device was able to stabilize the cryocooler temperature (( 40K 1 K) despite sudden heat bursts corresponding to twice the cooling power of the cryocooler. This thesis describes the construction of these devices as well as the tests performed. It is also shown that the thermal model developed to predict the thermal behaviour of these devices, implemented as a software,describes quite well the experimental results. Solutions to improve these devices are also proposed.
Resumo:
World population is increasing at an alarming rate while food productivity is decreasing due to the effect of various abiotic stresses. Soil salinity is one of the most important abiotic stress and a limiting factor for worldwide plant production. In addition to its important effects on yield, salt stress affects numerous cellular activities, including cell wall composition, photosynthesis, protein synthesis, ions and organic solutes. Up to 20% of the irrigated arable land in arid and semiarid regions is already salt affected and is still expanding. Improving salt tolerant varieties is of major importance, and efforts should be focused on finding adaptive mechanisms which are involved in salinity tolerance. In this study, several spelt wheat (Triticum aestivum var. Spelta) genotypes and one cultivar of modern bread wheat were used to screen them for salt tolerance. Spelt is an old-European cereal crop currently attracting renewed interest as a food grain because it is said to be harder than wheat and requires less fertilizer. Spelt wheat is also becoming very attractive genetic source by plant breeders due to its wide adaptation ability to various stressful conditions such as soil salinity. In this study morphological parameters (e.g., leaf appearance; shoot elongation), dry matter production, mineral nutrients (especially Na and K), and activity of antioxidative enzymes were measured to select superior genotypes of spelt for salt tolerance. The results showed that Spelt genotype Sp41 is a salt sensitive genotype and genotypes Sp69, Sp96 and Sp912 are good candidates for salt tolerant genotypes.
Resumo:
Field Lab Entrepreneurial Innovative Ventures
