Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-13T17:36:44Z No. of bitstreams: 1 andersondutrademelo.pdf: 664118 bytes, checksum: 3695f89c95d5ed609cc3f5bd69a7f406 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T12:58:01Z (GMT) No. of bitstreams: 1 andersondutrademelo.pdf: 664118 bytes, checksum: 3695f89c95d5ed609cc3f5bd69a7f406 (MD5) / Made available in DSpace on 2016-10-22T12:58:01Z (GMT). No. of bitstreams: 1 andersondutrademelo.pdf: 664118 bytes, checksum: 3695f89c95d5ed609cc3f5bd69a7f406 (MD5) Previous issue date: 2008-02-22 / O diabetes reduz a secreção de catecolaminas em resposta a variações glicêmicas, acentuando o quadro de descontrole metabólico dos indivíduos doentes. Diversos estudos têm demonstrado que a hiperglicemia é a principal causa dos problemas decorrentes da instalação do diabetes. Desta forma, o objetivo deste trabalho foi analisar o efeito do diabetes sobre o processo de síntese e secreção de catecolaminas e se o tratamento com insulina reverte as modificações causadas pela doença. Métodos e Resultados: Foram usados ratos Wistar, machos, com 60 dias. O diabetes foi induzido pela injeção intravenosa de estreptozotocina na proporção de 50 mg/Kg de animal. O grupo controle recebeu injeção de solução tampão. Para determinar o protocolo de tratamento com insulina, estudamos o padrão de ingestão alimentar e de variações glicêmicas durante um dia, medindo estes dois parâmetros de hora em hora, durante 24h consecutivas. Foi estabelecido o tratamento com insulina NPH humana na dose de 5U diárias, 1 U às 13 h e 4 U às 19h. Após 15 dias da indução, os animais foram sacrificados e as glândulas adrenais foram retiradas. Com a intenção de caracterizar o efeito do diabetes sobre alguns parâmetros bioquímicos correntemente utilizados como marcadores da doença, foram medidos os níveis de frutosamina, triglicerídeos e colesterol e suas frações. Foi quantificado o conteúdo total de catecolaminas e a secreção basal e a estimulada por altas concentrações de potássio, carbamilcolina e cafeína. As catecolaminas foram dosadas por método fluorimétrico. A expressão de tirosina hidroxilase (TH), enzima reguladora da via de síntese de catecolaminas, foi avaliada por Western Blot. A glicemia foi de 82,82 ± 1,24 mg/dl, 405,74 ± 23,35 mg/dl e 103,72 ± 6,79 mg/dl nos animais controle, diabéticos e diabéticos tratados com insulina (DTI). A variação na massa corporal durante o período experimental foi negativa nos ratos diabéticos, ou seja, eles emagreceram 6,1 ± 3,84 g, enquanto que os animais controles e os diabéticos tratados, aumentaram seus pesos, em média, 36,34 ± 1,8 g e 43,32 ± 3,79 g, respectivamente. O diabetes modificou os níveis de colesterol total, LDL e VLDL, modificações que foram corrigidas pelo tratamento com insulina. Não houve diferença, entre controles e diabéticos, nos níveis de triglicérides, frutosamina e LDH. O tratamento com insulina reduziu significativamente os níveis de frutosamina. O conteúdo total de catecolaminas foi 21,14% menor nos diabéticos sem tratamento, quando comparado aos controles (p<0,05). O tratamento com insulina recuperou os estoques de catecolaminas dos ratos diabéticos. A expressão de TH foi similar em todos os grupos experimentais. A secreção basal e a estimulada por altas concentrações de K+ e por carbamilcolina foi reduzida pelo diabetes em 24,3%, 42,28% e 28,9%, respectivamente. Este efeito não foi corrigido pelo tratamento com insulina. A secreção estimulada pela mobilização de Ca2+ de pools intracelulares sensíveis à cafeína não é afetada pelo diabetes. Os nossos resultados nos permitem concluir que o diabetes afeta a secreção basal de catecolaminas e a estimulada via membrana plasmática e que isto não é determinado pela redução dos estoques de catecolaminas, nem é revertido pelo tratamento com insulina exógena. / The diabetes reduces the catecholamine secretion with hypoglycemic episodes, to turning worse the metabolic disorder of diabetic people. Several studies have shown that hyperglycemia has pivotal role in diabetic complication development. This work studied the effect of diabetes on catecholamine synthesis and secretion and the effects of insulin treatment. Methods and results: 60 days old, male Wistar rats were used. The diabetes was induced by a single intravenous injection of streptozotocin (50mg/Kg body weight). The control group received buffer injection. To establish the protocol of insulin treatment the food consumption and the blood glucose levels were measured during 24h from hour to hour. The insulintreated diabetic rats received human NPH insulin at 1pm (1U) and 7pm (4U). After 15 days of the streptozotocin injection, the rats were sacrificed and the adrenal glands withdrew. To evaluate the effect of diabetes and the insulin treatment, fructosamine, triglycerides, total cholesterol, HDL, LDL and VLDL were measured. The total catecholamine content of adrenal gland and the basal and stimulated catecholamine secretion was quantified. The experiments of stimulated catecholamine secretion were performed with high potassium, carbachol and caffeine. The catecholamines measurement was done by fluorimetric method. The expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, was analyzed by western blotting. The glicemia was 82.82 ± 1.24 mg/dl, 405.74 ± 23.35 mg/dl and 103.72 ± 6.79 mg/dl in control, diabetic and insulin-treated diabetic groups, respectively. The body mass of diabetic rats was reduced in 6.1 ± 3.84g and increased on control and insulin-treated diabetic rats, in 36.34 ± 1.8g and 43.32 ± 3.79g, respectively. The diabetes changed total cholesterol, LDL and VLDL plasma levels, alteration reversed by insulin treatment. The triglycerides, fructosamine and LDH levels were not affected by diabetes. The insulin-treated rats showed significant reduction of fructosamine levels. The diabetic rats presented a significant reduction, 21.14% on the catecholamine content when compared to the control group, p<0.05. The insulin treatment recovered the catecholamine stores. The TH expression was similar in all three experimental groups. The diabetes reduced the basal and stimulated catecholamine secretion by 24.3%, 42.28% (high K+) e 28.9% (carbachol).The catecholamine secretion stimulated by mobilization of intracellular Ca2+ pools was not affected by diabetes or insulin treatment. Our results show that diabetes reduces the catecholamine secretion, and this is not consequence of reduction on cell catecholamine stores and it is not reversed by insulin therapy.

Estudos apontam a neuroinflamação como um dos possíveis agravantes do mal de Parkinson. Aqui investigamos a susceptibilidade de duas linhagens de camundongos selecionados quanto a suas capacidades de produzir alta (AIRmax) ou baixa (AIRmin) resposta inflamatória aguda aos agentes neurotóxicos 1-Metil-4-Fenil-1,2,3,6-Tetrahidropirimidina (MPTP) (Tratamento agudo: 4 x 20 mg/kg ou 5 x 20 mg/kg s.c. a cada 2 horas em um único dia) e rotenona (tratamento crônico: infusão s.c. contínua por bomba osmótica Alzet a 3, 6 e 12 mg/kg/dia por 28 dias ou sub-crônico: injeção i.p na dose de 3 mg/kg/dia durante 10 dias). A avaliação motora contou com Rotarod empregando paradigma de 5 minutos e rotação crescente de 5 a 50 rpm. A lesão foi quantificada por imunohistoquímica para tirosina hidroxilase em cortes de estriado e substância negra. Os resultados sugerem que ambas as linhagens AIRmax e AIRmin, são resistentes a lesão neuronal por MPTP ou rotenona. Conjecturamos que as linhagens se diferenciam quanto a resposta inflamatória periférica, mas não de origem central (neuroinflamação). / Studies suggest that neuroinflammatory processes are involved in Parkinson\'s disease. Here we investigated the susceptibility of two inbred strains of mice selected for their ability to produce high (AIRmax) or low (AIRmin) acute inflammatory response to neurotoxins 1-Methyl-4-phenyl-1,2,3,6-Tetrahydropyridine (MPTP) (4 x 20 mg/kg or 5 x 20 mg/kg s.c. every 2 hours in a single day) and rotenone (chronic treatment: continuous s.c. infusion of 3, 6 and 12 mg/kg/day by Alzet osmotic pump for 28 days or sub-chronic treatment: i.p. injection at a dose of 3 mg/kg/day for 10 days). The animals were evaluated by rotarod apparatus in sessions of 5 minutes and constantly increasing speed from 5 to 50 rpm. The lesion was quantified by tyrosine hydroxylase immunohistochemistry in sections of striatum and substantia nigra. The results suggest that both strains AIRmax and AIRmin are resistant to neuronal damage by MPTP or rotenone. We conjecture that the strains differ concerning the peripheral inflammatory response, but not in neuroinflammatory mechanisms.

O objetivo central do presente estudo foi avaliar se os agonistas adrenérgicos a2 e imidazólicos, importantes drogas de ação anti-hipertensiva utilizadas na clínica médica, podem atuar também na região comissural do núcleo do trato solitário (NTSc), o qual constituí uma importante região do bulbo envolvida no controle cardiovascular. Em ratos Wistar adultos, observamos que a hipotensão produzida pela injeção de moxonidina no 4º V foi reduzida após a lesão eletrolítica do NTSc. Ademais, a injeção de moxonidina no NTSc reduziu a pressão arterial média (PAM), a frequência cardíaca (FC) e a atividade simpática (AS). A injeção de antagonistas adrenérgicos (ioimbina ou RX821002) no NTSc foi capaz de bloquear as respostas hipotensora e de simpatoinibição produzida pela moxonidina no NTSc. A injeção bilateral de moxonidina na região RVL/C1 reduziu PAM e AS de maneira mais intensa do que as injeções no NTSc. Em concordância com os resultados apresentados, mostramos que a atividade elétrica dos neurônios da região do RVL/C1 foi reduzida após a injeção de moxonidina no NTSc. Concluímos que a moxonidina pode produzir os seus efeitos anti-hipertensivos atuando também sobre o NTSc. / The main objective of this study was to evaluate whether the a2 adrenergic and imidazoline agonists, important antihypertensive drugs used in clinical medicine, may also act in the commissural region of the nucleus of the solitary tract (cNTS), which constitutes an important region of brainstem involved in cardiovascular control. In adult rats, the hypotension elicited by central injections of moxonidine was reduced after electrolytic lesion of cNTS. Furthermore, injection of moxonidine into the cNTS reduced mean arterial pressure (MAP), heart rate (HR) and sympathetic activity (SNA). Injection of the a2 adrenergic antagonist (RX821002 or yohimbine) into the cNTS completely blocked the hypotension and sympathoinhibition responses produced by moxonidine into the cNTS. Bilateral injection of moxonidine in the RVLM/C1 produced huge effects on MAP and SNA in comparison of cNTS injections. In agreement with our results, moxonidine-injected into the cNTS also elicited a reduction in the activity of RVLM/C1 neurons. Our conclusion is that moxonidine may produce their antihypertensive effects also acting on cNTS neurons.

A glândula pineal de mamíferos sintetiza o hormônio melatonina exclusivamente durante o período noturno. A síntese é regulada primordialmente pela via retino-hipotalâmico-pineal e modulada por vários fatores, incluindo o sistema peptidérgico. Assim, o papel da insulina na regulação da síntese de melatonina foi estudado a partir da realização de culturas de glândulas pineais estimuladas com noradrenalina, insulina e noradrenalina associada à insulina, em culturas temporizadas ou não pela noradrenalina, avaliando: a produção de melatonina por HPLC com detecção eletroquímica; as atividades das enzimas envolvidas na síntese da melatonina, por radiometria; assim como, a expressão gênica das enzimas quantificada por Real-Time PCR. Os resultados sugerem uma interação entre as vias de sinalização da noradrenalina e da insulina, com a respectiva potencialização da síntese da melatonina, induzida por noradrenalina, observada pela adição da insulina, efeito esse, que se dá, provavelmente através de mecanismos pós-transcricionais. / The mammalian pineal gland synthesizes the neurohormone melatonin exclusively during the dark phase. Its synthesis is primarily regulated via a retino-hypothalamic-pineal pathway and modulated by many factors, including the peptidergic system. Thus, the role of insulin on the regulation of melatonin synthesis was studied using cultured gland treated with norepinephrine, insulin and norepinephrine associated to insulin. The cultures were also synchronized or not by norepinephrine. Melatonin content was assayed by HPLC (High Performance Liquid Chromatography) with electrochemical detection, melatonin synthesis enzymes activities by radiometry and enzymes gene expressions by Real-Time PCR. The results suggest an interaction between norepinephrine and insulin signaling pathway, with insulinic potentialization on melatonin synthesis norepinephrine-mediated, and this effect, seems to accurs potentially through post-transcriptional events.

Orientador: Andre Luís Berteli Ambrósio / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:57:05Z (GMT). No. of bitstreams: 1 Fala_AngelaMaria_M.pdf: 4666864 bytes, checksum: 3bcb145c1dc353b15b43b75e5723a310 (MD5) Previous issue date: 2013 / Resumo: A adaptação das células cancerosas ao microambiente é o ponto central que leva ao fenótipo invasivo e metastático, e é garantida principalmente através do controle preciso da expressão gênica. A resposta às necessidades energéticas e biossintéticas e principalmente à disponibilidade de oxigênio intracelular, por exemplo, é em grande parte mediada pelo fator de transcrição induzido por hipóxia 1 (HIF-1). HIF-1 é um heterodímero composto pelas subunidades ? e ?, que respondem a sequência consenso (5'-RCGTG-3') e ativam a transcrição de mais de 100 genes envolvidos em diversos aspectos cruciais da biologia tumoral, incluindo angiogênese, metabolismo de glicose, diferenciação celular, apoptose e resistência a radio e quimioterapias. São conhecidas três isoformas da subunidade ? (1 a 3) e todas se heterodimerizam com a subunidade ?. No geral, HIFs são constituídas de diferentes domínios funcionais, como de ligação ao DNA, de heterodimerização, transativação e degradação. Atualmente, pouco se sabe sobre os mecanismos estruturais e funcionais dos domínios da HIF- 1, deste modo este trabalho objetivou o estudo estrutural destes domínios. Os domínios bHLH, Pas-1 e Pac de HIF-1? e HIF-? em diferentes combinações entre si e o domínio Pac da HIF-3? foram clonados, as proteínas foram expressas em sistema bacteriano e purificadas por diferentes técnicas cromatográficas. Diversas destas construções se mostraram insolúveis ou suscetíveis a degradação, enquanto outras foram purificadas com sucesso. As construções Pac, um exemplo de sucesso na produção, foram submetidas a ensaios de anisotropia de fluorescência e ressonância magnética nuclear, o que nos permitiu a caracterização dos perfis de interação entre as várias combinações de heterodimerização. Neste contexto, os resultados mostram que o equilíbrio dinâmico da interação entre Pac-1? com a subunidade -1? é alcançado imediatamente, enquanto que para a interação entre Pac-3? e -1?, são necessários pelo menos 30 horas de incubação. O mesmo pode ser extraído da caracterização da interação direta entre Pac-1? e Pac-3?. Nos experimentos de RMN, foi possível identificar a região de interação entre as subunidades -1? e -3? com a subunidade ?, separadamente. Ambas as subunidades ? interagem com a Pac-1? na região das fitas-beta 1 e 5 e no loop entre as fitas 4 e 5. Em conjunto, estes resultados impactam no mecanismo de antagonização de HIF-3? na atividade transcricional de HIF-1?. Houve ainda a formação de monocristais da subunidade Pac-3?, que foram submetidos a experimentos preliminares de difração de raios X, que apesar de resultar em dados anisotrópicos e insuficientes para resolução estrutural, permitiram a caracterização dos parâmetros cristalinos, incluindo a presença de um alto conteúdo de solvente. Adicionalmente, são também apresentados os resultados obtidos visando a expressão e cristalização das Prolilhidroxilases (PHDs) isoformas 1 a 4, durante estágio de seis meses no Structural Genomics Consortium (SGC), da Universidade de Oxford, na Inglaterra. Foram expressas de maneira solúvel e purificadas, diversas construções das isoformas 1 e 3 das PHDs humanas. Cristais foram obtidos, porém estes foram determinados como sendo de compostos inorgânicos presentes na condição de cristalização. Como resultado final, está sendo estabelecida uma colaboração entre o nosso grupo e o SGC para que os estudos estruturais com PHDs se estendam e sejam realizados em nosso laboratório aqui no Brasil / Abstract: The adaptation process of cancer cells to the microenvironment is the central point leading to the invasive and metastatic phenotypes, and is guaranteed mainly through the precise control of gene expression. The cell response to the energetic and biosynthetic needs and especially to the availability of intracellular oxygen is mediated by the hypoxia inducible transcription factor 1, or HIF-1. HIF-1 functions as a heterodimer composed by subunits ? and ?, binding to responsive elements with the consensus sequence 5'-RCGTG-3 ', thus activating the transcription of more than 100 genes involved in many crucial aspects of tumor biology, including angiogenesis, metabolism glucose, cell differentiation, apoptosis, and resistance to radiotherapy and chemotherapy. There are three known isoforms of the ? subunit (1, 2 and 3) and all heterodimerize with the ? subunit. HIFs are composed of different functional domains, such as the DNA binding domain, the heterodimerization, transactivation and the oxygen-dependent degradation domains. Currently, little is known about the mechanisms of structural and functional domains of HIF-1, thus this work was to study these structural domains. The domain (bHLH, Pas-1 and Pac) of HIF-1? and HIF-? in different combinations with each other and Pac domain of HIF-3? were cloned, the proteins were expressed in bacterial system and purified by various chromatographic techniques. Several of these constructs proved insoluble or susceptible to degradation, while others were purified successfully. The constructs Pac, an example of success in production, were tested for fluorescence anisotropy and nuclear magnetic resonance, which allowed us to characterize the profiles of the interaction between the various combinations of heterodimers. In this context, the results show that the dynamic equilibrium of the interaction between the Pac-1? and -1? subunits is achieved immediately, whereas for the interaction between Pac-3? and -1?, it takes at least 30 hours of incubation. The same can be observed from the characterization of direct interaction between Pac-1? and -3?. From the NMR experiments, it was possible to identify the region of interaction between the subunits -1? and -3? with the -1? subunit. Both ? subunits interact with Pac-1? via betastrands 1 and 5 and the loop between the strands 4 and 5. Overall, these results impact in the mechanism of HIF-3? antagonizing the transcriptional activity of HIF-1?. We also obtained single crystals for Pac-3? subunit, which were subjected to preliminary experiments of X-ray diffraction. Although resulting in anisotropic, insufficient data for and structural resolution, it has allowed the characterization of crystalline parameters, including the presence of a high solvent content. Additionally, we also present the results targeting the expression and crystallization of Prolyl-hydroxylases (PHDs) human isoforms 1-4, during the six-month period at the Structural Genomics Consortium (SGC), the University of Oxford in England. Several construct from of isoforms 1 and 3 were successfully expressed and purified in the soluble form. Likewise, crystals were obtained, but these were determined to be composed by inorganic compounds present in the crystallization conditions. At the end, a collaboration was established between our and the SGC group for the structural studies with the PHDs to extend and carry out the experiments in our lab here in Brazil / Mestrado / Clinica Medica / Mestra em Clínica Médica

Abstract Oxygen deprivation (hypoxia) is related to many disease conditions, such as anemia, but is also a critical regulatory signal during normal development. Cellular responses to hypoxia are largely mediated through alterations in gene regulation brought about by the transcription factor known as hypoxia inducible facor (HIF). One of the most extensively studied systemic consequences of hypoxia is the induction of red blood cell production, erythropoiesis, which occurs through a HIF-dependent increase in erythropoietin (EPO) gene expression. The amount of HIF in cells is regulated by three HIF prolyl 4-hydroxylases (HIF-P4Hs) while a fourth P4H possessing a transmembrane domain (P4H-TM) is able to act on HIF at least in vitro. The putative role of P4H-TM in regulating erythropoiesis is studied here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm null and wild-type mice. By comparing the observed effects with those seen in FG-4497 treated hypomorphic Hif-p4h-2 and Hif-p4h-3 null mice, it is demonstrated for the first time that P4H-TM is involved in the regulation of Epo production in the mammalian kidney, but not in the liver. Long bones are formed via endochondral ossification, in which a cartilaginous template, the growth plate, is first laid down and then replaced with bone. The growth plate is rich in extracellular matrix (ECM) and contains a hypoxic central region in which HIF has been shown to regulate chondrocyte function. Importantly, growth plate chondrocytes are highly active in collagen synthesis. Collagen prolyl 4-hydroxylases (C-P4Hs I-III) provide collagen molecules with thermal stability and are thus necessary for the formation of a proper ECM. Through an in vitro approach it is demonstrated that hypoxia increases the amount and activity of C-P4H in primary mouse epiphyseal growth plate chondrocytes in a HIF-1-dependent manner. Lastly, it was set out to characterize mouse lines with complete inactivation of C-P4H-II with or without partial inactivation of C-P4H-I. A significant reduction in the total amount of C-P4H and its activity was found to result in mild chondrodysplasia and altered bone properties. The above mouse models provided new information on the specific in vivo roles of the C-P4H isoenzymes I and II. / Tiivistelmä Kudosten alentunut happipitoisuus (hypoksia) liittyy osana moniin elimistön patologisiin tiloihin, kuten anemiaan. Lisäksi se on tärkeä säätelytekijä normaalin yksilönkehityksen aikana. Jotta solut havaitsisivat hypoksian ja reagoidakseen siihen, on niille kehittynyt säätelyjärjestelmä, jossa hypoksiassa indusoituva transkriptiotekijä, HIF, on tärkeässä asemassa. Yksi merkittävin HIF:n indusoima systeeminen vaikutus elimistössä on punasolujen tuotannon, erytropoieesin, kiihtyminen. Sitä tapahtuu erytropoietiinia koodittavan geenin (EPO) lisääntyneen ilmentymisen kautta. HIF-tekijän määrää soluissa säätelee kolme HIF-prolyyli-4-hydroksylaasientsyymiä (HIF-P4Ht 1-3). Transmembraanisen prolyyli-4-hydroksylaasin (P4H-TM) tiedetään myös vaikuttavan HIF-tekijän määrään soluissa in vitro, mutta sen vaikutusta nisäkkään erytropoieesiin ei ole aiemmin tutkittu. Käyttämällä hyväksi kolmea eri transgeenista hiirilinjaa (P4h-tm-/-, Hif-P4h-2gt/gt, Hif-p4h-3-/-) ja HIF-P4H entsyymeitä inhiboivaa lääkeainetta, FG-4497, tässä työssä osoitettiin ensimmäistä kertaa, että P4H-TM osallistuu nisäkkään Epo-hormonin tuoton säätelyyn. Pitkät luut muodostuvat endokondraalisen luutumisen kautta. Siinä ensin muodostuu rustoinen malli, kasvulevy, joka vähitellen korvaantuu luukudoksella. Kasvulevyn sisin kerros on sen soluille, kondrosyyteille, hypoksinen kasvuympäristö. HIF:illä on todettu olevan tärkeä rooli kondrosyyttien toiminnan säätelijänä. Kasvulevyn soluvälitila sisältää runsaasti kollageeneja. Kollageenin prolyyli-4-hydroksylaasit (C-P4Ht I-III) ovat avainasemassa kollageenien biosynteesissä ja siten niiden toiminta on välttämätöntä kestävän soluvälitilan muodostumiselle. Käyttämällä in vitro menetelmiä, tässä työssä osoitettiin, että hiiren epifyseaalisten kasvulevyjen kondrosyyteissä hypoksia lisää C-P4H:n määrää ja aktiivisuutta HIF-tekijästä riippuvalla mekanismilla. Eri C-P4H-isoentsyymeiden toiminnasta ja merkityksestä in vivo tiedetään vain vähän. Tässä työssä karakterisoitiin hiirilinja, jossa C-P4H-II on täysin inaktiivinen, ja hiirilinja, jossa lisäksi C-P4H-I on osittain inaktiivinen. Merkittävästi alentuneen C-P4H:n aktiivisuuden todettiin aiheuttavan hiirimallissa lievän kondrodysplasian sekä heikentyneet luun ominaisuudet.

Carnitine is an essential metabolite in the human body. It carries out several roles in human metabolism, including that in fatty acid metabolism. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate dependent oxygenase, which catalyses the final step of carnitine biosynthesis, i.e. hydroxylation of γ-butyrobetaine (GBB) to carnitine. Inhibition of BBOX has potential in the treatment for cardiovascular diseases. The work described in this thesis focussed on mechanistic and inhibition aspects of BBOX catalysis. Firstly, a set of analytical tools for BBOX activity measurements was developed. The synthesis of fluorinated substrate analogues provided the basis for development of two assays for use in vitro with the isolated protein and in lysates, with detection by fluorescence or ¹⁹F NMR, respectively. Furthermore, the use of ¹⁹F NMR to monitor protein-ligand interactions was exemplified with the work on metallo-β-lactamases. The developed fluoride-release assay was then used to screen a library of small molecules and led to recognition of scaffolds with potential applications as inhibitors. Further structure-activity relationship studies led to the identification of potent BBOX inhibitors, which were then evaluated for their activity in cells. The crystal structure of human BBOX with one of the lead inhibitors revealed that BBOX can undergo significant conformational changes, involving a movement of an active site loop. BBOX conformational flexibility may have a role in the GBB mediated substrate inhibition observed both with isolated protein and in cells. In addition to the mechanistic and functional studies, the potential of BBOX as a biocatalytic tool was examined. BBOX has been shown to catalyse a hydroxylation of the symmetrical dialkyl piperidine carboxylic acids, leading to formation of up to three stereocentres in one reaction. In the last part of this work properties of human BBOX were compared to BBOX from Pseudomonas sp. AK1, revealing differences in kinetic behaviour and substrate specificity. Novel substrates for bacterial BBOX were identified. Pseudomonas sp AK1 BBOX was shown to hydroxylate amino acid analogues leading to formation of 1,2-amino alcohols.

The gene encoding cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt - 149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139- AGTTCAaggccGGGTAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter- transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription.

It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

1.Adult specimens of Ascaris lumbricoides of similar weights were incubated under nitrogen for 24 hours in a synthetic medium with 1 mM 2,2′-dipyridyl.2.Under these conditions, the viability of the parasites was not affected as evidenced by the amount of ATP in the whole sample and the mobility after mechanical stimulus.3.Incorporation of [14C]proline into non-collagenous proteins in the body wall and cuticle was reproducibly higher in 2,2′-dipyridyl-treated specimens than in untreated worms. Synthesis of collagenous hydroxyproline was inhibited in the cuticle and, to a greater extent, in the muscle layer.4.After transferring the specimens into a fresh medium enriched with 0·1 mM ferrous ions and incubated under 70% oxygen, the muscle collagen remained underhydroxylated. The synthesis of hydroxyproline, however, was almost completely normalized in the cuticle collagen.5.We interpret the data as further evidence of the existence of at least two different enzymes hydroxylating collagenous proline, one located in the subcuticle and the other in the muscle layer of Ascaris lumbricoides.