Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-motor symptoms, of which cognitive dysfunction is an example, also frequently occur. Current therapy provides symptomatic relief mainly by augmentation of dopaminergic signalling (levodopa, dopamine agonists, MAO and COMT enzyme inhibitors), but disease progression is not adequately addressed. New therapies that can prevent further neurodegeneration in addition to providing symptomatic relief are therefore urgently required. Adenosine has an important function as neuromodulator in the central nervous system. The adenosine A2A receptor in particular plays an essential role in the regulation of movement. This, coupled to the fact that it is uniquely distributed in the basal ganglia, contributes to its attractiveness as non-dopaminergic target in the treatment of movement disorders, such as Parkinson’s disease. The efficacy of adenosine receptor antagonists has been illustrated in animal models of Parkinson’s disease and several adenosine receptor antagonists have also reached clinical trials. The neuroprotective properties of adenosine A2A receptor antagonists are further attributed to their ability to modulate neuro-inflammation and decrease the release of the excitatory neurotransmitter glutamate, which is implicated in neurotoxicity. While adenosine A1 receptor antagonism has a synergistic effect on the motor effects of adenosine A2A receptor antagonism, it has the additional benefit of improving cognitive dysfunction, a cardinal non-motor symptom of Parkinson’s disease. Dual antagonism of adenosine A1 and A2A receptors therefore offers the potential of providing symptomatic relief as well as the neuroprotection so desperately needed in the clinical environment. Amino substituted heterocyclic scaffolds, such as those containing the 2-aminopyrimidine motif, have been shown to exhibit good efficacy as dual adenosine receptor antagonists. Since the structure activity relationships of 2-aminopyrimidines have not been comprehensively explored, it is in this regard that this study aimed to make a contribution. Results - Fourteen 2-aminopyrimidines were synthesised successfully over three steps, (although in low yields) and characterised by nuclear magnetic resonance and infrared spectroscopy, mass spectrometry, by determination of melting points and high performance liquid chromatography. Structure modifications explored included variation of the aromatic substituent on position 4, as well as variations in the substituents of the phenyl ring, present on position 6 of the pyrimidine ring. Radioligand binding assays were performed to determine the affinities of the synthesised compounds for the adenosine A1 and A2A receptor subtypes. Several high dual affinity derivatives were identified during this study; the compound with the highest affinity was 4-(5- methylthiophen-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (39f) with Ki values of 0.5 nM and 2.3 nM for the adenosine A2A and adenosine A1 receptors, respectively. A few general structure activity relationships were derived, which included: The effect of the aromatic substituent (position 4) on A2A affinity could be summarised (in order of declining affinity) as follows: 5-methylthiophene > phenyl > furan > pyridine > p-fluorophenyl > benzofuran. On the other hand, the effect of this substituent on A1 receptor affinity could be summarised (in order of declining affinity) as follows: phenyl > 5-methylthiophene > pfluorophenyl > benzofuran > pyridine. The affinities as exhibited by the methylthiophene derivatives 39f, 39h – 39j, further showed that while piperidine substitution (39f) resulted in optimal A2A and A1 affinity, pyrrolidine substitution (39j) was less favourable. Substitution at the 4ʹ position of the phenyl ring, as well as thiazole substitution, generally resulted in poor adenosine A1 and A2A receptor affinity. However, 4-[2-amino-6-(5-methylfuran-2-yl)pyrimidin- 4-yl]-N-(1,3-benzothiazol-2-yl)benzamide (39l) surprisingly demonstrated good affinity and selectivity for the adenosine A1 receptor. The results obtained during radioligand binding assays were rationalised by QSAR and molecular modelling (Discovery Studio 3.1, Accelrys) studies. The inverse relationship seen between log Ki (as indicator of affinity) and polar surface area, illustrated the importance of this physico-chemical property in the design of 2-aminopyrimidine A2A antagonists. The results from the docking study further showed that the orientation adopted by derivatives in the binding cavity (and particular hydrogen bonding to Asn 253 and Glu 169) is of importance. Results from the MTT cell viability assay indicated that none of the high affinity derivatives had a significant effect on cell viability at 1 μM, a concentration much higher than their Ki values. However, incorporation of the furan, benzofuran and p-fluorophenyl groups as aromatic substituent and a pyrrolidine as amine substituent, presented liabilities. Lastly, the haloperidol induced catalepsy assay (in rats) was used to give a preliminary indication of adenosine receptor antagonism or agonism. Compound 39f failed to reverse catalepsy under standard conditions, but showed some reversal after an increased time period. Indications therefore exist that 39f is an adenosine receptor antagonist that suffers from bioavailability issues. Compound (39c), 4-phenyl-6-[3-(piperidine-1- carbonyl)phenyl]pyrimidin-2-amine which also demonstrated promising affinity in the radioligand binding assays however showed a statistically significant reduction in catalepsy, indicating adenosine A2A receptor antagonism, and in vivo efficacy. Highly potent, dual affinity aminopyrimidine derivatives with acceptable toxicity profiles were identified in this study, with compound 39c demonstrating in vivo activity. The aim of designing and synthesising a promising dual adenosine A1/A2A receptor antagonist is therefore realised, with compound 39c as the most favourable example. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

<p>Uvod: Pu&scaron;enje predstavlja jedan od najznačajnijih uzroka lo&scaron;e kontrole astme, zbog iritativnog dejstva duvanskog dima na disajne puteve i razvoja rezistencije na inhalatorne kortikosteroide. Stoga je pu&scaron;ače sa astmom često potrebno lečiti kombinovanom antiinflamatornom terapijom, iako je efikasnost ovakvog tretmana jo&scaron; uvek nedovoljno ispitana. Cilj: utvrditi efikasnost kombinovane antiinflamatorne terapije: inhalatorni kortikosteroidi (ICS) u kombinaciji sa dugodelujućim beta2-adrenergičkim agonistima (DDBA) u odnosu na ICS u kombinaciji sa antagonistima leukotrijenskih receptora (ALTR) u postizanju dobre kontrole astme, pobolj&scaron;anju kvaliteta života i plućne funkcije kod pu&scaron;ača u odnosu na nepu&scaron;ače sa astmom. Metod: Pacijenti starosti od 18-50 godina sa astmom (&ge;6meseci), FEV1 većim od 60%, podeljeni su u grupu nepu&scaron;ača &ndash;NP (N=60) i aktivnih pu&scaron;ača &ndash;PU (&le;2 &ge;15 p/g i &ge;10&le;40 cigareta na dan; N=60). Obe grupe su randomizovane u jednu od dve, otvorene, terapijske grupe (ICS uz dodatak DDBA ili ALTR) u trajanju od 24 nedelje. Rezultati: u svakoj od 4 randomizovane grupe (NP-DDBA, NP-ALTR, PU-DDBA, PU-ALTR) je bilo po 30 pacijenata. Tokom 24 nedelje, PU su imali lo&scaron;ije kontrolisanu astmu od NP (p=0,02), bez ralizke između DDBA vs ALTR (0,677 vs 0,634). Konstantno dobru kontrolu astme (ACQ&lt;0,75) tokom 24 nedelje je postiglo 48% NP i 32% PU (p=0,094), bez značajne razlike u odnosu na terapiju (DDBA vs ALTR; p=1,000). NP su imali bolji kvalitet života od PU, ali razlika nije dostigla statističku značajnost (p=0,056)- Kod NP i kod PU u oba modaliteta lečenja (LABA, ALTR) je do&scaron;lo do statistički značajne promene srednjeg skora AQLQ (p&lt;0,001). Povećanje FEV1(%) je bilo statistički značajno i u grupi NP i u grupi PU (p=0,001 vs. p=0,002). Kod pacijenata lečenih DDBA povećenje FEV(%) je bilo na nivou p=0,001, dok je u grupu ALTR bilo na nivou p=0,005. Multivarijantnom analizom je utvrđeno da su nezavisni faktori postizanja dobre kontrole astme BMI&ge;24, nepu&scaron;ač, FEV1&ge;90%, ACQ&le;2,2 i AQLQ&ge;4,2 Zaključak: Kombinovana antiinflamatorna terapija je efikasnija kod NP u odnosu na PU, dok su u populaciji aktivnih pu&scaron;ača, oba dodatna leka (DDBA, ALTR) bila podjednako efikasna u pobolj&scaron;anju kontrole astme, kvaliteta života i plućne funkcije.</p> / <p>Introduction: Smoking is one of the major causes of a bad asthma control, due to negative effects of the tobacco smoke on the airways and consequent resistance to inhalant corticosteroids. Smoking asthmatics should therefore often be treated with combined anti-inflammatory therapy, although the efficacy of this treatment regimen has not been completely examined yet. Objective: To examine the efficacy of the combined anti-inflammatory therapy (ICS combined to LABA vs.LTRA) in achieving a good asthma control, better quality of life and improved lung function in smoking vs. nonsmoking asthmatics. Method: The patients at 18-50 years of age with asthma (&ge;6 months), FEV1 &gt; 60%, were subclassified into the group of nonsmokers &ndash;NS (N=60), and the group of active smokers - SM (&le;2 &ge;15 p/g and &ge;10&le;40 cigarettes a day; N=60). Both groups were randomized into one of the two open therapy groups (ICS combined to DDBA or ALTR), receiving the selected treatment for 24 weeks. Results: Any of the four randomized groups (NS-LABA, NS-LTRA, SM-LABA, SM-LTRA) consisted of 30 patients. During the 24-week period, SM had a worse control of their asthma than NS (p=0.02), but no difference was registered between DDBA vs. ALTR therapy subgroups (0.677 vs. 0.634). Over the 24-week period, a constantly good asthma control (ACQ&le;0,75) was achieved by 48% of NS and 32% of SM (p=0.094), and no significant difference related to the applied therapy regimen (LABA vs. LTRA; p=1.000). NS had a better life quality than SM, but this difference remained statistically insignificant (p=0.056). Both the NS and the SM group in either treatment modality (LABA, ALTR) had a statistically significant change of the AQLQ score (p&lt;0.001). FEV1 (%) improvement was statistically significant t in both the NS and the SM group (p=0.001 vs. p=0.002). The LABA and LTRA treated patients had their FEV (%) improvement at the level of p=0.001, and p=0.005 respectively. The multivariate analysis has established the following independent factors of a good asthma control: BMI&ge;24, nonsmoker, FEV1&ge;90%, ACQ&le;2.2, and AQLQ&ge;4.2. Conclusion: The combined anti-inflammatory therapy is more efficient in NS than in SM asthmatics, while in the population of active smokers, both additional drugs (LABA, LTRA) were equally efficient in improving asthma control, life quality, and lung function.</p>

Glutamate excitotoxicity is the predominant mechanism implicated in neuronal cell death associated with neurological disorders such as stroke, epilepsy, traumatic brain injury and ALS. Excessive stimulation of NMDA subtypes of glutamate receptors leads to protracted intracellular calcium elevations triggering calcium mediated neurotoxic mechanisms culminating in delayed neuronal cell death. In addition, glutamate excitotoxicity induces a NMDA dependent extended neuronal depolarization mediated by continuous calcium influx that correlates with delayed neuronal death. Attempts to prevent neuronal death by blocking calcium entry into the neurons using calcium channel blockers or NMDA receptor antagonists have failed to provide any beneficial effects in clinical trials. Thus, calcium continues to enter the neurons despite the presence of calcium entry blockers. This phenomenon is known as the "calcium paradox of stroke" and represents a major problem in developing effective therapies for treatment of stroke. Here employing a combination of patch clamp recordings, fluorescent calcium imaging and neuronal cell death assays in well-characterized in vivo and in vitro models of glutamate excitotoxicity, we report the discovery of a novel calcium permeable ion channel that is activated by excitotoxic glutamate injury and mediates a calcium current that is an early initiating step in causing neuronal death. Blocking this calcium permeable channel with high concentrations of Zn2+ or Gd3+ by removing extracellular calcium for a significant time period after the initial injury is effective in preventing calcium entry, apoptosis and neuronal death, thus accounting for the calcium paradox. This injury induced-calcium permeable channel provides a unique mechanism for calcium entry following stroke and offers a new target for extending the therapeutic window for preventing neuronal death after the initial excitotoxic (stroke) injury.

INTRODUÇÃO: A importância do parênquima pulmonar na piora funcional da asma tem sido recentemente investigada. Embora a ativação da enzima óxido nítrico sintase induzida (iNOS) amplifique a responsividade e o remodelamento das vias aéreas induzidos pela inflamação crônica, seu efeito no parênquima pulmonar não foi previamente estudado. OBJETIVO: Avaliar a influência do óxido nítrico derivado da iNOS na mecânica pulmonar, na inflamação e no processo de remodelamento no tecido pulmonar periférico de cobaias com inflamação pulmonar alérgica. MÉTODOS: Os animais foram submetidos a sete inalações com doses crescentes de ovalbumina (1~5 mg/mL) ou soro fisiológico por 4 semanas. As cobaias receberam 1400-W (inibidor específico de iNOS, intraperitoneal) ou veículo por 4 dias, iniciando 30 minutos antes da sétima inalação. Após 72h da sétima inalação, os animais foram anestesiados, exsanguinados e fatias de tecido pulmonar periférico foram retiradas e suspensas em banho orgânico de Krebs, e a resistência e elastância tecidual foram avaliadas em condição basal e após desafio com ovalbumina. Após, as fatias de tecido pulmonar periférico foram submetidas à avaliação histopatológica. RESULTADOS: Os animais expostos às inalações com ovalbumina apresentaram valores maiores de porcentagem de aumento da resistência e da elastância tecidual em relação ao basal após desafio com ovoalbumina no banho (p<0.05). Houve aumento no número de eosinófilos (p<0.001), nas células iNOS positivas (p<0.001), na deposição de fibras elásticas e colágenas (p<0.05), na densidade de actina (p<0.05) e na expressão de 8-epi-PGF2a (p<0.001) no septo alveolar. A administração de 1400-W reduziu todos estes parâmetros funcionais e morfológicos (p<0.05). CONCLUSÕES: Neste modelo experimental, o bloqueio específico da iNOS atenuou a constrição, a inflamação e o remodelamento no parênquima pulmonar. Estas alterações podem estar relacionadas aos efeitos do óxido nítrico na modulação da via do estresse oxidativo. O presente estudo sugere que a inibição específica da iNOS pode amplificar as estratégias terapêuticas utilizadas na abordagem de doenças inflamatórias crônicas pulmonares. / INTRODUCTION: The importance of lung parenchyma in functional asthma impairment has been recently addressed. Although the inducible nitric oxide synthase (iNOS) activation amplifies chronic inflammation-induced airway responsiveness and remodeling, its effect on lung parenchyma has not been previously investigated. OBJECTIVE: To evaluate the influence of iNOSderived NO in the pulmonary mechanics, inflammation, and remodeling processes in peripheral lung tissue of guinea pigs with pulmonary allergic inflammation. METHODS: Animals were submitted to seven ovalbumin exposures with increasing doses (1~5 mg/mL) or saline for 4 weeks. The guinea pigs received 1400-W (iNOS-specific inhibitor, intraperitoneal) or vehicle for 4 days, beginning 30 minutes before the 7th inhalation. At 72h after the 7th inhalation, animals were anesthetized, exsanguinated and peripheral lung tissue strips were retreat and suspended in a Krebs organ bath, and the tissue resistance and elastance were evaluated at baseline condition and after ovalbumin challenge. After that, strips were submitted to histopathological evaluation. RESULTS: The ovalbumin-exposed animals presented greater values of percentage of increase of tissue resistance and elastance related to baseline after ovalbumin challenge in the bath (p<0.05). There were increase in the number of eosinophils (p<0.001) and iNOSpositive cells (p<0.001), in collagen and elastic fiber deposition (p<0.05), in actin density (p<0.05) and in 8-epi-PGF2a expression (p<0.001) in the alveolar septa. The 1400-W administration reduced all these functional and morphological parameters (p<0.05). CONCLUSIONS: In this experimental model, the iNOS-specific blockage attenuated constriction, inflammation, and remodeling in the lung parenchyma. These alterations may be related to NO effects in the modulation of the oxidative stress pathway. The present study suggests that specific iNOS inhibition can amplify the therapeutics strategies used in the management in chronic inflammatory lung diseases.

O ergosterol é um componente responsável por manter a integridade e a fluidez das membranas de Leishmania spp. A partir de uma metodologia que consiste em seleção por superexpressão gênica, foram isolados nove diferentes loci de L. (L.) major relacionados com a resistência a dois inibidores da via de biossíntese do ergosterol: Terbinafina (TBF) e Itraconazol (ITZ). Análises funcionais individuais desses nove loci na presença de TBF e ITZ (ou do análogo Cetoconazol - CTZ) apresentaram níveis significantes de resistência após transfecção em células selvagens de L. (L.) major. Nesse trabalho apresentamos a metodologia de isolamento de um desses loci (cItz2), bem como a análise in silico das regiões cromossômicas correspondentes aos insertos dos nove cosmídios no genoma de L. (L.) major / Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated nine different loci of L. (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, Terbinafine (TBF) and Itraconazole (ITZ). Individual functional analysis of these nine loci in the presence of TBF and/or ITZ (or the ITZ analog Ketoconazole, CTZ), have showed significant levels of resistance after transfection into L. (L.) major wild-type cells, followed by over expression induction. In this work, we show the insert mapping and chromosomal identification of one of these loci (cItz2), as well as discuss the in silico chromosomal analysis of the nine correspondent inserts in the L. (L.) major genome

O Fator de Necrose Tumoral-alfa (TNF-alfa) possui um importante papel na imunopatogênese da psoríase e agentes biológicos, como os inibidores de TNF-alfa, têm apresentado bons resultados no tratamento desta. No entanto, estes agentes foram associados ao aumento de casos de reativação de tuberculose entre os pacientes que os utilizaram. Este estudo foi realizado com o intuito de avaliar a resposta imune de pacientes com psoríase grave, ativa, sem tratamento, frente a antígenos de Mycobacterium tuberculosis (Mtb), e o efeito dos inibidores de TNF-alfa nesta resposta. Estudamos 24 pacientes com psoríase grave divididos em 2 grupos: não reatores (n = 14) e reatores (n = 10) ao teste intradérmico com PPD. Como controle, utilizamos um total de 26 indivíduos sadios, também separados em 2 grupos segundo a reatividade ao PPD (PPD-, n = 13; PPD+, n = 13). Em uma segunda etapa estudamos 11 pacientes com psoríase leve a moderada, também sem tratamento, PPD (-) para avaliarmos a importância da gravidade da psoríase na resposta aos antígenos micobacterianos. Avaliamos a resposta imunológica in vitro através da linfoproliferação, quantificação da produção de IFN-gama (ELISA) e quantificação de células produtoras de IFN-gama (ELISPOT), na presença e ausência dos inibidores de TNF-alfa (infliximab e etanercepte), utilizando os antígenos purificados ESAT-6, Ag85B e o antígeno bruto sonicado da cepa H37Rv (AgSMtb), e o mitógeno fitohemaglutinina (PHA). Os pacientes com psoríase grave PPD (-) apresentaram reposta linfoproliferativa e níveis de IFN-gama menores que nos controles PPD (-). Os pacientes com psoríase leve a moderada apresentaram resposta imune intermediária entre controles e pacientes graves. Em relação aos inibidores de TNF- alfa, verificou-se que infliximab e etanercepte apresentaram diferença em suas capacidades de inibição, sendo que somente o infliximab ocasionou a inibição total de TNF-alfa. Em contrapartida o etanercept manteve a produção de TNF-alfa, e em alguns casos elevou sua produção. Estes diminuíram apenas parcialmente a reatividade in vitro dos pacientes com psoríase, uma vez que a secreção de IFN-gama e o número de células produtoras de IFN-gama não foram alterados na presença dos inibidores. A secreção de IL-10 foi diminuída tanto na presença do infliximab, quanto na presença do etanercepte. Os dados obtidos permitem concluir que (a) os pacientes com psoríase grave PPD (-) apresentam uma baixa reatividade in vitro, principalmente das respostas que avaliam linfócitos T de memória central, aos antígenos de Mtb, sendo que essa baixa reatividade não está totalmente relacionada com a gravidade da doença, uma vez que os pacientes com psoríase leve a moderada apresentaram resposta intermediária a dos controles e pacientes com psoríase grave; (b) e que apesar dos inibidores de TNF- alfa promoverem uma inibição parcial da resposta imune, a reativação da tuberculose estaria mais relacionada à própria ausência de TNF-alfa, não compensada pela atuação isolada, e provavelmente insuficiente, de IFN-gama na manutenção do granuloma, do que a outras substanciais modificações na resposta imunológica frente aos antígenos micobacterianos. / Tumor necrosis factor alpha (TNF-alpha) has a pivotal role in psoriasis pathogenesis and biologic agents, such as TNF-alpha inhibitors, have provided good results in its treatment. However, the use of these agents has been associated with an increase in the number of cases of tuberculosis reactivation. This study aimed to evaluate the immune response of severe psoriasis patients, with active, untreated disease to relevant Mycobcterium tuberculosis antigens, and the effect of the TNF-alpha inhibitors (infliximab and etanercept) in this response. Twenty four severe psoriasis patients were enrolled and divided in two groups according to their reactivity to the tuberculin skin test: TST (n= 14) and TST + (n=10). As controls, we studied 26 healthy donors, also divided in two groups to the TST reactivity (TST -, n=13; TST+, n=13). Eleven mild to moderate psoriasis patients, untreated, TST (-) were studied to evaluated the role of psoriasis severity in the immune response to the mycobacterial antigens. Immune responses were evaluated in vitro by the lymphocyte proliferative response (LPR) assay, ELISA for IFN-? secretion by peripheral blood mononuclear cells and enumeration of IFN-? secreted cells (ELISPOT) induced in response to the purified antigens ESAT-6, Ag85B and a crude sonicated antigen preparation from H37Rv Mtb strain (AgSMtb), as well as to the mitogen phytohemagglutinin (PHA), in the presence or absenceinflimab/etanercept. The LPR and IFN-g secretion to Mtb antigens were lower in TST- severe psoriasis patients than TST- controls. Mild to moderate psoriasis patients had intermediate responses, between controls and severe psoriasis patients. The TNF-a inhibitors infliximab and etanercept showed differences in their inhibitiory activity, since only infliximab was capable to neutralize all TNF-a. On the other hand, etanercept kept TNF-alpha production, and in some cases even increased its production. The TNF-alpha inhibitors diminished partially the in vitro patients immune responses, since the IFN-? secretion and enumeration of IFN-? secreted cells were not affected. IL-10 secretion was diminished with both TNF-a inhibitors. In conclusion: (a) TST(-) severe psoriasis patients have decreased in vitro reactivity, mainly in those responses that evaluate central memory T-cell responses, to Mtb antigens, and this decrease could not be fully explained by disease severity, since mild psotiasis patients had intermediate responses; (b) and despite the fact that TNF-alpha inhibitors promote a partial immune response inhibition, tuberculosis reactivation could be related more with the lack of TNF-alpha, which was probably not compensated by the IFN-g activity alone, probably insufficient, to the support granuloma formation, than other defects of the immune response to Mtb antigens.

Introdução: A disfunção cardíaca relacionada à cirrose acomete pacientes com insuficiência hepática avançada e pode estar associada a complicações como a síndrome hepatorrenal. Diferente do que ocorre na insuficiência cardíaca, em que o tratamento farmacológico com betabloqueadores é reconhecidamente eficaz em reverter o remodelamento cardíaco e aumentar a sobrevida, ainda não foi testada nenhuma modalidade terapêutica que possa bloquear o efeito remodelador e a progressão da disfunção cardíaca nos pacientes com cirrose. Formulamos a hipótese que o uso de betabloqueador poderia ter efeito benéfico sobre as alterações cardíacas morfológicas e funcionais observadas em pacientes com cirrose. Objetivos: O objetivo primário foi avaliar a eficácia do metoprolol na reversão da disfunção cardíaca em pacientes com cirrose não alcoólica. Os objetivos secundários compreenderam: reversão das alterações ecocardiográficas, biomarcadores da ativação neuro-humoral, eletrofisiológicas, eventos clínicos e efeitos adversos. Avaliou-se também o impacto da resposta inotrópica anormal no prognóstico da cirrose. Material e métodos: Conduzimos um estudo prospectivo, randomizado, duplo-cego, placebo-controlado, fase II, com análise \"por intenção de tratar\". Os critérios de inclusão foram cirrose de etiologia não alcoólica, escore MELD entre 10 e 20 pontos e idade entre 18 e 60 anos. Os critérios de exclusão foram doença cardiovascular prévia, doenças sistêmicas com acometimento cardíaco e contraindicação ao uso de betabloqueadores. No momento da inclusão e após 180 dias de tratamento, realizamos avaliação clínica, dosagem de biomarcadores (noradrenalina, troponina, peptídeo natriurético tipo B e atividade da renina plasmática), mensuração indireta da atividade simpática (ECG dinâmico de 24 horas) e ecocardiograma sob estresse com dobutamina. A disfunção cardíaca foi caracterizada pela resposta inotrópica anormal ao eco-estresse (incremento do débito cardíaco <= 30% após o estresse em relação ao basal, medido pela integral velocidadetempo na via de saída do ventrículo esquerdo). O desfecho primário foi definido como o aumento >=30% da resposta inotrópica ao estresse. Os eventos clínicos avaliados como desfecho foram: ascite, síndrome hepatorrenal, encefalopatia, infecções, hemorragia digestiva varicosa, internações e mortalidade. Considerando a possibilidade de erro alfa de 0,05, erro beta de 0,2 e diferença de proporções na melhora da VTI ( >= de 30%) entre os grupos, o tamanho amostral foi estimado em 72 pacientes. O protocolo foi aprovado pela Comissão de ética institucional e registrado na base de dados internacional ClinicalTrials.gov (NCT01676285, acrônimo \"Cardiac Remodeling in cirrhosis - CARE cirrhosis\"). Resultados: No período de junho de 2011 a dezembro de 2013, 478 pacientes com cirrose foram prospectivamente avaliados e 190 preencheram os critérios de elegibilidade, sendo incluídos 125 pacientes. Desses, 78 (62%) apresentaram resposta inotrópica anormal ao estresse farmacológico. Os demais 47 (38%) pacientes com resposta inotrópica normal foram seguidos sem intervenção farmacológica. Os pacientes com resposta anormal foram randomizados para receber tratamento (succinato de metoprolol, n=41 ou placebo, n=37). Três (7,3%) dos pacientes no grupo metoprolol e 9 (24,3%) no grupo placebo apresentaram normalização da resposta inotrópica ao estresse, diferença não estatisticamente significante (p=0,057). Não houve diferença entre os grupos metoprolol e placebo em relação à reversão das alterações ecocardiográficas, laboratoriais e eletrofisiológicas. Não houve diferença quanto aos desfechos clínicos isolados ou combinados na comparação entre metoprolol e placebo: síndrome hepatorrenal n=1 (2,4%) versus n=0, p=0,99; ascite n= 4 (12%) versus n=2 (5,4%), p=0,27; infecções bacterianas n= 2 (4,8%) versus n=2 (5,4%), p=0,94; encefalopatia hepática n= 5 (12,1%) versus n=6 (16.2%), p=0,59, hemorragia varicosa n=0 em ambos os grupos; internações n=6 (14,6%) versus n=8 (21,6%), p=0,45; morte n=5 (12,1%) versus n= 2 (5,4%), p=0,94; respectivamente. Não houve diferença entre metoprolol e placebo em relação ao surgimento de efeitos adversos n=4 (9,7%) versus n=6 (16,2%), respectivamente, p=0,5. Os pacientes no grupo com resposta inotrópica anormal apresentaram maiores taxas de desfechos combinados (26 (33,3%) versus 8 (17,0%), p=0,03) em relação àqueles com resposta inotrópica normal. Conclusão: embora seguro, o metoprolol não foi eficaz na reversão da disfunção cardíaca em pacientes com cirrose não alcoólica. Não houve benefício na terapia com metoprolol em relação aos desfechos ecocardiográficos, laboratoriais, eletrofisiológicos e clínicos, incluindo mortalidade. Os pacientes com resposta inotrópica normal tiveram melhor evolução em relação àqueles com resposta inotrópica anormal / Background: Cardiac dysfunction is found in patients with end-stage liver disease and is implicated in complications such as hepatorenal syndrome. Unlike heart failure, in which beta-blockers are admittedly effective in reversing cardiac remodeling and improving survival, the effect of beta-blockade on the cardiac dysfunction of patients with cirrhotic cardiomyopathy is unknown. We hypothesized that beta-blockers could have a beneficial effect on the morphological and the functional cardiac changes seen in patients with cirrhosis. Aim: To assess the efficacy of metoprolol in the reversal of the cardiac dysfunction in patients with non-alcoholic cirrhosis. Methods: We conducted a prospective, randomized, double-blind, placebocontrolled, with an \"intention to treat\" analysis, phase II study. Inclusion criteria were cirrhosis of non-alcoholic etiology, MELD score between 10 and 20 points and age between 18 and 60 years old. Exclusion criteria were previous cardiovascular disease, systemic diseases with cardiac involvement and contraindications to beta-blockers. Clinical assessment, measurements of biomarkers (norepinephrine, troponin, B-type natriuretic peptide and plasma renin activity), 24-hours Holter and stress echocardiography with dobutamine were performed at inclusion and after 180 days of treatment. Cardiac dysfunction was defined by an abnormal inotropic response to stress echo (an increasing in cardiac output <= 30% during peak stress in relation to baseline values, as measured by the left ventricular outflow tract velocity-time integral). Primary end-point was an increase of 30% in the inotropic response. The frequency of ascites, hepatorenal syndrome, encephalopathy, bacterial infections, variceal bleeding, hospitalization and mortality were also assessed. Considering an alpha error of 0.05, a beta error of 0.2, a difference between proportions of improvement of VTI between the groups of 20%, and an anticipated dropout rate of 10%, the sample size was estimated in 72 patients. The protocol was approved by the institutional ethics board and registered in the database ClinicalTrials.gov (NCT01676285 acronym \"Cardiac Remodeling in cirrhosis - cirrhosis CARE\"). Results: From June 2011 to December 2013, 478 patients with cirrhosis were prospectively evaluated. 190 were eligible to participate in the study. From these, 125 met the inclusion criteria and 78 had abnormal inotropic response to pharmacological stress. These 78 patients, who comprise the present series, were randomly assigned to receive treatment (metoprolol succinate, n = 41 or placebo, n = 37). The remaining 47 (38%) patients with normal inotropic response were followed without pharmacological intervention. Three (7.3%) patients in the metoprolol group and 9 (24.3%) in the placebo group achieved normalization of the inotropic response to stress (p = 0.057). There was no difference between metoprolol or placebo with respect to the reversal of the echocardiographic, electrophysiological and laboratory changes. There was no difference in clinical outcomes between the groups: hepatorenal syndrome n = 1 (2.4%) versus n = 0, p = 0.99; ascites n = 4 (12%) versus n = 2 (5.4%), p = 0.27; bacterial infections n = 2 (4.8%) versus n = 2 (5.4%), p = 0.94; hepatic encephalopathy n = 5 (12.1%) versus n = 6 (16.2%), p = 0.59), variceal bleeding n = 0 in both groups; admissions n = 6 (14.6%) versus n=8 (21.6%), p = 0.45; death n=5 (12.1%) versus n=2 (5.4%), p = 0.94; for metoprolol and placebo, respectively. Adverse effects were similar in both groups: metoprolol n = 4 (9.7%) versus placebo n = 6 (16.2%), p = 0.5. Patients in the group with abnormal inotropic response showed higher rates of combined outcomes (26 (33.3%) versus 8 (17.0%), p = 0.03) compared to those with normal inotropic response. Conclusion: Although safe, metoprolol was not effective in reversing the cardiac dysfunction in patients with nonalcoholic cirrhosis. There was neither improvement in echocardiographic, laboratory, and electrophysiological parameters nor in clinical outcomes, including mortality. Abnormal inotropic response was associated with a higher incidence of clinical events

Nuclear hormone receptors (NHR) constitute a superfamily of ligand inducible transcriptional activators that enable an organism to regulate development and homeostasis through switching on or off target genes in response to stimuli reflecting changes in environment as well as endocrine. NHRs include classical steroid hormone receptors (GR, AR, ER and MR) and retinoid, thyroid hormone receptors. One long-term goal of our lab is to understand the molecular mechanisms through which the transcriptional activity of NHRs is regulated. Extensive studies in the past few years have revealed that in addition to the dependence on ligand availability, the transcriptional activity of NHRs is also regulated by two types of proteins: co activators and corepressors. In the absence of ligand, many NHRs, including TR and RAR can actively repress target gene transcription with the help of corepressors, proteins that physically interact with both NHRs and histone deacetylases (HDACs). Functional interactions between NHRs and corepressors therefore lead to tightly compact and transcriptionally non-permissive chromatin structures after the removal of obstructive acetyl groups from histone tails by HDACs. On the other hand, ligand binding stabilizes NHRs in a conformation that favors interaction with proteins other than corepressors; many of these proteins are able to potentiate the transcriptional activity of NHRs through various mechanisms, such as histone acetylation, chromatin remodeling and recruitment of basal transcription machinery and are collectively termed coactivators. Two highly related corepressors, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and N-CoR (nuclear receptor corepressor), have been cloned. This research in corepressor SMRT started by a systematic study of its subcellular localization. We found that SMRT predominantly forms a specific nuclear punctuate structure that does not appear to overlap with any other well-known subnuclear domains/speckles. Although our searching for specific sequence signals that may determine the specific speckle localization of SMRT did not yield conclusive results, we discovered the colocalization of unliganded RAR and certain HDACs, including HDAC1, 3,4 and 5, in the SMRT nuclear speckles. Moreover, SMRT is likely to be the organizer of such speckles since it appears to be able to recruit other proteins into these speckles. The presence of HDAC1 in the SMRT speckles suggests a direct association between these two proteins, which has not been detected by previous biochemical analyses. Interestingly, HDAC1 point mutants that are completely defective in deacetylase activity failed to locate to SMRT nuclear speckles, while another partially active mutant maintained the colocalization. These discoveries may indicate SMRT nuclear speckles as novel nuclear domains involved in transcriptional repression. More physiologically relevant support for this hypothesis arises from study of HDAC4 and 5. HDAC4 and 5 are potent inhibitors of transcriptional activator MEF2C. Nuclear presence of HDAC4/5 can block the activation of MEF2C, which is required during muscle differentiation. Normally, HDAC4 is predominantly located in cytoplasm. However, we found that in the presence of SMRT overexpression, HDAC4 was found mostly in SMRT nuclear speckles. This accumulation enhanced HDAC4 mediated inhibition on MEF2C transcriptional activity in a transient transfection assay. SMRT overexpression also resulted in accumulation of HDAC5 in the SMRT nuclear speckles compared to the nuclear diffuse distribution in the absence of SMRT. Again, this accumulation of HDAC5 in nuclear speckles correlated with enhanced inhibition of MEF2C. Taken together, our study suggested that instead of being merely a corepressor for NHRs, SMRT might function as an organizer of a nuclear repression domain, which may be involved in a broad array of cellular processes. In contrast to the limited number of corepressors, numerous co activators have been identified; the SRC (or p160) family is relatively well studied. This family includes three highly related members, SRC-1, TIF2/GRIP1, RAC3/AIB1/ACTR/p/CIP. Similar domain structures are shared among these factors, with the most highly conserved region, the bHLH-PAS domain found within the N terminal ~400 amino acid residues. This study of RAC3 aims to identify the function of the highly conserved N terminal bHLH-PAS domain by isolating interacting proteins through yeast two-hybrid screening. One candidate gene isolated encodes the C terminal fragment of the human homologue of the yeast protein MMS19. Functional studies of this small fragment revealed that it specifically interacted with human estrogen receptors (ERs) and inhibited ligand induced transcriptional activity of ERs in the transient transfection assay. Then we cloned the full-length human MMS19 cDNA and characterized the hMMS19 as a weak coactivator for estrogen receptors in the transient transfection assay. Furthermore, when tested on separate AF-1 or AF-2 of ERs, hMMS19 specifically enhanced AF-1 but had no effect on AF-2. These results identified hMMS19 as a specific coactivator for ER AF-1.

A doença de Chagas, descrita há quase 100 anos por Carlos Chagas, continua sendo um problema de saúde pública na América Latina, com 15 milhões de infectados e 28 milhões de indivíduos susceptíveis. Vários mecanismos fisiopatológicos foram propostos para a progressão da miocardiopatia chagásica crônica, entre eles a denervação simpática. O uso do betabloqueador na insuficiência cardíaca congestiva diminuiu morbidade e mortalidade. Entretanto, nenhum trabalho conclusivo incluiu a miocardiopatia chagásica como etiologia. OBJETIVO: Avaliar o papel do carvedilol na sobrevida e remodelamento miocárdico na miocardiopatia chagásica experimental. MATERIAL E MÉTODOS: Foram avaliados 55 Hamsters Sirius, divididos em grupo controle, grupo infectado com Trypanosoma cruzi com 105 formas tripomastigotas via intraperitoneal e grupo infectado, tratado inicialmente com carvedilol 10 mg/Kg/dia e após 6 meses com 15 mg/Kg/dia em dose única por gavagem. Foi analisado o peso do animal; os diâmetros ventriculares, função sistólica e diastólica obtidas pelo ecocardiograma; a freqüência cardíaca, a duração do QRS, a presença de extra-sístoles e o ritmo cardíaco usando o eletrocardiograma e a avaliação da mortalidade. Após 12 meses os animais sobreviventes foram sacrificados e realizada a quantificação da fração do volume de colágeno intersticial e perivascular no miocárdio. RESULTADO: A razão dos diâmetros diastólico e sistólico pelo tamanho da tíbia não mostrou diferenças estatísticas, apesar de maiores nos grupos infectados. A fração de encurtamento também não mostrou diferença estatística, apesar de menor nos grupos infectados. A fração do volume de colágeno do ventrículo esquerdo e perivascular mostrou maior acúmulo nos grupos infectado e carvedilol em relação ao controle de forma significativa (p<0,001), mas sem diferença nos grupos infectados tratados ou não. A mortalidade foi maior nos grupos infectado e carvedilol (p = 0,001) e não ocorreu diferença entre estes grupos após 12 meses. Na fase aguda (até 100 dias) o grupo carvedilol apresentou melhor sobrevida (p = 0,001) em relação ao infectado. CONCLUSÃO: O carvedilol não mostrou atenuação no remodelamento miocárdico e demonstrou benefício na mortalidade na fase aguda da doença nesse modelo de miocardiopatia chagásica crônica experimental. / Chagas\' disease was described 100 years ago by Carlos Chagas and nowadays it is a public health problem in Latin America yet. There are about 15 million infected people and another 28 million at risk to be infected. Several pathophysiologic mechanisms are suggested to be responsible for the progression of chronic Chagas\' cardiomyopathy. Among them the sympathetic denervation seems to be an important issue. The use of beta blockade regarding heart failure has been proving to improve morbidity and mortality. However, none of these papers included Chagas\' cardiomyopathy as etiology. OBJECTIVES: To evaluate the role of carvedilol upon survival and myocardial remodeling in a Chagas\' cardiomyopathy animal model. MATERIAL AND METHODS: 55 Hamsters Sirius were studied and divided into control group, intraperitoneously infected group with Trypanosoma cruzi and infected group treated with carvedilol 10 mg/Kg/day by gavage which was increased to 15 mg/Kg/day after 6 months. Ventricular diameters, systolic and diastolic left ventricular function were evaluated by 2D-echocardiogram. The heart rate, QRS lasting, premature ventricular complex and cardiac rhythm were analyzed by ECG. We evaluated clinical parameters as the case for body weight. The survival curve was also studied. After 12 months the survivors were sacrificed and the myocardial interstitial and perivascular collagen volume fraction were analyzed. RESULTS: The ratio of diastolic or systolic diameters over tibia length did not show statistical differences although it was larger on infected groups. The fractional shortening also did not show statistical differences although it was decreased on infected groups. The left ventricular collagen volume fraction at the interstitial and perivascular area showed a higher accumulation on infected groups compared to control (p<0.001) but no differences were observed between infected and carvedilol treated animals. The mortality was higher in the infected groups compared to control (p = 0.001) but no differences were observed between infected and carvedilol group during the total time of the experiment. However, when we divided in acute phase (until 100 days) the carvedilol significantly attenuated mortality compared to infected group (p = 0.001). CONCLUSION: Carvedilol did not show benefits regarding myocardial remodeling or total mortality and it did demonstrate a mortality reduction on acute phase of the disease in this experimental model of Chagas\' cardiomyopathy.

by Wai Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 123-157). / Abstracts in English and Chinese. / Acknowledegment --- p.iv / Abstract --- p.v / List of Abbreviations --- p.ix / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Parkinson's Disease (PD) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Neuropathology --- p.2 / Chapter 1.1.3 --- Clinical Symptoms --- p.3 / Chapter 1.1.4 --- Treatment --- p.6 / Chapter 1.2 --- Proposed Mechanisms of Neurodegeneration in PD --- p.11 / Chapter 1.2.1 --- Oxidative Stress --- p.11 / Chapter 1.2.2 --- Mitochondrial Dysfunction --- p.13 / Chapter 1.2.3 --- Genetic Factors --- p.15 / Chapter 1.2.4 --- Environmental Factors --- p.17 / Chapter 1.2.5 --- Ageing --- p.20 / Chapter 1.3 --- "1-Methy-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) as a PD Model" --- p.22 / Chapter 1.3.1 --- Discovery of MPTP --- p.22 / Chapter 1.3.2 --- The Mechanisms of MPTP-induced Neurotoxicity --- p.23 / Chapter 1.4 --- Antioxidants in the Central Nervous System --- p.26 / Chapter 1.4.1 --- Superoxide Dismutase --- p.26 / Chapter 1.4.2 --- Glutathione --- p.27 / Chapter 1.5 --- Metallothioneins (MTs) --- p.29 / Chapter 1.5.1 --- Characteristics of MTs --- p.29 / Chapter 1.5.2 --- Functions of Astrocytes --- p.31 / Chapter 1.6 --- Astrocytes --- p.34 / Chapter 1.6.1 --- Characteristics of Astrocytes --- p.34 / Chapter 1.6.2 --- Functions of Astrocytes --- p.35 / Chapter 1.6.3 --- Role of Astrocytes in Parkinson's Disease --- p.39 / Chapter 1.7 --- Aim of Project --- p.41 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- In Vitro Study --- p.44 / Chapter 2.1.1 --- Astrocyte Cultures --- p.44 / Chapter 2.1.2 --- Treatment Regimen --- p.46 / Chapter 2.1.2.1 --- 1 -methyl-4-phenyl-pyridinium (MPP+) Treatment --- p.46 / Chapter 2.1.2.2 --- Induction of Metallothioneins (MTs) and Glutathione (GSH) --- p.46 / Chapter 2.1.2.2.1 --- Northern Blot Analysis --- p.47 / Chapter 2.1.2.2.2 --- Immunocytochemical Staining for MTs --- p.48 / Chapter 2.1.2.2.3 --- GSH Assay --- p.49 / Chapter 2.1.2.3 --- Iron Chelation --- p.51 / Chapter 2.1.2.4 --- Combined Pretreatment --- p.51 / Chapter 2.1.3 --- Lactate Dehydrogenase (LDH) Assay --- p.51 / Chapter 2.1.4 --- "3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) Assay" --- p.53 / Chapter 2.1.5 --- Reactive Oxygen Species (ROS) Assay --- p.55 / Chapter 2.1.6 --- Protein Assay --- p.56 / Chapter 2.1.7 --- Statistics --- p.57 / Chapter 2.2 --- In Vivo Study --- p.57 / Chapter 2.2.1 --- "Administration of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)" --- p.57 / Chapter 2.2.2 --- Tyrosine Hydroxylase (TH) Immunocytochemical Staining --- p.58 / Chapter 2.2.3 --- DAT Receptor Binding Assay --- p.59 / Chapter 2.2.4 --- Dopamine (DA) and DA metabolites - High Performance Liquid Chromatography (HPLC) --- p.60 / Chapter 2.2.5 --- Statistics --- p.61 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- In Vitro Study --- p.62 / Chapter 3.1.1. --- Induction of Metallothioneins (MTs) in Astrocytes with Zinc Sulfate (ZnS04) --- p.62 / Chapter 3.1.1.1 --- Immunocytochemical changes --- p.62 / Chapter 3.1.1.2 --- Northern Blot Analysis --- p.62 / Chapter 3.1.1.3 --- The Effects of ZnSO4 Pretreatment on 1 -methyl-4-phenyl- pyridinium (MPP+)-treated Astrocytes --- p.63 / Chapter 3.1.1.3.1 --- Lactate Dehydrogenase (LDH) Activities --- p.63 / Chapter 3.1.1.3.2 --- "3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl- tetrazolium bromide (MTT) Activities" --- p.67 / Chapter 3.1.1.3.3 --- Reactive Oxygen Species (ROS) Production --- p.71 / Chapter 3.1.2 --- The Effects of NAc Pretreatment on MPP+-treated Astrocytes --- p.75 / Chapter 3.1.2.1 --- Glutathione (GSH) levels --- p.75 / Chapter 3.1.2.2 --- LDH Activities --- p.77 / Chapter 3.1.2.3 --- MTT Activities --- p.80 / Chapter 3.1.2.4 --- ROS Production --- p.83 / Chapter 3.1.3 --- The Effects of Deferoxamine on MPP+-treated Astrocytes --- p.87 / Chapter 3.1.3.1 --- LDH Activities --- p.87 / Chapter 3.1.3.2 --- ROS Production --- p.89 / Chapter 3.1.4 --- The Effects of ZnSO4 and NAc Combined Treatment on MPP+-treated Astrocytes --- p.92 / Chapter 3.1.4.1 --- LDH Activities --- p.92 / Chapter 3.1.4.2 --- ROS Production --- p.95 / Chapter 3.2 --- "Effects of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on MT-I, -II Knock-out Mice" --- p.99 / Chapter 3.2.1 --- The Effects of MPTP on Substantia Nigral (SN) Cell Loss --- p.99 / Chapter 3.2.2 --- The Effects of MPTP on Striatal (ST) and SN Dopamine Transporter (DAT) Binding --- p.99 / Chapter 3.2.3 --- The Effects of MPTP on ST Dopamine (DA) Metabolites --- p.100 / Chapter CHAPTER FOUR: --- DISCUSSION AND CONCLUSION --- p.102 / REFERENCES --- p.123