Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.

Influenza A virus (IAV) exhibits a high mutation frequency. Mutations in primary viral targets such as hemagglutinin, neuraminidase, and the proton channel M2 have afforded IAV with substantial drug resistance to previously effective drugs such as oseltamivir and amantadine (AMT) along with their analogs. One of the main drug-resistant alterations in the M2 protein that has become ubiquitous is the mutation S31N. Divalent copper has previously been suggested to have anti-IAV properties using in vitro assays involving Xenopus oocytes and SSNMR. In this work, the EC50 of AMT, two AMT analogs, CuCl2 · 2H2O, and four previously published Cu2+ complexes were tested for antiviral activity against the California/07/2009 (H1N1) IAV strain containing the S31N M2 protein in viral mini-plaque assays. AMT, CuCl2 · 2H2O, and two previously published divalent copper complexes were tested for M2 proton-transport blocking efficacy in liposomes using truncated M2 (22-62, S31N). Two novel divalent copper species, NAG101 and NAG107, both derived from AMT analogs were developed and tested for both antiviral activity and M2 blocking efficacy. Cell integrity was maintained at concentrations up to 1 mM (48 hours of exposure) with all compounds except Cu(II) 3. In the viral mini-plaque assay the novel divalent copper complexes NAG101 (EC50 25.7 +/-7.7 µM) and NAG107 (EC50 2.91 +/- 0.29 µM) were 2.5-fold and 21-fold better than AMT (EC50 64.3 +/- 9.3 µM) respectively. In the liposome assay NAG101 (EC50 18.9 +/- 1.5 µM) and NAG107 (EC50 4.5 +/- 0.6 µM) were 2.5-fold and 11-fold better than AMT (EC50 49.3 +/- 2.9 µM respectively. In the viral mini-plaque assay, CuCl2 · 2H2O (EC50 57.2 +/- 10.1 µM) was comparably effective to AMT, but was more than ten times more effective in the liposome assay targeting S31N M2 with an EC50 6.1 +/- 0.8 µM. Divalent copper species possess anti-IAV activity against the ubiquitous S31N M2-containing viral strain California/07/2009 (H1N1) and may prove to be effective against other IAV strains by blocking M2.

Post-traumatic epilepsy is a common outcome of severe traumatic brain injury (TBI). The development of spontaneous seizures after traumatic brain injury generally follows a latent period of little to no symptoms. The series of events occurring in this latent period are not well understood. Additionally, there is no current treatment to prevent the development of epilepsy after TBI (i.e. antiepileptogenics). One cell signaling pathway activated in models of TBI and in models of epilepsy is the mammalian target of rapamycin (mTOR). mTOR activity is sustained for weeks after the initial insult in models of TBI, and the inhibition of mTOR using rapamycin has shown promising pre-clinical outcomes in rodent models. This makes rapamycin an ideal therapeutic to test various outcomes associated with epileptogenesis after TBI. The results from this study suggest that rapamycin treatment after controlled cortical impact reduces aberrant axonal sprouting of ipsilateral dentate granule cells, prevents increased neurogenesis in the subgranular zone, and differentially alters phasic and tonic inhibition in dentate granule cells. However, rapamycin treatment did not prevent all forms of axon sprouting in the dentate gyrus or cell loss in selected regions of the hippocampus. Collectively these results support a role of mTOR activity in both excitatory and inhibitory plasticity in the mouse dentate gyrus after TBI.

Fear and stigmatization are often used to motivate overweight individuals to engage in healthy behaviors, but these strategies are often counterproductive and can lead to undesirable outcomes. In the present study, I examined the impact of weight-based stigma on cognitive ability (i.e., inhibitory control) and food selection (i.e., calories selected) in individuals who consider themselves to be overweight. I predicted that participants higher in perceived weight stigma would perform more poorly on an inhibitory control task and order more calories on a menu task when they read about discrimination against the overweight versus discrimination against a self-irrelevant out-group. Additionally, I expected that inhibitory control would mediate the relationship between perceived weight stigma and calories ordered for participants who read about discrimination against the overweight, but not for control participants. Participants completed online prescreen measures assessing whether or not they considered themselves to be overweight and their perceptions of weight stigma. Those individuals who considered themselves overweight were then invited into the laboratory to complete tasks to (1) manipulate weight-based discrimination, (2) measure inhibitory control, and (3) measure food selection. As predicted, participants higher in perceived weight stigma performed more poorly on the inhibitory control task and ordered more calories when they read about discrimination against the overweight, but not when they read about discrimination against an out-group. Conversely, inhibitory control did not mediate the relationship between perceived weight stigma and number of calories ordered by participants. Importantly, the present findings provide evidence that perceptions of weight stigma are critical in our understanding of the impact of weight discrimination. Additionally, these results have theoretical and practical implications for both understanding and addressing the psychological and physical consequences of weight-based stigma.

Os neutrófilos estão entre as principais células da imunidade inata e são as primeiras células a migrarem para o sítio de infecção. O BAY 41-2272, estimulador de guanilato ciclase solúvel, é capaz de ativar fagócitos mononucleares e em neutrófilos diminuir migração in vivo e in vitro. O presente trabalho teve como objetivo avaliar o potencial do BAY 41-2272, e sua via, como uma ferramenta farmacológica para modulação da função dos neutrófilos. Para isso foi realizado o tratamento in vitro dos PMNs com o BAY 41-2272. A viabilidade celular, quimiotaxia e funções efetoras como burst oxidativo e produção de citocinas foram avaliadas, observando-se que o BAY 41-2272, como ativador direto, não altera estas funções. No entanto, o pré-tratamento com BAY 41-2272 nas doses de 3 M e 30M por uma hora, com subsequente ativação com PMA, mostrou perfil inibitório na quimiotaxia, produção da citocina IL-8 e no burst oxidativo . Foram avaliados também a expressão de moléculas como CD15, CD31, CD35, CD49d, CD63, CD66b, CD162 e a produção de GMPc. As moléculas de superfícies não mostraram alterações após tratamento direto com BAY 41-2272. A produção de GMPc foi induzida na dose de 30 M de BAY 41-2272 e pelo SNAP (doador de NO). Esses dados sugerem um potencial inibitório do BAY 41-2272 sobre a ação dos neutrófilos, e uma possível alternativa a ser explorada em seus aspectos translacionais na busca por novas terapias destinadas ao controle de doenças ligadas a inflamações crônicas e doenças auto-imunes. / Neutrophils are the major cells of innate immunity and are the first cells to migrate to the site of infection. BAY 41-2272, a soluble guanylate cyclase stimulator, is capable of activating mononuclear phagocytes and in neutrophils decreasing migration in vivo and in vitro. The present work aimed to evaluate the potential of BAY 41-2272, and its pathway, as a pharmacological tool for modulating neutrophil function. For this, the in vitro treatment of PMNs with BAY 41-2272 was performed. Cell viability, chemotaxis and effector functions such as oxidative burst and cytokine production were evaluated, observing that BAY 41-2272, as a direct activator, does not alter these functions. However, pretreatment with BAY 41-2272 at the doses of 3 M and 30 M for one hour, with subsequent activation with PMA, showed an inhibitory profile in chemotaxis, IL-8 cytokine production and in the \"oxidative burst\". We also evaluated the expression of molecules such as CD15, CD31, CD35, CD49d, CD63, CD66b, CD162 and cGMP production. Surface molecules did not show changes after direct treatment with BAY 41-2272. The cGMP production was induced at the dose of 30 M BAY 41-2272 and for SNAP (NO donor). These data suggest an inhibitory potential of BAY 41-2272 on the action of neutrophils, and a possible alternative to be explored in its translational aspects in the search for new therapies aimed at the control of diseases linked to chronic inflammation and autoimmune diseases.

Abstract not available

<p>Bacillus anthracis is one of the most feared agents of biological warfare and causes the</p><p>deadly disease called anthrax. SVA (statens veterinärmedicinska anstalt) is working on a</p><p>project together with SLV (statens livsmedelsverk) where the target is to find rapid and</p><p>effective detection methods for Bacillus anthracis in animal feed and food samples. Bacillus</p><p>subtilis, which is harmless, was used in this study as a model organism to Bacillus anthracis.</p><p>A known concentration of vegetative Bacillus subtilis was spiked in animal feed and food</p><p>samples. The genomic work flow was based on automated DNA isolation and real time PCR.</p><p>The aim of the study was to screen for inhibitory components in the animal feed and food</p><p>samples using two different DNA isolation robots; Magnatrix 8000 and Biorobot EZ1. The</p><p>results showed that DNA of high quality was extracted from the samples with both robots.</p><p>However, the CT-value generated by the real time PCR showed considerable variation</p><p>depending on the sample matrix. Some samples, for instance egg and liver, were problematic</p><p>and gave low concentrations and high CT-values probably due to inhibitory components in the</p><p>samples. Further studies will be needed to solve these problems and optimize the methods that</p><p>were used in this study.</p>

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 22, 2007). Includes bibliographical references (p. 92-107).

Bacillus anthracis is one of the most feared agents of biological warfare and causes the deadly disease called anthrax. SVA (statens veterinärmedicinska anstalt) is working on a project together with SLV (statens livsmedelsverk) where the target is to find rapid and effective detection methods for Bacillus anthracis in animal feed and food samples. Bacillus subtilis, which is harmless, was used in this study as a model organism to Bacillus anthracis. A known concentration of vegetative Bacillus subtilis was spiked in animal feed and food samples. The genomic work flow was based on automated DNA isolation and real time PCR. The aim of the study was to screen for inhibitory components in the animal feed and food samples using two different DNA isolation robots; Magnatrix 8000 and Biorobot EZ1. The results showed that DNA of high quality was extracted from the samples with both robots. However, the CT-value generated by the real time PCR showed considerable variation depending on the sample matrix. Some samples, for instance egg and liver, were problematic and gave low concentrations and high CT-values probably due to inhibitory components in the samples. Further studies will be needed to solve these problems and optimize the methods that were used in this study.

Extensive evidence suggests that glucose has both positive and negative effects on memory and these effects likely involve an influence on the brain. For instance, direct infusions of glucose into the septum (MS) or hippocampus can enhance or impair memory. The present set of experiments attempted to determine the different conditions that dissociate the memory-enhancing and -impairing effects of glucose in rats. Specifically, these experiments examined the effects of glucose in spontaneous alternation, a measure of spatial working memory and shock avoidance, an index of emontional long-term memory. The results showed that the memory-impairing effects of MS infusions of glucose are not concentration-dependent. These data also indicated that the memory-impairing effects of MS glucose elevations are specific to gamma-aminobutyric acid GABA receptor activation but do not depend on increases in MS GABA synthesis or release. Importantly, we showed that the memory-impairing interaction between MS glucose and GABA agonists does not generalize to the hippocampus, suggesting the memory-modulating effects of glucose are brain region-dependent. We showed further that these brain region-dependent effects of glucose are not due to difference in basal extracellular glucose levels. Moreover, these findings showed that the memory-enhancing effects of hippocampus glucose override the memory-impairing interaction between MS glucose and GABA. These findings are important because they are the first to show that the memory-modulating effects of glucose are both neurotransmitter- and brain region-dependent. Furthermore, these findings provide preliminary evidence suggesting that the memory-impairing effects of MS glucose may involve compromised hippocampal function. These data also suggest the memory-impairing effects of MS co-infusions of glucose with GABA agonists likely involve an influence on the GABAergic SH projection. Finally, these findings demonstrate the mnemonic and neurochemical consequences of glucose in the MS and hippocampus, two brain regions affected by normal aging, Alzheimer’s disease, and diabetes.