Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (E-MSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in E-MSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic E-MSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, Mtb Delta whiB3 displayed intramacrophage survival defect, which can be rescued by pharmacological inhibition of phagosomal acidification. Last, Mtb Delta whiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning

Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor.

1-42 beta-Amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death

1-42 beta-Amyloid (A beta(1-42)) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of A beta(1-42) peptide activation of the neurodegenerative program is still poorly understood. Here, A beta(1-42) peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol- dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of A beta(1-42) peptide in neurons.

Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

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Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

Molecular mechanisms of interleukin-2 gene inducibility: developmental control and combinatorial action of transcription factors

Interleukin-2 is one of the lymphokines secreted by T helper type 1 cells upon activation mediated by T-cell receptor (TCR) and accessory molecules. The ability to express IL-2 is correlated with T-lineage commitment and is regulated during T cell development and differentiation. Understanding the molecular mechanism of how IL-2 gene inducibility is controlled at each transition and each differentiation process of T-cell development is to understand one aspect of T-cell development. In the present study, we first attempted to elucidate the molecular basis for the developmental changes of IL-2 gene inducibility. We showed that IL-2 gene inducibility is acquired early in immature CD4- CD8-TCR- thymocytes prior to TCR gene rearrangement. Similar to mature T cells, a complete set of transcription factors can be induced at this early stage to activate IL-2 gene expression. The progression of these cells to cortical CD4^+CD8^+TCR^(1o) cells is accompanied by the loss of IL-2 gene inducibility. We demonstrated that DNA binding activities of two transcription factors AP-1 and NF-AT are reduced in cells at this stage. Further, the loss of factor binding, especially AP-1, is attributable to the reduced ability to activate expression of three potential components of AP-1 and NF-AT, including c-Fos, FosB, and Fra-2. We next examined the interaction of transcription factors and the IL-2 promoter in vivo by using the EL4 T cell line and two non-T cell lines. We showed an all-or-none phenomenon regarding the factor-DNA interaction, i.e., in activated T cells, the IL-2 promoter is occupied by sequence-specific transcription factors when all the transcription factors are available; in resting T cells or non-T cells, no specific protein-DNA interaction is observed when only a subset of factors are present in the nuclei. Purposefully reducing a particular set of factor binding activities in stimulated T cells using pharmacological agents cyclosporin A or forskolin also abolished all interactions. The results suggest that a combinatorial and coordinated protein-DNA interaction is required for IL-2 gene activation. The thymocyte experiments clearly illustrated that multiple transcription factors are regulated during intrathymic T-cell development, and this regulation in tum controls the inducibility of the lineage-specific IL-2 gene. The in vivo study of protein-DNA interaction stressed the combinatorial action of transcription factors to stably occupy the IL-2 promoter and to initiate its transcription, and provided a molecular mechanism for changes in IL-2 gene inducibility in T cells undergoing integration of multiple environmental signals.

Octopamine neurons mediate flight-induced modulation of visual processing in Drosophila melanogaster

Activity-dependent modulation of sensory systems has been documented in many organisms, and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena, and often their functional consequences, remain poorly characterized. I investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in the fruit fly Drosophila melanogaster. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine neurons that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, I find that they are both necessary and sufficient for the flight-induced visual boost. This work provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies. Further, I investigated the role of a single pair of octopamine neurons that project to the optic lobes, and found no evidence that chemical synaptic transmission via these neurons is necessary for the flight boost. However, I found some evidence that activation of these neurons may contribute to the flight boost. Wind stimuli alone are sufficient to generate transient increases in the VS cell response to motion vision, but result in no increase in baseline membrane potential. These results suggest that the flight boost originates not from a central command signal during flight, but from mechanosensory stimuli relayed via the octopamine system. Lastly, in an attempt to understand the functional consequences of the flight boost observed in visual interneurons, we measured the effect of inactivating octopamine neurons in freely flying flies. We found that flies whose octopamine neurons we silenced accelerate less than wild-type flies, consistent with the hypothesis that the flight boost we observe in VS cells is indicative of a gain control mechanism mediated by octopamine neurons. Together, this work serves as the basis for a mechanistic and functional understanding of octopaminergic modulation of vision in flying flies.

Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

Fluorescence microscopy of nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.

Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.

Lynx1 and the β2V287L mutation affect the stoichiometry of the α4β2 nicotinic acetylcholine receptor

GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent α4 and β2 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein.

Produção in vitro e criopreservação de raízes de Cleome rosea Vahl (Capparaceae)

Cleome rosea é uma espécie nativa, de porte herbáceo, ocorrente em restingas brasileiras. Estudos recentes têm revelado o potencial medicinal da espécie para importantes propriedades farmacológicas, como por exemplo, as atividades anti-inflamatória, antigenotóxica, antiviral e antibacteriana. Porém, nos últimos anos, C. rosea não tem sido encontrada em várias regiões de seu ambiente natural, devido, principalmente, às ações antrópicas. Dessa forma, torna-se relevante o desenvolvimento de métodos de conservação que permitam o estudo e exploração das propriedades medicinais da espécie. O cultivo in vitro de raízes representa uma forma eficiente para produção de biomassa, devido ao rápido crescimento, produção estável de metabólitos, além de representar uma potencial fonte de explantes para a propagação em massa de diferentes espécies. O presente trabalho teve como objetivo a produção in vitro de culturas de raízes de C. rosea, associada à criopreservação, como forma de manutenção em longo prazo das culturas, monitorada através da análise de estabilidade genética. As culturas estabelecidas a partir de explantes radiculares de plantas propagadas in vitro de C. rosea demonstraram excelente capacidade de multiplicação de raízes em meio de cultura suplementado com o fitorregulador ANA, com manutenção dessa capacidade ao longo de sucessivas subculturas. Associado a esses resultados, o estabelecimento de protocolos de criopreservação pelo método de vitrificação resultou em elevados valores de frequência de recuperação do material após congelamento em nitrogênio líquido com as soluções de vitrificação PVS2 e PVS3. Os estudos de monitoramento da estabilidade genética, pela técnica de marcadores moleculares RAPD, revelaram a presença de polimorfismos significativos em uma das três culturas iniciadas a partir de raízes de C. rosea criopreservadas. Esses resultados demonstram as possibilidades de produção de raízes de C. rosea e conservação em longo prazo através da criopreservação, iniciando estudos inéditos para a espécie.

Excreção urinária de glicosaminoglicanos em crianças com bexiga neurogênica secundária a mielomeningocele

A excreção urinária de glicosaminoglicanos (GAG) está alterada em várias patologias do trato urinário; o padrão de excreção pode estar associado com o estado da doença. A excreção urinária de GAG em crianças com bexiga neurogênica (BN) secundária a mielomeningocele (MMC) pode também estar alterada, mas até a presente data não há detalhamento epidemiológico dos pacientes e não se correlacionou o padrão de excreção com grau de disfunção vesical. Analisamos a excreção urinária de um grupo bem definido de crianças com MMC e correlacionamos os resultados com escore cistométrico. As amostras de urina de 17 pacientes com MMC, 10 meninos e 7 meninas (média de idade DP de 4,6 2,9 anos) foram obtidas durante o exame cistométrico. As amostras do grupo controle foram obtidas de 18 crianças normais, 13 meninos e 5 meninas (6,9 2,2 anos). Todas as crianças não estavam com infecção urinária, tinham função renal normal e não estavam sob tratamento farmacológico. A quantificação do GAG urinário total foi expressa em μg de ácido hexurônico / mg de creatinina e a proporção dos diferentes tipos de GAGs sulfatados foi obtida por eletroforese em gel de agarose. A avaliação cistométrica foi realizada utilizando aparelho de urodinâmica Dynapack modelo MPX816 (Dynamed, São Paulo, Brasil), a partir da qual o escore cistométrico foi calculado de acordo com procedimento recente publicado. [14]. Não observamos diferença significativa na excreção urinária de GAG total entre meninos e meninas tanto no grupo com MMC ( 0,913 0,528 vs 0,867 0,434, p>0,05) como no grupo controle (0,546 0,240 vs 0,699 0,296, p>0,05). Os resultados mostraram também que a excreção de GAG urinário não se correlacionou com a idade tanto no grupo com MMC ( r = -0,28, p> 0,05) como no grupo controle (r = -0,40, p> 0,05). Entretanto, a comparação dos dois grupos mostrou que o grupo com MMC excretava 52% a mais de GAG total que o grupo controle (0,894 0,477 vs 0,588 0,257, p <0,04). Nesses pacientes a excreção de GAG total não se correlacionou com a complacência vesical isoladamente (r = -0,18, p> 0,05) mas foi significativa e negativamente correlacionada ao escore cistométrico (r= -0,56, p<0,05). Em média, os pacientes com piores escores (<9) excretaram 81% a mais de GAG que os pacientes com melhor escore (>9) (1,157 0,467 vs 0,639 0,133, p<0,04). O sulfato de condroitin foi o GAG sulfatado predominante nos grupos neurogênico e controles (92,5 7,6% vs 96,4 4,8%, respectivamente, p> 0,05), enquanto o sulfato do heparan estava presente em quantidades marcadamente menores; o dermatam sulfato não foi detectado. A excreção urinária de GAG em pacientes com MMC é significativamente maior que a excreção das crianças normais e os altos valores encontrados estão correlacionados a um maior compromentimento da função vesical. Evidências em modelos animais com MMC induzida sugerem que alterações no detrusor estão associadas a um elevado turnover da matriz extra celular (MEC) vesical, o que pode explicar a elevada excreção de GAG nos pacientes com MMC. Além disso, esses resultados indicam que a excreção urinária de GAG pode ser usada como fator adjuvante para a caracterização da disfunção vesical em pacientes com MMC.

Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.

Interfaces e lacunas na assistência prestada ao adolescente soropositivo

O objetivo desta dissertação foi estudar as várias interfaces e possíveis insuficiências no atendimento prestado aos adolescentes que vivem com HIV / AIDS. Assim, a área de pesquisa concentrou-se em algumas unidades de saúde da área do Rio de Janeiro - Programa municipal 2.2. O primeiro capítulo descreve um esboço histórico da formação do Estado moderno e as bases para a política social sustentável do Estado capitalista contemporâneo culminando com a análise da construção de um modelo de proteção social no Brasil, depois dos anos trinta. Desta forma, o Estado é visto como uma área atravessada por paradoxal interesses conflitantes e as políticas sociais, administradas no interior do estado, sendo fruto de processos históricos, econômicos e políticos. No segundo capítulo, os problemas da política de saúde no Brasil são discutidos, enfocando as orientações das políticas sobre a AIDS e a adolescência. Em primeiro lugar, os aspectos históricos sobre as políticas de AIDS são analisados e, em seguida, há uma investigação do conceito de adolescência e os princípios norteadores do Estatuto da Criança e do Adolescente e do Programa Saúde do Adolescente. No terceiro capítulo o material coletado na pesquisa por profissionais de saúde é analisado e relacionado com o estudo documental com a crítica das políticas destinadas. A conclusão mostra que, apesar de todo o progresso clínico e / ou farmacológico para o tratamento de pessoas vivendo com AIDS e na formulação de políticas públicas para garantir os direitos, os adolescentes precisam de espaços de boas-vindas nas relações sociais, onde nem a família, a religião, a escola e seus pares estão preparados para essa proximidade. Outra questão importante é a incapacidade dos profissionais de saúde para lidar com os vários aspectos da doença. Contas de contenção e abuso são comuns, revelando que as unidades de saúde nem sempre desenvolvem o seu potencial para cuidar.