Made available in DSpace on 2016-06-02T20:19:10Z (GMT). No. of bitstreams: 1 2258.pdf: 1895332 bytes, checksum: 2fb8eb6332f875b5c6927d676c54a134 (MD5) Previous issue date: 2009-02-16 / Universidade Federal de Minas Gerais / This study investigated the effect of post-training treatment with L-histidine, Pyrilamine and Zolantidine on the memory consolidation of inhibitory avoidance in Carassius auratus submitted to cerebellar ablation. Cerebellar ablation was performed 3 days before the experiment. The inhibitory avoidance procedure included one habituation day, one training day (5 trials: T1, T2, T3, T4, T5) and a test day (TE). On the training day, each fish was individually placed in a white compartment separated from a black compartment by a sliding door. After 30s, the door was opened. When the fish crossed into the black compartment, a weight was dropped in front of it (aversive stimulus). An intraperitoneal injection with saline or L-histidine (100 mg/kg), Pyrilamine (35 mg/ml) and Zolantidina (ZO) was administered 10 minutes after the trials. The difference in the time elapsed until the fish entered the black compartment (latency) during training and testing was taken as an indicator of behavioral learning and memory. Data were analyzed by ANOVA and the Student Newman-Keuls test (p< 0.05). The three experiments indicated that in T5, all groups had similar latencies that were significantly higher than T1 latencies, indicate that all groups were able to acquire inhibitory avoidance, suggesting that cerebellar ablation does not impair learning. However, twenty four hours after the trainings, the animals with ablation indicating retention of the task while that the animals of the Sham group do not consolidate memory. The results of experiment I showed that the L-histidine reverted the effect of injury. Experiment II demonstrated that the Pyrilamine does not influence in the consolidation of the memory and the data of experiment III indicated that the zolantidina facilitates the acquisition of inhibitory avoidance independent of the cerebellum. These results suggest that the cerebellum is involved in the memory consolidation of inhibitory avoidance and/or interpretation of the aversive value and that this process is mediated by H2 receptors. / Esse trabalho teve como objetivo investigar o efeito farmacológico do precursor da histamina (L-histidina) e dos antagonistas dos receptores H1 (Pirilamina) e H2 (Zolantidina) na consolidação da memória de esquiva inibitória em Carassius auratus após ablação cerebelar. Para isso, foram realizados três experimentos, sendo empregados os mesmos procedimentos experimentais, no entanto, diferindo nos tratamentos farmacológicos realizados, que foram: I -L-histidina (dose: 100mg/kg) II - Pirilamina (dose: 35 mg/kg) e III - Zolantidina (dose: 20mg/kg), sendo todos acompanhados de dois grupos controle (Sham), um injetado com solução salina e outro da cirurgia (Lesão Fictícia). Um aquário foi dividido em dois compartimentos iguais por uma porta tipo guilhotina, sendo um compartimento branco e o outro preto. No compartimento preto foi associado um sistema de polia, do qual um peso era liberado na sua extremidade quando o animal entrava neste compartimento. Após três dias de recuperação da cirurgia, foi realizado o procedimento comportamental composto por três dias consecutivos, sendo eles: Habituação, Treino cinco treinos (T1, T2, T3, T4 e T5) e Teste. O tratamento farmacológico era realizado via intraperitoneal após dez minutos do último treino (T5) e em seguida os peixes foram recolocados no aquário de origem. O tempo utilizado pelo animal para entrar no compartimento escuro (latência) foi registrado em segundos nos treinos e no teste. Os dados foram analisados pelo teste de homogeneidade (Levene) e quando necessário foram transformados para log10 (Experimento I). As latências dos treinos e do teste de cada experimento foram submetidas a análise de Variância (ANOVA), seguido pelo teste de comparação múltipla Student Newman Keuls (SNK). Os três experimentos indicaram que a ablação do cerebelo não impede que o animal aprenda o condicionamento de esquiva inibitória, no entanto, vinte quatro horas após o treino, os animais com ablação indicam retenção da tarefa enquanto os animais do grupo Sham não consolidam a mesma. Os resultados do experimento I mostraram que a L-histidina reverteu o efeito da lesão. O experimento II demonstrou que a Pirilamina não influencia na consolidação da memória e os dados do experimento III indicam que a zolantidina facilita a aquisição de esquiva inibitória independente do cerebelo. Estes resultados sugerem que o cerebelo está envolvido na consolidação da memória de esquiva inibitória e/ou interpretação do valor aversivo e que este processo é mediado via receptores H2.

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

Orientador: Lisandro Pavie Cardoso / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-07T10:55:28Z (GMT). No. of bitstreams: 1 Menezes_AlanSilvade_M.pdf: 6146664 bytes, checksum: 0379ab9f914d1e2c189aba25560a6217 (MD5) Previous issue date: 2006 / Resumo: Neste trabalho, o método baseado na variação nas posições angulares dos picos secundários em uma varredura Renninger da difração múltipla de raios-X usando radiação síncrotron foi utilizado para a determinação dos coeficientes piezelétricos da -histidina. HCl. H2O. A grande sensibilidade da técnica a pequenas deformações na célula unitária dos cristais analisados permite esta determinação, desde que o campo elétrico seja aplicado nas direções específicas escolhidas nas amostras. Foram determinados os parâmetros de rede da -histidina.HCl. H2O pura e dopada com Ni 2+, com boa precisão, a partir da escolha adequada dos picos secundários nas varreduras Renninger, e, também determinou-se o coeficiente d14 para a amostra dopada. Destes resultados foi observada uma contração do volume da célula unitária (0,04%) do cristal dopado em relação ao puro, resultado também obtido através do refinamento das amostras em forma de pó com o método de Rietveld. O efeito do Ni 2+ na rede da -histidina.HCl. H2O foi evidenciado na comparação entre as varreduras Renninger para os dois cristais, puro e dopado, através de mudanças na intensidade, posição angular e assimetria dos picos secundários, principalmente no pico correspondente ao caso de quatro-feixes (604)(404) simultâneos para a reflexão primária (10 0 0), que praticamente desapareceu no cristal dopado, indicando possivelmente uma mudança de fase nas reflexões deste pico secundário. Observamos ainda, a mudança no hábito de crescimento e na coloração do cristal quando dopado com Ni 2+ , e a clara diminuição na taxa de crescimento da face [1 0 0]. Por fim, a difração múltipla de raios-X usando a radiação síncrotron permitiu a determinação, pela primeira vez, de todos os coeficientes piezelétricos do cristal de -histidina.HCl. H2O: [ d14] = 295(9) pC/N; [ d25] = 41(5) pC/N; [ d36] = 230(20) pC/N, assim como do coeficiente [ d14] = 93(5) pC/N para o cristal dopado com Ni 2+ / Abstract: In this work, the method based on the variation in the secondary peak angular positions of the Renninger scanning of the X-ray multiple diffraction using synchrotron radiation was used in the determination of -histidine.HCl. H2O piezoelectric coefficients. The high sensitivity of this technique to subtle distortions in the analyzed crystal unit cells allows this determination since the electric field be applied towards the previously chosen specific directions in the samples. Pure and Ni 2+ -histidine. HCl. H2O lattice parameters were measured with good precision from the adequate choice of Renninger scanning secondary peaks as well as, the d14 piezoelectric coefficient of the doped sample. From these results a contraction of the doped unit cell volume (0.04%) regarding the pure one was obtained, which is in agreement with that contraction obtained from the refinement of powder samples using the Rietveld method. The effect of Ni 2+ in the -histidine. HCl. H2O lattice was evidenced in the comparison between the Renninger scanning for both pure and doped crystals through the intensity, angular position and asymmetry of the secondary peaks, mainly the one corresponding to the (604)(404) four-beam simultaneous case for (10 0 0 ) primary reflection, that has practically disappeared for the doped crystal scanning. It indicates a possible change in the phase of the secondary peak reflections. It was also observed a change in the growth habit and color of the crystal when doped as well as a clear decrease in the [100] face growth ratio. At last, the x-ray multiple diffraction using synchrotron radiation allowed, for he firs ime, the determination of all piezoelectric coefficients for the -histidine. HCl. H2O crystal: [ d14] = 295(9) pC/N; [d25] = 41(5) pC/N; [d3]? = 230(20) pC/N, and the [d14] = 93(5) pC/N for the Ni 2+ doped crystal coefficient / Mestrado / Estrutura de Liquidos e Solidos ; Cristalografia / Mestre em Física

Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation. KEYWORDS Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation

No description available.

Résumé : Grâce à des caractéristiques physiques particulières, le [indice supérieur 64]Cu (T[indice inférieur 1/2]= 12.7 h; β[indice supérieur+], 0.65 MeV [17.8 %]; β[indice supérieur −], 0.58 MeV [38.4 %]) est un candidat idéal pour l’imagerie TEP et la radiothérapie ciblée du cancer. Son utilisation est actuellement limitée par la disponibilité de chélateurs bi-fonctionnels (CBFs) offrant une résistance élevée aux réactions de transmétallation in vivo. Récemment nous avons développés deux nouveaux CBFs cycliques, DOTHA[indice inférieur 2] et NOTHA[indice inférieur 2], portant des ligands hydroxamates pour la complexation au [indice supérieur 64]Cu. Ces CBFs possèdent une cinétique de marquage rapide dans des conditions très douces, une stabilité élevée in vivo et un profil de biodistribution favorable avec une clairance rapide. Nous proposons maintenant d’étendre notre approche à la préparation de CBFs acycliques plus flexibles et compacts afin de moduler les propriétés biologiques et la pharmacocinétique des traceurs peptidiques. Le but de mon projet de maîtrise est de développer une série de chélateurs acycliques dérivés de l'histidine et de l'acide glutamique et fonctionalisés avec des groupements hydroxamates pour identifier un CBF offrant un complexe stable in vivo avec le [indice supérieur 64]Cu(II). Les CBFs ont été préparés en solution pour faciliter l’optimisation de chaque étape réactionnelle. Les groupements chélatants hydroxamates ont été sélectionnés pour leur habilité à former des complexes stables avec différents métaux et ils ont été liés en position N-terminale et sur la chaîne latérale des acides aminés grâce à des réactions de substitution nucléophile. Les groupements para-methoxy-benzyles ont été judicieusement sélectionnés pour la protection des groupements hydroxamates afin de faciliter, au besoin, une déprotection sélective sous des conditions très douces. L’optimisation du marquage a été effectuée avec l’isotope stable du cuivre et ensuite avec le [indice supérieur 64]Cu en faisant varier le contre ion métallique, le pH, la concentration, et la température. Le CBF offrant la plus grande stabilité, soit celui dérivé de l’histidine, a été conjugué à un peptide, le H[indice inférieur 2]N-PEG-[D-Tyr[indice supérieur 6],βAla[indice supérieur 11],Thi[indice supérieur 13],Nle[indice supérieur 14]]bombesin(6-14) (BBN), se liant fortement aux récepteurs de la relâche de la gastrine surexprimés dans les cancers du sein et de la prostate. La stabilité et l’activité spécifique du CBF-histidine et du radiotraceur marqués au [indice supérieur 64]Cu s’est avérée faible in vitro. Il est connu que l’activité antibactérienne de ligands hydroxamates est associée à leur capacité à complexer le fer. En perspective, comme nos chélateurs complexent très fortement le Fe(III), une alternative pour ces composés serait d’évaluer leur capacité à inhiber la croissance et la prolifération des bactéries. || Abstract : Thanks to its particular physical characteristics, [superscript 64]Cu (T[subscript ½= 12.7 h; β[superscript +], 0.65MeV [17.8 %]; β[superscript −], 0.58MeV [38.4 %]) is an ideal candidate for PET imaging and targeted cancer radiotherapy. Currently, its use is limited by the availability of bi-functional chelators (BFCs) which give high resistance to in vivo transmetallation reactions. Recently, we developed two new cyclic BFCs, DOTHA[subscript 2] and NOTHA[subscript 2], bearing hydroxamate pendant arms for the complexation with [superscript 64]Cu. Those BFCs have fast labeling kinetics under very mild conditions, a high in vivo stability and a biodistribution profile which is favorable with a fast clearance. Now, we propose to expand our approach to the preparation of acyclic BFCs, which are more flexible and compact, in order to better modulate biological properties and the pharmacokinetics of the peptidic tracers. The goal of my Master’s degree project is to develop a series of acyclic chelators derived from histidine and glutamic acid and functionalized with hydroxamate pendant arms to identify a BFC that shows highly stable in vivo complexes with [superscript 64]Cu(II). BFCs have been prepared in solution to facilitate the optimization of each reactive step. Hydroxamate chelating groups have been selected for their ability to form stable complexes with different metals and they have been conjugated in N-terminal position and on the lateral chain of amino acids via nucleophilic substitution reactions. Para-methoxy-benzyl groups have been judiciously selected for the protection of the hydroxamate groups to facilitate, if needed, a selective deprotection under mild conditions. The labeling optimization has been performed with a stable copper isotope, and then with [superscript 64]Cu varying the metallic counter-ion, pH, concentration and temperature. The BFC having the highest stability, the one derived from histidine, was conjugated to a peptide, H[subscript 2]N-PEG-[D-Tyr[superscript 6],βAla[superscript 11],Thi[superscript 13],Nle[superscript 14]]bombesin(6-14) (BBN), strongly bounding the gastrin releasing peptide receptor, which is overexpressed in breast and prostate cancers. Both the stability and specific activity of BFC-histidine of the radiotracer labeled with [superscript 64]Cu were low in vitro. It is known that the antibacterial activity of hydroxamate ligands is associated with their ability to complex iron. In perspective, because our hydroxamate ligands strongly complex Fe(III), an alternative for these compounds would be to assess their ability to inhibit the growth and proliferation of bacteria.

Two-component signal systems serve as basic stimulus-response coupling mechanism to allow organisms (predominantly bacteria) to sense and respond to changes in many environmental conditions. The prototypical system consists of two proteins, namely a histidine kinase, containing a sensor domain and catalytic kinase core, and a response regulator protein (RR protein). Extracellular stimuli are sensed by a histidine kinase sensor domain. Then ATP is bound to the catalytic kinase core and the γ-phosphoryl group is transferred to the conserved histidine residue. This phosphoryl group is subsequently transferred to a conserved aspartate residue within the RR protein. Phosphotransfer to the RR protein results in activation of a downstream effector domain that elicits the specific response (usually it is transcription activity, but a few RR proteins function as enzymes). The histidine kinase sensor domain is designed for specific ligand interactions. This master thesis focused on the unique histidine kinase containing a sensor domain with a globine structure, which coordinates a heme molecule, namely globin-coupled histidine kinase from Anaeromyxobacter sp. Fw 109-5 (AfGcHK) and its appropriate RR protein. The aim of this thesis was to study and characterize the phosphorylation activity of AfGcHK and RR...

A exposição prolongada a estressores socio-ambientais induz alterações duradouras nos níveis afetivo, cognitivo e fisiológico característicos de transtornos de ansiedade e depressão. No paradigma de estresse crônico variado (ECV) é possível modelar essas alterações com base na exposição aleatória, intermitente e incerta dos roedores a vários estressores. Porém, alguns indivíduos também demostram uma capacidade notável de adaptação ativa e persistem diante de eventos imprevisíveis e incontroláveis. Sabe-se também que o sistema neural histaminérgico (SNH) é um indicador sensível do estresse e regula as reações defensivas relacionadas. Contudo, pouco se sabe sobre o papel da histamina no modelo de ECV. Considerando ainda que o perfil comportamental dos camundongos estressados pelo ECV seja contraditório, o presente estudo investigou se (1) duas linhagens de camundongos seriam susceptíveis a respostas relacionadas ao estresse; (2) a neurotransmissão histaminérgica estaria envolvida na ansiedade induzida pelo estresse; (3) o tratamento crônico com L-histidina (LH) combinado ou não ao ECV modificaria a expressão de Fos em áreas cerebrais límbicas. Para testar o impacto do protocolo ECV sobre respostas do tipo depressivas, os comportamentos de camundongos Suíços não estressados (NST) e estressados (ST) foram analisados na tarefa de esquiva ativa de duas vias e no teste de suspensão da cauda. Não foi detectado aumento significativo da imobilidade passiva, mas o grupo ST apresentou hiperreatividade na tarefa de esquiva. Como etapa seguinte, os efeitos do ECV no comportamento ansioso dos animais NST e ST foi verificado no labirinto em cruz elevado (LCE). Notavelmente, camundongos C57Bl/6 estressados desenvolveram respostas ansiogênicas, enquanto a linhagem de Suíço exibiu um perfil comportamental heterogêneo no LCE. Estes resultados indicam que o regime de ECV induz um efeito ansiogênico de modo consistente em animais C57Bl/6 adultos, enquanto os camundongos Suíço são resilientes ao protocolo. Além disso, a ansiedade induzida pelo ECV não foi revertida ou potencializada pela administração crônica de LH, enquanto que a estimulação farmacológica prolongada do SNH poderia representar um potencial estresse isoladamente. Adicionalmente, uma hipo-ativação das áreas corticais pré-limbicas e infralímbicas foi relacionada à condição de estresse crônico, sem efeitos resultantes do tratamento farmacológico. A expressão de Fos+ induzida pela exposição ao LCE foi detectada nos subnúcleos lateral, basolateral e central, porém não houve ativação diferencial destes subnúcleos amígdaloides influenciados pelo ECV e/ou tratamento. Assim, os resultados apresentadas corroboram evidências de que respostas ao estresse são genética e experiência-dependentes, resultando em resiliência ou má adaptação de indivíduos e linhagens. Além disso, o ECV foi capaz de causar uma resposta ansiogênica acompanhada de hipo-ativação de subáreas específicas do córtex pré-frontal medial, região cerebral importante na regulação dos comportamentos defensivos e nas respostas psicofisiológicas do estresse. Finalmente, o tratamento crônico com LH não alterou os parâmetros comportamentais e neuroanatômico-funcionais influenciados pelo estresse. / Chronic exposure to socio-environmental stressors leads to a myriad of long-term alterations in affective, cognitive and physiological levels, which typifies prevalent neuropsychiatric disorders. Importantly, chronic variable stress (CVS) is an experimental model for anxiety- and depressive-like disorders based on the random, intermittent, and uncertain exposure to various stressors. Some individuals also show a remarkable ability to adapt and actively cope and persist in the face of such unpredictable and uncontrollable events. Histamine is a sensitive indicator of stressful experiences and modulates the activation of neuroendocrine stress response to influence defensive reactions. However, little is known about the role of histamine on CVS model. While the behavioral profile of CUS-stressed mice is also contradictory, we investigated whether (1) two widely used mouse strains were susceptible to stress-related responses; (2) histaminergic neurotransmission is involved on stress-induced anxiety; (3) L-histidine (LH) chronic treatment combined to CVS changes Fos expression in limbic areas. To test the impact of the CVS protocol on depressive-like responses, the performance of non-stressed (NST) and stressed (ST) Swiss animals was analyzed in the two-way avoidance task and in the tail suspension test. No increased passive immobility was detected, but the ST group did display hyperreactivity in the avoidance task. Next, the effects of CVS on anxiety were examined in the elevated plus maze (EPM). Remarkably, stressed C57Bl/6 developed anxiogenic responses, while Swiss mice displayed a heterogeneous behavioral profile in the EPM. These results indicate that 2-week-long CVS regimen consistently induces anxiogenic-like response in adult C57Bl/6 mice, while Swiss animals seem to be resilient. Additionally, CVS-induced anxiety is not reversed or potentialized by the chronic administration of LH, but the histamine precursor appears to be a potential stressor per se. Importantly, a hypoactivation of the prelimbic and infralimbic cortical areas was related to the chronic stress condition, with no main effects of the pharmacological treatment. EPM induced Fos+ expression was detected in the lateral, basolateral and central subnuclei, without differential activation of these amygdaloid subnuclei provoked by CVS and/or histaminergic stimulation. The present evidences corroborate the concept that stress responses can be genetic- and experience-dependent, resulting in resilience or maladaptation of a particular strain. Also, stress-induced anxiety could be related to a hypoactivation of the medial prefrontal cortex, important brain region in regulating the defensive behaviors and HPA stress response.

A atividade biológica de aldeídos &#945;,&#914;-insaturados tem sido associada a diversos processos incluindo regulação gênica, envelhecimento Alzheimer e disfunções mitocondriais. Neste trabalho investigamos a formação de adutos do trans,trans-2,4-decadienal (DDE), um aldeído produzido endogenamente e presente como contaminante em alimentos e água, em lisina, histidina e citocromo c. Avaliamos também alterações na função de mitocôndrias de fígado de rato expostas ao aldeído. As análises por espectrometria de massas, LC-ESI/MS, indicaram a formação de diversos tipos de adutos de DDE nos aminoácidos lisina e histidina, entre eles bases de Schiff e enaminas. Os resultados obtidos por espectrometria de massas, MALDI-Tof, indicaram a formação de adutos de DDE formados via base de Schiff de maneira concentração do aldeído, tempo e pH dependentes. As análises da proteína digerida por ESI-Q-ToF, indicaram que os adutos foram formados nos resíduos H-33, K-39, 72 e 100, localizados em regiões ricas em resíduos básicos, cuja interação com membranas e citocromo e oxidase tem sido postulada. Observamos também o deslocamento da banda Soret (&#955; = 409 nm) e o desaparecimento da banda em &#955; = 695 nm, relativa a coordenação do sexto ligante do grupo heme (M-80). Este fenômeno está associado a abertura da cavidade do grupo heme e o deslocamento do ligante, indicando alterações nas estruturas secundária e terciária da proteína. Os experimentos realizados com mitocôndrias isoladas indicaram que DDE promove danos à membrana interna mitocondrial, demonstrando i pelo aumento no consumo de O2 em mitocôndrias em estado 4. Em decorrência destas lesões observamos também o intenso inchamento mitocondrial, indicado por experimentos de espalhamento de luz e microscopia eletrônica de transmissão. O inchamento não foi bloqueado por ciclosporina A, um inibidor do poro de transição mitocondrial. DDE também induziu a perda do potencial de membrana da organela, demonstrado pelo monitoramento do indicador fluorescente safranina O e aumento da peroxidação lipídica atestado pela quantificação de malondialdeido (MDA). Estes resultados indicam que DDE promove alterações estruturais no citocromo e podendo levar ao comprometimento da atividade da proteína, além de promover alterações em parâmetros mitocondriais, indicando um possível envolvimento na disfunção mitocondrial promovida por estresse oxidativo. / Lipid hydroperoxide-derived &#945;,&#946;-unsaturated aldehydes are involved in several cellular processes such as gene expression, aging, Alzheimer disease and mitochondrial dysfunction. In this work we have investigated adduct formation in lysine, histidine and cytochrome c by trans,trans-2,4-decadienal (DDE), an endogenously lipoperoxidation product. DDE is also a widespread environment aldehyde found, for example, in food and as a contaminant in water. Alterations in rat liver mitochondrial parameters such as oxygen consumption, membrane potentials, swelling and lipoperoxidation were also investigated. LC-ESI/MS experiments indicated that DDE react with aminoacids lysine and histidine producing adducts. In addition, MALDI-TOF experiments indicated increases in the molecular weight of cytochrome c consistent with the formation of DDE adducts via the Schiff base mechanism. Our data shows that the protein modification was time, pH and DDE-concentration dependent, leading to the formation of at least six adducts after 2 h incubation at pH 7.4. ESI-Q-TOF MS analysis of tryptic digests indicated that H-33, K-39, K-72 and K-100 were modified by DDE. These adducts are present in clusters of basic amino acid residues, which are believed to participate in the interaction of cytochrome c with mitochondrial membrane and cytochrome c oxidase. A blue shift in the Soret band from 409 to 406 nm was also observed, indicating heme crevice opening and displacement of heme sixth ligand (Met-80) coordination in modified protein. DDE (180 &#181;M) treatment increases the rate of mitochondrial oxygen consumption, suggesting a partial mitochondrial uncoupling. Moreover, extensive mitochondrial swelling upon treatment with DDE (900 nM-162 &#181M) was observed by light scattering and transmission electron microscopy experiments. These effects were not prevented by the mitochondrial permeability transition inhibitor cyclosporin A. A DDE concentration-dependent loss in the inner mitocondrial membrane potential, monitored by safranin O fluorescence and an increase in lipoperoxidation were also observed. All together, these results suggest that reactive aldehydes can induce inner mitochondrial membrane damage playing a role in mitochondrial dysfunction associated with oxidative stress.

Made available in DSpace on 2017-07-24T19:37:52Z (GMT). No. of bitstreams: 1 Robson Rodrigo Miranda.pdf: 3117830 bytes, checksum: 06e1c7195d09d214dbcc6eeb5412efe4 (MD5) Previous issue date: 2015-03-10 / Chagas disease is one of the seventeen neglected tropical diseases according to the World Health Organization. In the recent decades, new parasite metabolic pathways were identified, what brings perspectives for the development of more specific and less toxic drugs, towards crucial target pathways. Once the therapeutic target is identified, a structural and biochemical characterization of the enzymes involved becomes necessary. It may be speculated that one possible therapeutic target to combat Chagas disease is the Histidine Ammonia-Lyase enzyme, which participates in the catabolic pathway of histidine. Therefore, in order to contribute to the structural and biochemical understanding of this enzyme, their heterologous production in E. coli was performed. The product protein was purified by affinity chromatography and used in various techniques for initial characterization. The activity was determined by kinect assay, the thermal stability and secondary structure content were investigated by Circular Dichroism (CD) and the oligomerization stated in solution was analyzed by Dynamic Light Scattering (DLS). The X ray diffraction technique was used to elucidate the three dimensional structure. TcHAL was expressed and purified satisfactorily. The activity proved adequate protein folding and the Circular Dichroism indicated a predominance of α-helix secondary structure and the start of the thermal denaturation at 68°C. TcHAL was crystallized and provided suitable diffraction patterns for the 3D structure elucidation. These biochemical and structural studies advanced the understanding of this enzyme and of the inhibition potentialities. / A Doença de Chagas é uma das dezessete doenças tropicais negligenciadas de acordo com a Organização Mundial da Saúde. Nas últimas décadas foram descritas novas vias metabólicas deste parasita, o que abre perspectivas para o desenvolvimento de medicamentos mais específicos e menos tóxicos, para vias cruciais como alvo. Uma vez identificado o alvo terapêutico, passa a ser necessária a caracterização estrutural e bioquímica das enzimas envolvidas. Especula-se como alvo terapêutico para combater a Doença de Chagas a enzima Histidina Amônio Liase, que participa da via catabólica da histidina. Sendo assim, visando contribuir com o entendimento estrutural e bioquímico desta, foi realizada a sua produção heteróloga em E. coli. Esta foi purificada por cromatografia de afinidade e utilizada em diversas técnicas para caracterização inicial. A atividade foi determinada em ensaio cinético, a estabilidade térmica e as estruturas secundárias foram investigadas por Dicroísmo Circular (CD) e o estado de oligomerização em solução foi analisado por Espalhamento Dinâmico de Luz (DLS). A técnica de difração de raios X foi empregada para elucidação da estrutura tridimensional. A TcHAL foi expressa e purificada de maneira satisfatória. A atividade revelou um adequado enovelamento protéico e o Dicroísmo Circular indicou predominância de estruturas secundárias hélices-α e início da desnaturação térmica próximo a 68 °C. A TcHAL foi cristalizada e forneceu padrões de difração suficientes para elucidação da estrutura 3D. Os estudos bioquímicos e estruturais avançaram o entendimento desta enzima e das possibilidades de sua inibição.