Mais de 25% das mortes em humanos são causadas por doenças infecciosas. Muitas dessas doenças são emergentes e de importância zoonótica. A leptospirose é considerada uma das mais importantes doenças zoonóticas emergentes. Sua distribuição global e seu potencial epidêmico constituem um problema de Saúde Pública. Estima-se que ocorram anualmente 1.03 milhão de casos e 58.900 mortes por leptospirose em todo o mundo, mas, em se tratando de uma doença negligenciada, a real prevalência da doença é subestimada. O Rattus norvegicus é o principal reservatório associado a epidemias urbanas. Leptospiras possuem a capacidade de aderir às células dos túbulos renais e interagem com muitos componentes da matriz extracelular do hospedeiro, o que facilita a invasão e colonização. Possuem também mecanismos de evasão ao sistema complemento do hospedeiro. A identificação destes mecanismos tem sido alvo de pesquisas desenvolvidas por vários grupos. Enolase e Gliceraldeído-3-fosfato desidrogenase (GAPDH) pertencem à categoria de proteínas conhecidas como proteínas moonlighting. Estas englobam um grupo de proteínas multifuncionais. Enolases são metaloenzimas citossólicas que catalisam a conversão de 2-D-fosfo-glicerato em fosfoenolpiruvato. Apesar de não possuírem sequência clássica de ancoragem à membrana, são encontradas na superfície de uma variedade de células eucarióticas e procarióticas, tendo a capacidade de interagir com plasminogênio. GAPDH é uma enzima da via glicolítica responsável pela conversão de gliceraldeído 3-fosfato em D glicerato 1,3-bifosfato. Estudos recentes mostram que a GAPDH tem múltiplas funções independentes do seu papel no metabolismo de energia. Neste trabalho demonstrou-se que GAPDH de Leptospira está localizada na superfície da bactéria, e que tanto GAPDH como enolase interagem com plasminogênio, o qual é convertido em sua forma ativa, a plasmina, na presença do ativador exógeno uPA. A capacidade da plasmina gerada sobre a enolase de clivar substratos fisiológicos foi testada. A cadeia β do fibrinogênio foi totalmente degradada, e a molécula vitronectina parcialmente clivada em fragmentos de 61- 64 kDa. Ainda, mostrou-se que a enolase interage com os reguladores do complemento C4BP e FH. Ambos os reguladores permanecem funcionais, agindo como co-fatores de Fator I na degradação de C3b (FH) e C4b (C4BP). No que diz respeito à GAPDH, os dados claramente mostram que a plasmina ligada à GAPDH degrada as cadeias α e β do fibrinogênio, assim como a isoforma de 75 kDa da vitronectina, de forma tempodependente. Ainda, na presença de GAPDH, a plasmina degradou totalmente a cadeia α de C5, mas não degradou C3b. Por fim, resultados obtidos por Far Western Blot revelam que GAPDH interage com C1q, molécula-chave da via clássica do sistema complemento, e também com fibronectina plasmática, podendo, portanto, contribuir para a adesão da bactéria durante a colonização do hospedeiro. Em suma, no presente estudo caracterizamos duas novas proteínas moonlighting de Leptospira: enolase e GAPDH. A caracterização funcional destas proteínas, assim como a identificação das moléculas-alvo do hospedeiro com as quais essas proteínas são capazes de interagir, podem contribuir para a compreensão dos mecanismos de invasão, disseminação e evasão imune utilizados por leptospiras patogênicas. / More than 25% of human deaths are caused by infectious diseases, among which a large number are emerging and of zoonotic importance. Leptospirosis is considered one of the most important emerging zoonotic diseases. Its global distribution and its epidemic potential constitute a Public Health problem. It is estimated that approximately 1,03 million cases and 58,900 deaths from leptospirosis occur annually worldwide, but as a neglected disease, its actual prevalence is underestimated. Rattus norvegicus is the main reservoir associated with urban epidemics. Leptospires have the capacity to adhere to renal tubule cells which facilitates invasion and colonization. They also have mechanisms to evade the host\'s complement system. The identification of these mechanisms has been the object of research developed by several groups. Enolase and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) belong to the category of proteins known as moonlighting proteins. These encompass a group of multifunctional proteins. Enolases are cytosolic metalloenzymes that catalyze the conversion of 2-D-phosphoglycerate to phosphoenolpyruvate. Although they do not have a classic membrane anchor sequence, they are found on the surface of a variety of eukaryotic and prokaryotic cells, and have the capacity to interact with plasminogen. GAPDH is an enzyme of the glycolytic pathway responsible for the conversion of glyceraldehyde 3-phosphate to D-glyceryl 1,3-bisphosphate. Recent studies show that GAPDH has multiple functions independent of its role in energy metabolism. In this work we demonstrated that Leptospira GAPDH is located on the surface of the bacterium, and that both GAPDH and enolase interact with plasminogen, which is converted into its active form, plasmin, in the presence of the exogenous activator uPA. The capacity of plasmin-bound enolase to cleave physiological substrates was tested. The β-chain of fibrinogen was totally degraded, and vitronectin was partially cleaved into fragments of 61-64 kDa. Further, enolase interacts with the complement regulators C4BP and FH. Both regulators remain functional, acting as cofactors for Factor I on the degradation of C3b (FH) and C4b (C4BP). With regard to GAPDH, the date clearly show that plasmin bound to GAPDH degrades the α and β chains of fibrinogen as well as the 75-kDa isoform of vitronectin, in a time-dependent manner. Furthermore, in the presence of GAPDH, plasmin totally degraded C5 α-chain, but did not degrade C3b. Finally, our Far Western Blot data reveal that GAPDH interacts with C1q, a key molecule of the classical pathway of the complement system, and also interacts with plasma fibronectin, and may therefore contribute to bacterial adhesion during host colonization. Briefly, in the present study we characterized two novel moonlighting proteins of Leptospira: enolase and GAPDH. The functional characterization of these proteins, as well as the identification of the host target molecules with which these proteins are capable of interacting, may contribute to the understanding of the mechanisms of invasion, dissemination and immune evasion used by pathogenic leptospires.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by neurofibrillary tangles, senile plaques and loss of synapses. Many studies support the notion that oxidative stress plays an important role in AD pathogenesis. Previous studies from our laboratory employed redox proteomics to identify oxidatively modified proteins in the AD inferior parietal lobule (IPL). The proteins were consistent with AD pathology and have been central to further investigations of the disease. The present study was focused on the identification of specific targets of protein S-glutathionylation in AD, early AD (EAD), and mild cognative impairment (MCI) using a redox proteomics approach. In AD IPL we identified deoxyhemoglobin, α-crystallin B, glyceraldehyde phosphate dehydrogenase (GAPDH), and α-enolase as significantly S-glutathionylated relative to these brain proteins in control IPL. Both, GAPDH and α-enolase were also shown to have reduced activity in the AD IPL. With further investigation gammaenolase, dimethylarginine dimethylaminohyrdolase (DDAH), Cathepsin D, and 14-3-3 gamma were identified as significantly S-glutathionylated in the EAD IPL. Alpha enolase was also identified as significantly S-glutathionylated in MCI IPL. These results provide a correlation in proteins S-glutathionylated in the progression of AD even in the reversible conditions of amnestic MCI. Amyloid beta-peptide (1-42) [Aβ(1-42)], one of the main component of senile plaque, can induce in vitro and in vivo oxidative damage to neuronal cells through its ability to produce free-radicals. The aim of this study was to investigate the protective effect of the xanthate, D609, on Aβ(1-42)-induced protein oxidation using a redox proteomics approach. D609 was recently found to be a free radical scavenger and antioxidant. In the present study, rat primary neuronal cells were pretreated with 50 μM of D609 followed by incubation with 10 μM Aβ(1-42) for 24 hours. In the cells treated with Aβ(1-42) alone four proteins that were significantly oxidized were identified: Glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase, malate dehydrogenase, and 14-3-3 zeta. Pretreatment of neuronal cultures with D609 prior to Aβ (1-42) protects all the identified oxidized proteins in the present study against Abeta(1- 42)-mediated protein oxidation. Therefore, D609 may ameliorate the Aβ(1-42)-induced oxidative modification.

A cloned esterase and several cytoplasmic proteins which lack a classical cleavable signal-peptide were secreted during late stationary phase in B. subtilis. Several lines of evidence indicate that secretion of enolase, SodA, and Est55 is not due to cell lysis. The extent of possible release of these proteins mediated by membrane vesicles into the medium was also found to be minimal. We have identified a hydrophobic α–helical domain within enolase that contributes to the secretion specificity. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid-encoded mutant enolases is totally blocked, while that of the wild-type chromosomal enolase is not affected in the same cultures. However, mutations on the conserved basic residues flanking the hydrophobic core region show no effect. GFP fusion experiments demonstrate that minimal length of N-terminus 140 amino acids and its tertiary structure are required to serve as a functional signal for the export of enolase. Transcriptome analysis revealed several interesting patterns in gene expression when the cell growth switches from exponential phase into stationary phase. As expected, once cell growth enters the stationary phase, expressions of most SigA-dependent house-keeping genes (for syntheses of ATP, amino acids, nucleotides, ribosomes), and surprisingly secY and yidC homolog in the Sec-dependent general protein secretion system were significantly decreased; however, secA and sipT were found progressively induced in the stationary phase. The sigB gene and the SigB regulon exhibited a distinct pattern of transient induction with a peak in transition phase. A total of 62 genes were induced by three fold after cessation of SigB-dependent surge, which includes sigW and many of SigW-depedent genes specifically for antitoxin resistant genes, and some unknown function genes. In addition, oxidative stress response and damage repair genes also dominantly induced in stationary phase implied a high level of oxidant or thio-depleting agents in stationary phase. Besides, induction of fruRAB at T40 and gap operon at T100 suggested a sequential switch of carbon utilization from glucose to fructose. These results indicate a complex adaptation physiology as Bacillus cells change from the fast growing exponential phase toward the stationary phase.

Not available / text

Thesis (M.S.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Includes bibliographical references (leaves 30-40). Also issued on the Internet.

Thesis (M.Sc.) -- Memorial University of Newfoundland, 1987. / Restricted until November 1988. Typescript. Bibliography: leaves 102-110. Also available online.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 30-40). Also issued on the Internet.

Zugl.: Berlin, Humboldt-Universiẗat, Diss., 2003.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007. / Phillip Gibbs, Committee Member ; Rick Trebino, Committee Member ; Christoph Fahrni, Committee Member ; Donald Doyle, Committee Member ; Andreas Bommarius, Committee Chair.

Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-05Bitstream added on 2014-06-13T20:35:40Z : No. of bitstreams: 1 marcos_cm_me_arafcf.pdf: 2508470 bytes, checksum: bc1a1f1c4040c405fe6c122e87b067ff (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Paracoccidioides brasiliensis é um importante patógeno humano que causa a paracoccidioidomicose (PCM), uma micose sistêmica com ampla distribuição na América Latina. A adesão e invasão de células são eventos cruciais envolvidos na infecção e disseminação do patógeno. Além disso, patógenos utilizam suas moléculas de superfície para se ligar aos componentes da matriz extracelular para estabelecer a infecção. Uma proteína antigênica de P. brasiliensis foi isolada de géis de eletroforese bidimensional do cell-free do fungo e caracterizada. Peptídeos foram obtidos da proteína de 54 kDa e pI 5,6 e mostraram homologia com enolase de Paracoccidioides brasiliensis e outros fungos. A proteína foi purificada através de eletroeluição e utilizada para a produção de anticorpo policlonal em coelho. Por microscopia de fluorescência não foi possível observar a localização exata desta proteína, apenas que se encontra aparentemente distribuídapor todo o fungo, foi possível verificar alterações no citoesqueleto de pneumócitos durante a infecção por P. brasiliensis. A localização foi confirmada por microscopia imunoeletrônica a presença de enolase foi detectada principalmente na parede celular de leveduras de P. brasiliensis e também no citoplasma, ela se demonstrou mais expressa quando este fungo foi cultivado em meio onde houve acréscimo de sangue de carneiro e durante a situação de infecção a pneumócitos. A enolase purificada foi capaz de se ligar a fibronectina, fibrinogênio, laminina, plasminogênio, colágenos tipo I e IV. Foi confirmado que a ligação desta proteína à pneumócitos é influenciada pela sequência de aminoácidos Arg-Gly-Asp contida provavelmente nos receptores da matriz extracelular presentes na célula do hospedeiro. Essas informações indicam que a enolase possivelmente contribui para a adesão do microrganismo aos tecidos do... / Paracoccidioides brasiliensis is an important human pathogen that causes paracoccidioidomycosis (PCM), a systemic mycosis with a wide distribution in Latin America. The ability of P. brasiliensis to cause not only human diseases but also mycoses with a variety of clinical manifestations from localized forms to the disseminated disease progressing to lethality, probably depends on the relationship between the virulence of the fungus, its ability to interact with and to invade the surface structures of the host and the immune response of the host. The adhesion of the pathogen leads to the recognition of carbohydrate and protein ligands on the surface of the host cell or proteins of the extracellular matrix (ECM). The large number of tissue types that fungi can colonize and infect suggests that they use a variety of surface molecules for adhesion.Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis