Orientador: Maria Julia Marques / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-09-11T21:17:52Z (GMT). No. of bitstreams: 1 Taniguti_AnaPaulaTiemi_D.pdf: 2092152 bytes, checksum: 12213bebdeb4e5920c800ccf1d164d9a (MD5) Previous issue date: 2011 / Resumo: O camundongo mdx é o animal mais utilizado como modelo da distrofia muscular de Duchenne (DMD), diferindo dos humanos doentes por apresentar ciclos de regeneração muscular e reduzida fibrose. Este trabalho tem como objetivos: 1. desenvolver protocolo experimental para promover fibrose muscular através de exercício de corrida em esteira e 2. verificar se a suramina inibe a fibrose induzida experimentalmente. A suramina tem efeito anti-fibrótico, sendo um potencial agente farmacológico para tratamento da DMD visando sucesso de terapias celulares. Foram utilizados camundongos mdx (n=42) e C57BL/10 (n=11) com seis meses de idade. Os camundongos mdx foram divididos em quatro grupos experimentais: grupo sedentário e tratado com salina (n=11), grupo sedentário e tratado com suramina (n=11), grupo exercitado e tratado com salina (n=10) e grupo exercitado e tratado com suramina (n=10). Os animais foram submetidos à corrida em esteira diariamente e o tratamento com suramina (60mg/kg) foi realizado em dias alternados, via intra-peritoneal. Após sete semanas, os animais foram sacrificados e o músculo tibial anterior, bíceps braquial, diafragma e coração coletados e congelados para análise histológica e protéica por western blot. O plasma sanguíneo foi submetido à análise de creatina-quinase. A força de tração dos membros anteriores foi medida no início e no final do protocolo experimental utilizando-se Grip Strength Meter e o músculo diafragma submetido ao estudo in vitro para verificar a força de contração. Verificamos que o protocolo de corrida em esteira foi adequado para induzir a fibrose e inibir a regeneração nos músculos da pata dos camundongos mdx. O aumento da área de fibrose foi acompanhado pelo aumento dos níveis de TGF-?1, aumento de creatina-quinase e diminuição da força de tração. O tratamento com suramina diminuiu a fibrose nos músculos exercitados e acelerou o processo de regeneração. Adicionalmente, observamos que a suramina reduziu o número de fibras marcadas com azul de Evans, diminuiu os níveis da CK e impediu a perda da força de tração bem como a força de contração do músculo diafragma. Concluímos que o protocolo de corrida em esteira foi eficaz na indução de fibrose nos músculos tibial anterior e bíceps braquial. O efeito anti-fibrótico da suramina torna-a droga potencialmente útil para a terapia farmacológica da DMD / Abstract: The mdx mouse is commonly used as a model to study Duchenne muscular dystrophy (DMD) however shows cycles of muscle regeneration and reduced fibrosis. The purposes of this study were (1) to induce muscle fibrosis through eccentric running exercise in mdx mice and (2) to verify the effects of suramin on muscle fibrosis. Six-month-old mdx (n=42) and control (C57BL/10, n=11) mice were used. Mdx mice were divided in four groups: sedentary and saline-treated (n=11); sedentary and suramintreated (n=11); exercised and saline-treated (n=10) and exercised and suramin-treated (n=10). The mdx mice belonging to the exercised groups were placed on treadmill to run daily, for seven weeks. Suramin was injected at a dose of 60mg/kg i.p. on alternate days. At the end of the experimental protocol, the mice were sacrificed and the tibialis anterior, biceps brachii, diaphragm and heart muscles were dissected, frozen in liquid nitrogen and used to histological and western blot analysis. Blood was obtained to determine creatine-kinase (CK) levels. The forelimb force was measured by an adapted Grip Strength Meter. Force of contraction of diaphragm in vitro was verified. Our results showed that the experimental protocol was adequate to induce muscle fibrosis in mdx mice. The occurrence of fibrosis was accompanied by elevated levels of TGF- ?1 and serum CK and decreased forelimb force. Suramin reduced muscle fibrosis, decreased the number of muscle fibers stained with Evans blue, reduced serum CK levels and prevented the loss of muscle force in exercised mdx and diaphragm strips in vitro. We concluded that the downhill running protocol was effective for inducing fibrosis in tibialis anterior and biceps brachii muscles of mdx mice. Suramin seems to be a potential useful therapeutic alternative for DMD treatment / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Homologous recombination (HR) is conserved across all three domains of life and is associated with a number of key biological processes. Over the years, numerous genetic, biochemical and structural studies have uncovered important mechanistic details and established a role for HR in DNA damage repair, control of DNA replication fidelity and suppression of various types of cancer. Much of our current understanding of the mechanistic aspects of HR is gained from the study of Escherichia coli paradigm. E. coli RecA is the founding member of a nearly ubiquitous family of multifunctional proteins and is substantially conserved among eubacterial species. During HR, RecA protein promotes homologous pairing followed by strand exchange reaction leading to heteroduplex formation. In addition to HR, RecA is a central component of SOS response, recombinational DNA repair and rescue of collapsed replications forks. Moreover, recent work has suggested that DNA recombination/repair mechanisms might contribute to genome evolution and consequently to the generation of multidrug-resistant strains of the pathogen. The disease caused by Mycobacterium tuberculosis, endemic in certain regions of the world, is a leading cause of disability and death. A thorough knowledge of the function and interaction of specific HR proteins/enzymes involved in the maintenance of genome integrity is essential in order to elucidate the impact of genome perturbation effects on M. tuberculosis. Toward this end, modulation of RecA protein activity, a central component of HR, represents a potential novel target for design of new drugs because of its involvement in various processes of DNA metabolism. Additionally, small molecule modulators of RecA activity may offer novel insights into the regulation and its role in cellular physiology and pathology. Traditionally, antibiotics have been used to treat infections caused by bacteria. Despite their importance, the development of new antibiotics against M. tuberculosis has considerably decreased over the past several years due to disappointing results in clinical trials. These failures may be due the fact that they suffer from low potency or low cell permeability. Therefore, one of the aims of studies described in this thesis was to test the effect of suramin, a known inhibitor of E. coli RecA, on various biochemical activities of mycobacterial RecA proteins and determine its mechanism of action. Furthermore, the most crucial step in the HR pathway and rescue of collapsed DNA replication forks is the resolution of Holliday junctions and other branched intermediates. Because Holliday junction resolvases are essential for the resolution of different types of DNA recombination/repair intermediates, therefore, we considered it worthwhile to study the genomic expression and biochemical properties of HJRs in M. tuberculosis. Suramin is a commonly used antitrypanosomal and antifiliarial drug, and a novel experimental agent for the treatment of several cancers. A forward chemical screen assay identified several small molecule inhibitors of E. coli RecA. In this screen, suramin (also called germanin), a polysulfonated naphthylurea, and suramin-like agents were found to inhibit EcRecA catalyzed ATPase and DNA strand exchange activity. However, the mechanism underlying such inhibitory action of suramin and whether it can exert antibacterial activity under in vivo conditions remains largely unknown. In an attempt to delineate the range of suramin action, we reasoned that it might be useful to test its effect on mycobacterium RecA proteins. We found that suramin is a potent inhibitor of all known biochemical activities of mycobacterial RecA proteins with IC50 values in the low μM range. The mechanism of action involves, in part, its ability to disassemble the nucleoprotein filaments of RecA-ssDNA. To validate the above results and to obtain quantitative data, a pull-down assay was developed to assess the effect of suramin on RecA–ssDNA filaments. The data indicated that suramin was able to dissociate >80% of RecA bound to ssDNA. Altogether, these results indicated the effectiveness of suramin in the disassembly of RecA nucleoprotein filament. Next, we sought to test whether suramin binds to RecA by using a CD spectropolarimeter. Significant spectral changes were observed upon addition of increasing concentrations of suramin, indicating alterations in the secondary structure of RecA protein. Additional evidence revealed that suramin impaired RecA catalyzed proteolytic cleavage of LexA repressor and blocked ciprofloxacin-inducible recA gene expression and SOS response. More importantly, suramin potentiated the cidal action of ciprofloxacin and reduced the growth of Mycobacterium smegmatis recA+ strain but not its isogenic recA∆ mutant, consistent with the idea that it acts directly on RecA protein. This approach, which appears as an appealing concept, opens up new possibilities to chemically disrupt the pathways controlled by RecA and treat drug-sensitive as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies. The annotated genome sequence of M. tuberculosis revealed the presence of putative homologues of E. coli DNA recombination/repair genes. However, it is unknown whether these putative genes have the ability to encode catalytically active proteins or participate in biochemical reactions intrinsic to the process of HR or DNA repair. Studies in the second half of the thesis originated from an in silico analysis for genes that encode functional equivalents of E. coli RuvC HJ resolvase(s) in M. tuberculosis. The central intermediate formed during mitotic and meiotic recombination is a four-way DNA junction, also known as the Holliday junction (HJ), and its efficient resolution is essential for proper segregation of chromosomes. The resolution of HJ is mediated by a group of structure specific endonucleases known as the Holliday junction resolvases (HJR) which have been identified in a wide variety of organisms based on their shared biochemical characteristics. Bioinformatics analyses of the evolutionary relationships among HJ resolvases suggests that HJR function has arisen independently from four distinct structural folds, namely RNase H, endonuclease VII-colicin E, endonuclease and RusA. Furthermore, similar analyses of HJRs identified another family within the RNaseH fold, along with previously characterized RuvC family of junction resolvases. This new family of putative HJRs is typified by E. coli Yqgf protein. The yqgf gene is highly conserved among bacterial genomes. Nuclear magnetic resonance structural studies have disclosed notable structural similarities between E. coli RuvC and YqgF proteins. Utilizing homology-based molecular modelling, YqgF is predicted to function as a nuclease in various aspects of nucleic acid metabolism. Sequence analysis of M. tuberculosis genome has revealed the presence of two putative HJ resolvases, ruvC (Rv2594c) and ruvX (Rv2554c, yqgF homolog). Previous studies have demonstrated that M. tuberculosis ruvC is induced following DNA damage and ruvX is expressed during active growth phase of M. tuberculosis. More importantly, the absence of ruvC increased the potency of moxifloxacin in M. smegmatis. Although, these results imply that the ruv genes play crucial roles in DNA recombination and repair in M. tuberculosis, the biochemical properties of their gene products have not been characterized. In this study, we have isolated M. tuberculosis ruvC and yqgF genes and purified their encoded proteins, M. tuberculosis RuvC (MtRuvC) and M. tuberculosis RuvX (MtRuvX), respectively, to near homogeneity. Protein-DNA interaction assays conducted with purified MtRuvC and MtRuvX revealed that both can bind HJ, albeit with different affinities. However, in contrast to MtRuvC, MtRuvX showed robust HJ resolvase activity. The endonuclease activity of MtRuvX was completely dependent on Mg2+and Mn2+ partially substituted for Mg2+. Additional experiments showed that RuvX exhibits >2-fold higher binding affinity for HJ over other recombination/ replication intermediates. As demonstrated for other HJRs, MtRuvX failed to cleave static HJ and linear duplex DNA. The cleavage sites were mapped within the homologous core of a branch-migratable HJ. To identify catalytic residues in RuvX, we conducted mutational analysis of an acidic amino acid residue guided by the bioinformatics data. The product of MtRuvXD28N retained full HJ-binding activity, but showed extremely reduced HJ-specific endonuclease activity. Further biochemical characterization revealed that MtRuvX exists as a homodimer in solution. Notably, we found that disulfide-bond mediated intermolecular homodimerization is crucial for the ability of MtRuvX to cleave Holliday junctions, implicating that stable junction binding is necessary to promote branch migration and to create cleavable sites. Analysis of qPCR data suggested that the pattern of yqgF gene expression was similar to those of ruvC and recA genes following DNA damage. Together, these data indicate that ruvX expression is induced by DNA-damaging agents and that RuvX might be functionally involved in recombinational DNA repair in M. tuberculosis. These findings are all consistent with the idea that RuvX might be the bona fide HJ resolvase in M. tuberculosis analogous to that of E. coli RuvC. More importantly, we provide the first detailed characterization of RuvX and present important insights into the mechanism of HJ resolution, which could be directly linked to the regulation of different DNA metabolic processes, including HR, DNA replication and DNA repair. Overall, this study opens a new avenue in the understanding of HR in this human pathogen, together with elucidation of the function of some of the uncharacterized genes may represent a novel set of recombination enzymes.

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T07:19:08Z (GMT). No. of bitstreams: 1 Moreira_DrielendeOliveira_M.pdf: 2075338 bytes, checksum: 00bb5917e4f176b73c58453340cb3a5e (MD5) Previous issue date: 2012 / Resumo: A Distrofia muscular de Duchenne (DMD) é uma doença caracterizada pela fraqueza muscular progressiva que leva à insuficiência respiratória e cardíaca, resultando em morte por volta dos 30 anos de idade. No camundongo mdx, modelo experimental da DMD, os músculos diafragma e cardíaco são severamente afetados apresentando fibrose semelhante à observada na patologia humana. O objetivo deste trabalho foi investigar os efeitos do tratamento a longo prazo com suramina, uma droga anti-fibrótica, nos músculos diafragma e cardíaco de camundongos mdx idosos. Camundongos mdx (n=20; 8 meses de idade) receberam injeções intraperitoneais de suramina (60 mg/kg), durante 3 meses. Controles mdx (n=20; 8 meses) e C57BL/10 (n=18; 8 meses) foram injetados com solução salina. Os camundongos da linhagem C57BL/10 expressam distrofina e são utilizados como controle da linhagem mdx. A suramina diminuiu os níveis de CK e atenuou a perda da força muscular. No músculo diafragma, a suramina reduziu a área de fibrose e a mionecrose. No músculo cardíaco, houve redução da fibrose, da inflamação e melhora significativa de parâmetros funcionais cardíacos (amplitude das ondas P, Q, R e S do eletrocardiograma). Sugere-se que a suramina possa ser potencialmente útil nas distrofinopatias, atenuando a miopatia nos músculos mais afetados, o coração e o diafragma, nos estágios tardios da doença / Abstract: Duchenne muscular dystrophy (DMD) is a disease characterized by progressive muscle weakness leading to respiratory and cardiac failure, resulting in death around 30 years of age. In the mdx mice model of DMD, diaphragm and cardiac muscles are severely affected in the later stages of the disease, showing intense fibrosis similar to that observed in human pathology. The aim of the present study was to investigate the effects of long-term treatment with suramin, an anti-fibrotic agent, in the diaphragm and cardiac muscles of the mdx mice. Mdx mice (n=20; 8 months of age) received intraperitoneal injections of suramin (60 mg/kg) for 3 months. Mdx controls (n=20; 8 months) and C57BL/10 (n=18; 8 months old) were injected with saline. C57BL/10 mice express dystrophin and are the control strain for the mdx mice. Suramin decreased CK levels and reduced the loss of muscle strength. Suramin reduced fibrosis and myonecrosis in diaphragm. In the cardiac muscle, suramin decreased fibrosis, inflammation and improved cardiac functional parameters (P, Q, R and S waves of the electrocardiogram). It is suggested that suramin may be a potential therapy for distrophinopaties, attenuating the dystrophic phenotype of the most affected cardiac and diaphragm muscles of the mdx mice, during later stages of the disease / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

Made available in DSpace on 2016-06-02T19:22:55Z (GMT). No. of bitstreams: 1 4102.pdf: 1499777 bytes, checksum: 83d49633865ad84ab741b0091f168280 (MD5) Previous issue date: 2011-12-14 / Universidade Federal de Minas Gerais / Locus coeruleus (LC) is considered as a chemosensitive region to CO2/pH in mammals and amphibians, mainly its noradrenergic neurons. The LC purinergic neuromodulation is of particular interest since adenosine 5′-triphosphate (ATP) acts as a neuromodulator in many brainstem areas involved in cardiovascular and respiratory regulation, which includes Locus coeruleus (LC). ATP acting on LC P2 receptors influences the release of noradrenaline (NE) and the LC noradrenergic neurons are involved in the CO2-drive to breathing. Thus, the goal of the present study was to investigate the role of purinergic neuromodulation in the LC in the ventilatory, thermal and cardiovascular responses during normocapnia and hypercapnia in Wistar male unanesthetized rats. We assessed the purinergic modulation of cardiorespiratory and thermal responses by microinjecting ATP P2X receptor agonist (α,β-MeATP, 0.5 nmoL/40 nL and 1 nmoL/40 nL) and P2 receptor non selective antagonists (PPADS 0.5 nmoL/40 nL and 1 nmoL/40 nL; suramin, 1 nmoL/40 nL) into the LC. Pulmonary ventilation (VE, plethysmography), mean arterial pressure (MAP), heart rate (HR) and body core temperature (Tb, dataloggers) were measured before and after unilateral microinjection (40 nL) of α,β-MeATP, PPADS, suramin or 0.9% saline (vehicle) into the LC during 60 min normocapnia or 30 min period of 7% CO2 exposure followed by 30 min of normocapnia. Under normocapnic conditions, α,β-MeATP did not affect any parameter, whereas PPADS decreased respiratory frequency (f), increased MAP and HR and suramin increased Tb, MAP and HR and did not change ventilation. Hypercapnia induced an increase in ventilation, a fall in HR and did not change Tb in all groups. During hypercapnia, α,β-MeATP produced a further increase in ventilation and did not cause changes in cardiovascular and thermal parameters, PPADS caused an increase in MAP, did not alter ventilation and Tb and suramin elicited increases in ventilation, MAP and bradycardia and did not change Tb. Thus, our data suggest that purinergic neuromodulation in the LC plays an important role in the cardiorespiratory control during hypercapnia and modulates cardiorrespiratory and thermal control during normocapnic conditions in unanesthetized animals. / O LC é considerado uma região quimiossensível a CO2/pH em mamíferos e anfíbios, especificamente os neurônios noradrenérgicos. A neuromodulação purinérgica no LC desperta um interesse particular uma vez que a adenosina 5 -trifosfato (ATP) atua como neuromodulador em várias áreas do tronco encefálico envolvidas na regulação cardiorrespiratória, incluindo o LC e sua atuação em receptores P2 influencia a liberação de noradrenalina (NE) dos neurônios do LC. Portanto, o objetivo do presente estudo foi investigar a participação da neuromodulação purinérgica no LC nas respostas ventilatória, térmica e cardiovascular durante normocapnia e hipercapnia em ratos Wistar não anestesiados. A possível modulação do ATP nessas respostas foi realizada por meio da microinjeção do agonista de receptor P2X (α,β-MeATP, 0.5 nmol/40 nL e 1 nmol/40 nL) e dos antagonistas não seletivos de receptor P2 (PPADS 0.5 nmol/40 nL e 1 nmol/40 nL; suramin, 1nmol/40nL) no LC. Foram feitas medidas de ventilação pulmonar ( VE, pletismografia), temperatura corporal (TC) pressão arterial média (PAM) e frequência cardíaca (FC) antes da microinjeção unilateral de α,β--MeATP, PPADS, suramin ou salina (veículo, 40nL) no LC em condições basais, e após microinjeção durante 60 min de normocapnia ou 30 min de exposição a 7% CO2, seguido de 30 min de normocapnia. Em condições normocápnicas, a microinjeção de α,β-MeATP não afetou nenhuma das variáveis analisadas, enquanto que o PPADS promoveu uma redução da freqüência respiratória (fR), aumento da PAM e FC, e o suramin aumentou a TC, PAM e FC sem causar alterações na ventilação. A hipercapnia promoveu aumento da ventilação, uma redução na FC e não alterou a TC em todos os grupos. Durante hipercapnia, α,β-MeATP promoveu aumento da hiperpnéia sem causar alterações nas variáveis cardiovasculares e na temperatura, PPADS promoveu aumento da PAM sem alterar as variáveis respiratórias e a temperatura corporal e o suramin promoveu aumento da hiperventilação, aumento na PAM e bradicardia sem alterar a temperatura corporal. Portanto, nossos dados sugerem que a neuromodulação purinérgica no LC participa do controle cardiorrespiratório durante normocapnia e hipercapnia e modula a termorregulação em condições normocápnicas em animais não anestesiados.