Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 3 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 82-92; 128-134). Online version available via ProQuest Digital Dissertations.

Made available in DSpace on 2014-06-11T19:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:30:21Z : No. of bitstreams: 1 menezes_mf_me_arafo.pdf: 831526 bytes, checksum: 44cecb40bcf8a2490778eb7d99c815ee (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudos recentes demonstram que os receptores purinérgicos estão presentes no núcleo parabraquial lateral (NPBL), uma estrutura pontina envolvida no controle da ingestão de sódio. No presente estudo, investigamos os efeitos das injeções do , -methyleneadenosine 5 -triphosphate ( , -metileno ATP, agonista dos receptores P2X) sozinho ou combinado com o ácido piridoxalfosfato-6-azofenil-2',4'-disulfônico (PPADS, antagonista dos receptores P2X) ou suramin (antagonista não seletivo dos receptores P2) no NPBL sobre a ingestão de NaCl 1,8% induzida por depleção de sódio. Também investigamos os efeitos da injeção de , -metileno ATP sozinho ou combinado com o PPADS no NPBL sobre a pressão arterial média (PAM) e freqüência cardíaca (FC) em ratos saciados e depletados de sódio. Foram utilizados ratos Holtzman com implante de cânulas implantadas bilateralmente em direção ao NPBL. A depleção de sódio foi induzida pelo tratamento com o diurético furosemida (20 mg/kg do peso corporal) acompanhado de uma dieta deficiente em sódio por 24 horas. As injeções bilaterais de , -metileno ATP (2,0 e 4,0 nmol/0,2 μL) no NPBL aumentaram a ingestão de NaCl 1,8% induzida por depleção de sódio (25,3 ± 0,8 e 26,5 ± 0,9 mL/2 h, respectivamente, vs. salina: 15,2 ± 1,3 mL/2 h). O pré-tratamento com o suramin (2,0 nmol/0,2 μL) ou com o PPADS (4,0 nmol/0,2 μL) no NPBL aboliu os efeitos do , -metileno-ATP na ingestão de NaCl 1,8% (15,2 ± 1,2 e 16,9 ± 0,9 mL/2 h, respectivamente). As injeções de PPADS sozinho no NPBL não alteraram a ingestão de NaCl 1,8% (14,6 ± 0,8 mL/2 h vs. salina: 18,3 ± 1,8 mL/2 h). No entanto, as injeções de suramin sozinho no NPBL quase aboliram a ingestão de NaCl 1,8% (5,7 ± 1,9 mL/120 min, vs. salina: 15,5 ± 1,1 mL/120 min) e aumentaram a ingestão de sacarose 2% somente no tempo de 90 minutos (7,1 ± 1,3 vs. salina: 5,3 ± 0,8 mL/90 min) sem alterar... / Recent studies have shown that purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of , -methyleneadenosine 5 -triphosphate ( , - methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced by 1.8% NaCl intake. We also investigated the effects of , -methylene ATP alone or combined with PPADS injected into the LPBN on mean arterial pressure (MAP) and heart rate (HR) on replete and sodium depleted rats. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20 mg/kg of body weight) followed by 24 h of sodium-deficient diet. Bilateral injections of , -methylene ATP (2.0 and 4.0 nmol/0.2 μL) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3 ± 0.8 and 26.5 ± 0.9 mL/2 h, respectively, vs. saline: 15.2 ± 1.3 mL/2 h). Pre-treatment with suramin (2.0 nmol/0.2 μL) or PPADS (4 nmol/0.2 μL) into the LPBN abolished the effects of , - methylene-ATP on 1.8% NaCl intake (15.2 ± 1.2 and 16.9 ± 0.9 mL/2 h, respectively). Injections of PPADS alone into the LPBN did not change 1.8% NaCl intake (14.6 ± 0.8 ml/2 h vs. saline: 18.3 ± 1.8 mL/2 h). However, injections of suramin alone into the LPBN strongly reduced 1.8% NaCl intake (5.7 ± 1.9 mL/120 min, vs. saline: 15.5 ± 1.1 mL/2 h) and increased the 2% sucrose intake only at 90 min (7.1 ± 1.3 vs. saline: 5.3 ± 0.8 mL/90 min), without changing 24h water deprivation-induced water intake (16.7 ± 1.8 mL/2 h vs. saline: 15.0 ± 2.1 mL/2 h)... (Complete abstract click electronic access below)

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

Human erythrocyte glucose sugar transport displays a complexity that is not explained by available models. Sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions (intracellular [sugar] = extracellular [sugar]). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Two models for biphasic sugar transport are presented in which 3MG must overcome a sugar-specific, physical (diffusional) or chemical (anomerization) barrier to equilibrate with cell water. The anomerization model was rejected through several lines of direct experimental investigation. 1) The sizes of the fast and slow phases of sugar transport do not correlate with the equilibrium anomer distributions of all GLUT1 sugar substrates. 2) Increasing the rate of anomerization by addition of exogenous intracellular mutarotase has no effect on biphasic transport kinetics. 3) Direct measurement of initial rates of sugar uptake or exchange demonstrates that GLUT1 shows no anomer preference. The physical barrier model was further refined by the use of the counterflow condition (intracellular [sugar] >> extracellular [sugar]). The presence of a physical barrier alone was unable to explain the complex counterflow time courses observed. As a result, the model was modified to include the action of a specific sugar export that is compartmentalized from rapidly equilibrating, GLUT1-mediated uptake and exit.

Methylmercury (MeHg) is a potent neuro and nephrotoxicant in several animal species including humans, particularly during their development. The purpose of this study was to investigate the effects of aqueous seed extract of Syzygium cumini (L.) Skeels (Scc) on the acute MeHg treatment in neonatal rats. Neonatal rats (P2) received orally a single dose of MeHg (10 mg/kg) and also two doses of Scc. After two days, the effects of this treatment were investigated in the cerebral cortex, hippocampus, kidney, liver and urine samples of rats. We observed that N-Acetyl-β-d-glucosaminidase (NAG) activity in the kidney and urine was higher in MeHg-group when compared with the control group. Similarly, the lipid peroxidation levels were higher in the liver and kidney as well as the Adenosine deaminase (ADA) activity increased in the hippocampus, kidney and liver. These results indicate that increased NAG and ADA activities, as well as thiobarbituric acid reactive species (TBARS) levels may play a critical role in MeHg nephrotoxicity. The most relevant finding in our investigation was that acute MeHg treatment in neonatal rats caused liver and renal impairment and Scc was able to prevent such effects. It appears that mechanisms related to scavenging activity of Scc could be involved with its protection effect. Key words: Methylmercury; Syzygium cumini; N-Acetyl-β-d-glucosaminidase; rat; adenosine deaminase / O metilmercúrio (MeHg) é um agente tóxico potente tanto para o sistema nervoso central como para o renal. Provoca danos nos seres humanos e em ratos, particularmente durante o estágio de desenvolvimento. Neste estudo, ratos em desenvolvimento (P2) receberam uma dose única de MeHg (10 mg/kg), por via oral e/ou duas doses do extrato aquoso de sementes de Syzygium cumini (L.) Skeels (Scc). Após dois dias (P4), foram investigados os efeitos deste tratamento no córtex cerebral, hipocampo, rim, fígado e urina. Observamos que a atividade da N-acetil-β-d-glicosaminidase (NAG) nos rins e na urina foi maior no grupo que recebeu MeHg, quando comparado com o grupo controle. Da mesma forma, os níveis de peroxidação lipídica foram maiores no fígado e rim e a atividade da adenosina deaminase (ADA) encontrou-se elevada no hipocampo, rins e fígado, nos ratos tratados com MeHg. Estes resultados indicam que o aumento da atividade da NAG e da ADA, bem como os níveis das espécies reativas ao ácido tiobarbitúrico (TBARS), desempenham um papel importante como marcadores de nefrotoxicidade causada pelo MeHg. Assim, o achado mais relevante na nossa investigação foi a de que o tratamento agudo com MeHg em ratos neonatos pode provocar nefrotoxicidade e a administração do Scc pode reverter estes efeitos provavelmente devido as propriedades antioxidantes de Scc.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Metabolic syndrome (MetS) is a complex disorder represented by a set of cardiovascular risk factors, including endothelial dysfunction, oxidative stress and inflammation. Syzygium cumini has hypoglycemic, anti-inflammatory, antipyretic, hypolipidemic and antioxidant properties, besides antiviral and anticarcinogenic action. Considering the importance of MetS in the current economic and social context, morbidity and complications following this pathology, the aim of this study was to assess biochemical and inflammatory parameters in patients with MetS. Moreover, to check the effect of aqueous extract of Syzygium cumini (ASc), as well as its mechanism of action on the activity of the enzyme adenosine deaminase (ADA) and other biochemical parameters under hyperglycemic and oxidative conditions in vitro. To develop the first step of this work, were obtained samples of serum and whole blood of patients diagnosed with MetS (n=40) before initiating a physical activity program, in which we analyzed biochemical, oxidative and inflammatory parameters. The results showed an increase in ADA, acetylcholinesterase (AChE) and dipeptidyl peptidase IV (DPP-IV) activities in lymphocytes of patients with MetS. Further, in these patients, we observed an increase in the activity of butyrylcholinesterase (BuChE) and ˠ- glutamyltransferase (ˠ-GT) and in C-reactive protein (hsCRP) and nitric oxide (NOx) levels, as well as disturbances in antioxidant defenses and some correlations between the parameters analyzed were obtained. Thus, these results demonstrated that MetS affects the purinergic and cholinergic systems reflecting the inflammatory and immune status of these patients, besides altering the antioxidant defenses of the same. For in vitro determinations, first, erythrocytes (RBCs) from healthy individuals were used. ASc was able to prevent the increase in ADA activity caused by exposure of RBCs to hyperglycemic conditions for 2 hours. Also, ASc acted on the activity of ADA similarly caffeine and insulin, on the other hand, dipyridamole attenuated the effect of ASc by antagonizing its the effect or by competition with the extract. Thus, it can be suggested that the ASc act by influencing the metabolism of adenosine, and its effect is related to the presence of phenolic compounds and the antioxidant properties attributed to this plant. In the next step of this work , we observed an increase in the ADA activity and lipid peroxidation and decreased cell viability after exposure of lymphocytes from healthy subjects to 2,2 ' -azobis -2- amidinopropane dihydrochloride ( AAPH ) by 2 hours in vitro. ASc and gallic acid were able to reduce the ADA activity, but did not alter the lipid peroxidation caused by AAPH . The ASc increased cell viability and reduced the activity of the enzyme lactate dehydrogenase ( LDH ). ASc, by reducing the activity of ADA, may be increasing adenosine levels and helping to maintain the beneficial effects caused by the same, such as antioxidant, anti-inflammatory and antithrombotic actions. Furthermore, the results demonstrate the cytoprotective effect evidenced by the extract. We conclude that the changes found in patients with MetS are related to inflammatory and oxidative processes and may favor the prevention and control of this clinical situation Also, the protective effects demonstrated by ASc contribute to the understanding of the wide use of this plant and its therapeutic value in the treatment of various clinical conditions. / A Síndrome Metabólica (SMet) é um transtorno complexo representado por um conjunto de fatores de risco cardiovascular, entre eles a disfunção endotelial, o estresse oxidativo e a inflamação. O Syzygium cumini, conhecido popularmente como jambolão, é uma planta que apresenta propriedades hipoglicêmicas, antiinflamatórias, antipiréticas, hipolipidêmicas e antioxidantes, além de ação antiviral e anticarcinogência. Considerando a importância da SMet no contexto social e econômico atual, a morbidade e as complicações consequentes desta situação clínica, o objetivo deste estudo foi avaliar parâmetros bioquímicos e inflamatórios em pacientes com SMet. Além disso, verificar o efeito do extrato aquoso de Syzygium cumini (ASc), bem como o seu mecanismo de ação sobre a atividade da enzima Adenosina desaminase (ADA) e outros parâmetros bioquímicos sob condições hiperglicêmicas e oxidativas, in vitro. Para o desenvolvimento da primeira etapa deste trabalho, foram utilizadas amostras de soro e sangue total de pacientes com diagnóstico de SMet (40 pessoas) antes de iniciarem um programa de atividades físicas, nos quais foram analisados parâmetros bioquímicos, inflamatórios e oxidativos. Os resultados demonstraram um aumento na atividade das enzimas ADA, acetilcolinesterase (AChE) e dipeptidil peptidase IV (DPP-IV) em linfócitos de pacientes com SMet. Ainda, nesses pacientes, observou-se um aumento na atividade das enzimas Butirilcolinesterase (BuChE) e gama-glutamiltransferase (GGT), e nos níveis de proteína C reativa (PCR) e óxido nítrico (NOx), bem como alterações nas defesas antioxidantes e algumas correlações entre os parâmetros analisados foram obtidos. Assim, esses resultados demonstraram que a SMet afeta a atividade da ADA e o sistema colinérgico refletindo o estado imune e inflamatório desses pacientes, além de alterar as defesas antioxidantes dos mesmos. Para as análises in vitro, primeiramente, foram utilizados eritrócitos (RBCs) de indivíduos saudáveis. O ASc foi capaz de prevenir o aumento na atividade da ADA causado pela exposição dos RBCs a condições hiperglicêmicas por 2 horas. Ainda, o ASc atuou sobre a atividade da ADA de maneira semelhante a cafeína e a insulina; por outro lado, o dipiridamol atenuou o efeito do ASc por antagonizar o efeito do mesmo ou por competição com o extrato. Assim,pode-se sugerir que o ASc influencia no metabolismo da adenosina, além de seu efeito estar relacionado a presença de compostos fenólicos e às propriedades antioxidantes atribuídas a essa planta. Na etapa seguinte da realização deste trabalho, observou-se um aumento na atividade da ADA e na lipoperoxidação, e redução da viabilidade celular após a exposição de linfócitos de indivíduos saudáveis ao 2,2 -azobis (aminodipropano) dihidrocloreto (AAPH) por 2 horas, in vitro. O ASc e ácido gálico foram capazes de reduzir a atividade da ADA, mas não alteraram a lipoperoxidação causada pelo AAPH. O ASc aumentou a viabilidade celular e reduziu a atividade da enzima Lactato desidrogenase (LDH). O ASc ao reduzir a atividade da ADA, pode estar aumentando os níveis de adenosina e colaborando para a manutenção dos efeitos benéficos provocados pela mesma, como ações antioxidantes, antiinflamatórias e antitrombóticas. Além disso, os resultados demonstraram o efeito citoprotetor evidenciado pelo extrato. Podemos concluir que as alterações encontradas nos pacientes com SMet estão relacionadas aos processos inflamatórios e oxidativos e podem favorecer medidas de prevenção e controle desta situação clínica. Também, os efeitos protetores demonstrados pelo ASc contribuem para o entendimento da ampla utilização desta planta e de seu valor terapêutico no tratamento de diversas patologias clínicas.

The inherently repressive nature of chromatin presents a sizeable barrier for all nuclear processes in which access to DNA is required. Therefore, eukaryotic organisms ranging from yeast to humans rely on a battery of enzymes that disrupt the chromatin structure as a means of regulating DNA transactions. These enzymes can be divided into two broad classes: those that covalently modify histone proteins, and those that actively disrupt nucleosomal structure using the free energy derived from ATP hydrolysis. The latter group, huge, multisubunit ATP-dependent chromatin remodeling factors, are emerging as a common theme in all nuclear processes in which access to DNA is essential. Although transcription is the process for which a requirement for chromatin remodeling is best documented, it is now becoming clear that other processes like replication, recombination and DNA repair rely on it as well. A growing number of ATP-dependent remodeling machines has been uncovered in the last 10 years. Although they differ in their subunit composition, organism or tissue restriction, substrate specificity, and regulating/recruiting partners, it has become increasingly evident that all ATP-dependent chromatin remodeling factors share a similar underlying mechanism. This mechanism is the subject of the studies presented in this thesis. Chromatin-remodeling factors seem to bind both the histone and DNA components of nucleosomes. From a fixed position on nucleosomes, the remodeling factors appear to translocate on the DNA, generating torsional stress on the double helix. This activity has several consequences, including the distortion of the DNA structure on the surface of the histone octamer, the disruption of histone-DNA interactions, and the mobilization of the nucleosome core with respect to the DNA. The work presented in this thesis, along with data reported by other groups, supports the hypothesis that yeast SWI/SNF chromatin remodeling complex and the recombinational repair factor, Rad54p, both employ similar mechanisms to regulate gene transcription, and facilitate homologous DNA pairing and recombination, respectively.