Thesis (Ph.D.)--Cleveland State University, 2009. / Abstract. Title from PDF t.p. (viewed on Sept.8, 2009). Includes bibliographical references (p. 101-104). Available online via the OhioLINK ETD Center and also available in print.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2008 Aug 15.

Theses (M.S.)--Marshall University, 2006. / Title from document title page. Includes abstract. Document formatted into pages: contains xii, 76 pages. Bibliography: p. 71-76.

This thesis focuses on the nature of heme metabolism in various eukaryotes. One of the aims was the elucidation of the origin of the unique heme biosynthesis pathway in apicomplexan parasites through a comparative study of their photosynthetic relative Chromera velia combining molecular biology, biochemistry and bioinformatics approach. Using similar strategy, I have also investigated the origin and spatial organization of tetrapyrrole biosynthesis in Euglena gracilis. Based on the phylogenetic data I described the complex evolution of heme metabolism in kinetoplastid flagellates including pathogenic trypanosomes. I revealed that one of them (Phytomonas) does not require heme for viability by the combination of various biochemical and molecular biology experiments and bioinformatic analyses.

A long standing global scientific challenge has been the activation of O2 at a single metal center, and use of the subsequent metal-based oxidant for a variety of difficult chemical transformations. Towards this end, computational and synthetic methods have been utilized in an approach to develop model compounds capable of this type of chemistry, and to better understand the electronic and mechanistic properties of the observed catalytic reactivity. We have developed a first generation catalyst that has been shown to be fully functional in utilizing α-keto acids for the catalytic activation of O2 and oxidation of organic substrates in a highly conserved manner. This reactivity takes place at room temperature and standard pressure, and resembles the type of chemistry performed by mononuclear non-heme enzymes, which inspired the design of the catalyst. However, these solution-phase reactions do not benefit from the controlled environment provided by a protein active site, and solution studies and DFT simulations demonstrate an isomeric family of reactive species that ultimately deactivate via a dimerization pathway. A second generation catalyst, which incorporates ligand aromatic functionality, has been developed. This complex has been shown to catalytically oxide methanol to formaldehyde in the presence of α-ketoglutarate using O2. The aromatic group provides a synthetic platform, allowing a variety of substituents geared toward increasing complex solubility and the tuning of the redox properties of the metal center. Additionally, the ligand has been functionalized to allow for the immobilization of the catalyst using an azido-functionalized solid support, by means of 'click' chemistry. A procedure for the immobilization of the catalyst has been developed that sets the stage for the preparation of a material that will diminish dimerization and inactivation. Additional insights into potential reaction pathways of the first generation catalyst have been obtained from DFT studies. These simulations have provided energetic comparisons of proposed intermediates and set the stage for future computational and spectroscopic studies. This synergistic approach will not only allow for detailed electronic and mechanistic descriptions of the intimate mechanism, but will be used in the development of next generation catalysts that that can be tuned for desired reactivity properties.

Made available in DSpace on 2016-02-26T13:34:40Z (GMT). No. of bitstreams: 2 pedro_rodrigues_ioc_dout_2007.pdf: 495339 bytes, checksum: 0791930416bc740981dac64b0ead8959 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A heme oxigenase-1 (HO-1), uma enzima induzida sob diversas condições de estresse celular, cataboliza o heme em monóxido de carbono (CO), biliverdina (convertida posteriormente a bilirrubina) e ferro livre. A deficiência dessa enzima resulta em inflamação crônica e morte prematura. Por outro lado, o aumento da HO-1 e de seus produtos resulta em efeitos antiinflamatórios e antioxidantes. As injúrias inflamatória e oxidativa desempenham um papel importante na fisiopatologia da sepse. Nesse contexto, a HO-1 vem sendo caracterizada como um gene protetor. Recentemente, estudos demonstraram que a indução da HO-1 ou a terapia com o CO e a biliverdina, isoladamente ou em associação, são capazes de diminuir a disfunção orgânica e a mortalidade em modelos animais de endotoxemia letal. Nossa proposta foi estudar o efeito da modulação da HO-1 e do tratamento com a biliverdina em um modelo mais clinicamente relevante de sepse, a ligadura e perfuração cecal (CLP). Nossos resultados apontam para um efeito benéfico da HO-1 no tratamento da sepse. Demonstramos que o tratamento com a estanho protoporfirina (SnPP), um supressor da HO-1, aumenta a mortalidade da CLP. Já nos animais tratados com a cobalto protoporfirina (CoPP), um indutor da HO-1, há um aumento da sobrevida. O tratamento com a biliverdina também teve um impacto significativo, tanto em um modelo de endotoxemia letal como no modelo de CLP, reduzindo a mortalidade em aproximadamente 60% e 40%, respectivamente. Esse efeito protetor observado na CLP foi associado a uma modulação da resposta inflamatória, constatada pela redução do acúmulo de leucócitos e dos níveis de mediadores inflamatórios (TNF-a, IL-6, KC e IL-10) na cavidade peritoneal. Ao mesmo tempo, os animais tratados com a biliverdina apresentaram um decréscimo no número de unidades formadoras de colônias no lavado peritoneal, sugerindo um melhor controle local da infecção. / Heme oxygenase-1 (HO-1), an enzyme induced under va rious situations of cellular stress, catabolyses heme into carbon monoxide (CO), biliver din (subsequently converted to bilirubin) and free iron. The deficiency of this en zyme results in chronic inflammation and premature death. On the other hand, an increase in HO-1 and its products results in anti- inflammatory and antioxidant effects. Inflammatory and oxidative injuries play an important role in sepsis pathophysiology. In this c ontext, HO-1 has been characterized as a protective gene. Recently, studies have shown that the induction of HO-1 or therapy with CO and biliverdin, isolated or in association, is c apable of reducing organic dysfunction and mortality in animal models of lethal endotoxemia. O ur goal was to study the effects of the modulation of HO-1 and the treatment with biliverdi n in a more clinically relevant model of sepsis, the cecal ligation and puncture (CLP) model . Our results suggest a beneficial effect of HO-1 in sepsis treatment. We demonstrated that t he treatment with tin protoporphyrin (SnPP), a suppressor of HO-1, increases CLP mortali ty. On the other hand, animals treated with cobalt protoporphyrin (CoPP), an inducer of HO -1, had an increased survival. Treatment with biliverdin also had a significant im pact over both lethal endotoxemia and CLP, reducing mortality in approximately 60% and 40 %, respectively. This protective effect of biliverdin on CLP was associated with a m odulation of the inflammatory response, observed by the reduction of leukocyte accumulation and levels of inflammatory mediators (TNF, IL-6, KC and IL-10) in the peritoneal cavity. At the same time, the animals treated with biliverdin had decreased numbers of colony-for ming units in the peritoneal lavage fluid, suggesting a better local control of infecti on

MAGALHÃES, P. A. F. Acidose metabólica agrava a lesão renal aguda isquêmica em ratos. 2016. 110 f. Tese (Doutorado em Ciências Farmacêuticas) - Faculdade de Farmácia, Odontologia e Enfermagem, Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Erika Fernandes (erikaleitefernandes@gmail.com) on 2016-03-29T13:57:35Z No. of bitstreams: 1 2016_tese_pafmagalhaes.pdf: 3724835 bytes, checksum: 2211058119db6e128e61846550417799 (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2016-03-29T13:57:48Z (GMT) No. of bitstreams: 1 2016_tese_pafmagalhaes.pdf: 3724835 bytes, checksum: 2211058119db6e128e61846550417799 (MD5) / Made available in DSpace on 2016-03-29T13:57:48Z (GMT). No. of bitstreams: 1 2016_tese_pafmagalhaes.pdf: 3724835 bytes, checksum: 2211058119db6e128e61846550417799 (MD5) Previous issue date: 2016-02-18 / Lesão renal por isquemia/reperfusão (I/R) e acidose metabólica (MA) são duas condições críticas que podem ocorrer simultaneamente na prática clínica. O resultado dessa combinação pode ser prejudicial para os rins, mas esta questão não tem sido exaustivamente estudada até hoje. O presente estudo avaliou em ratos a influência do baixo pH sistêmico em vários parâmetros da função renal mediante lesão renal por I/R. A acidose metabólica foi induzida em ratos Wistar machos através da ingestão de cloreto de amônio (NH4CI) dissolvido em água da torneira, começando 2 dias antes da agressão isquêmica e mantida durante todo o estudo. Isquemia/reperfusão renal foi induzida por clampeamento de ambas as artérias renais durante 45 min, seguido por 48 h de reperfusão. Foram estudados quatro grupos de animais: controle (submetido à cirurgia sham, n = 8), I/R (n = 8), acidose metabólica (AM; solução de NH4CI 0,28 M e cirurgia sham, n = 6), e AM+I/R (solução de NH4CI 0,28 M + I/R, n = 9). Em comparação com grupo I/R, ratos AM+I/R exibiram redução significativa de pH sanguíneo, bicarbonato plasmático (pBic), e excesso de base (SBE), com declínio no ritmo de filtração glomerular e função tubular. Foram detectados sinais de lesão tubular microscópica. Imunofluorescência mostrou que a combinação entre acidose metabólica e isquemia/reperpusão renal aumentou nitidamente a expressão do fator nuclear kappa B (NF-B) e da heme oxigenase-1 (HO-1), mas não interferiu na diminuição da expressão da óxido nítrico sintase endotelial (eNOS) causada por I/R. Os resultados sugerem que a lesão renal induzida por isquemia/reperfusão é agravada pela acidose.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Gram-positive bacteria are characterized by a single lipid bilayer with a thick peptidoglycan layer. This group of organisms contains bacteria commonly associated with human infection, including: Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis and Streptococcus pneumoniae among others. These bacteria have a common system for importing iron in the form of heme, which is acquired by proteins containing heme-binding NEAT (NEAr iron Transporter) domains. The heme acquisition system in S. aureus is termed the Iron Surface Determinant (Isd) system and in L. monocytogenes is termed Heme Binding Protein (Hbp) and Heme/Hemoglobin Uptake Protein (Hup). These proteins work together to obtain heme from hemoglobin and then transport the heme into the cytoplasm via well characterized ABC-transporters. Although there have been clinical trials with antibodies directed against Isd proteins, there are currently no antibiotics targeting iron uptake systems in bacteria in general. Building upon fluorescent approaches for detection of iron uptake in Gram negative organisms, this work develops fluorescent heme acquisition detection in Gram positive organisms. The spectrofluorimetric methodology facilitates the understanding of heme acquisition protein interactions and mechanisms in bacteria. This work could subsequently be used to identify inhibitors of Gram positive bacterial iron uptake systems, and develop a new target for antibiotic action.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Objective: To evaluate the protective effect of the heme-oxygenase 1 (HO-1)/biliverdin/CO pathway in models of gastropathy in mice, evaluating the role of the soluble guanylate cyclase (GCs) and of the constitutive NOS in this event. Methods: Protocol 1: Mice were pre-treated with hemin (HO-1 inducer; 1,3,10 mg/Kg, i.p.), biliverdin (HO-1 product; 1,3 or 10 mg/Kg., i.p.), DMDC (CO donor; 2.5, 7.5, 12.5 or 10 Âmol/Kg, i.p.) or ZnPP IX (HO-1 antagonist; 0,3, 1 or 3 mg/kg. i.p.), one hour before, gastric damage was induced by ethanol 50% (hemin, biliverdin, DMDC) or 25% (ZnPP IX). In another group, the animals were pre-treated with ODQ (12.5 mg/kg, v.o) or L-NAME (3 mg/Kg, v.o), thirty minutes before of the treatments cited previously. After 1h, the mice were sacrificed and the stomachs removed for evaluation of the gastric lesions (Image J). Protocol 2: Mice were pre-treated with hemin (3 mg/Kg, i.p.), biliverdin (3 mg/Kg., i.p.), DMDC (12,5 Âmol/Kg) or ZnPP IX (3,0 mg/kg), one hour before of the administration of INDO 30 mg/Kg (hemin, biliverdin, DMDC) or 10 mg/Kg (ZnPP IX). In another group, the animals were pre-treated with ODQ (12.5 mg/kg, v.o) or L-NAME (3 mg/Kg, v.o), thirty minutes before of the treatments cited previously. Three hours after, the mice were sacrificed and the stomachs removed for evaluation of the gastric lesions, utilizing a digital paquimetry. In all of the experimental groups, fragments of the gastric mucous were collected for determination of the concentration of MDA, GSH or bilirubin. Another samples of tissue was removed for microscopic analyzes and HO-1 expression by immunohistochemistry. The detection of the TNF-α, IL-1β, IL-10 and MPO activity were evaluated only in the INDO gastropathy. Results: Ethanol increased the expression of HO-1 and the levels of bilirrubin in the gastric tissue. Hemin, biliverdin and DMDC reduced gastric damage, MDA levels and GSH consume in ethanol 50%- induced gastropathy. The histological parameters, edema, hemorrhage and loses of epithelial cells, were diminished in the presence of hemin, biliverdin or DMDC. ZnPP IX amplified the ethanol-induced gastric lesion, increased MDA formation and decreased the GSH concentration in gastric mucosa. The histological parameters also were amplified after the handling with ZnPP IX. Bilirubin concentration was elevated during the protection induced by hemin and biliverdin, but not DMDC. INDO increased the HO-1 expression and the bilirrubin levels in the gastric mucosa. Hemin, biliverdin or DMDC reduced the gastric lesion, the MPO activity, and the MDA levels and increased the GSH concentration in the gastropathy INDO- induced. The histological parameters, edema, hemorrhage, loss of epithelial cells and the presence of inflammatory cells, were inhibited by hemin, biliverdin or DMDC. ZnPP IX amplified the effect of the INDO increasing the gastric lesion, the MPO activity, the MDA levels and the GSH consume. The histological parameters also were amplified after the handling with ZnPP IX. Bilirubin was shown elevated during the protection induced by hemin and biliverdin, but not DMDC. Hemin, biliverdin and DMDC diminished the TNF-α and IL-1β concentrations and increased the IL-10. ODQ and L-NAME completely abolished the DMDC protective gastric effect, but not biliverdin in the gastropathy ethanol or INDO- induced. Conclusion: HO-1/biliverdin/CO pathway plays a protective effect against ethanol or INDO-induced gastric damage. In the gastropathy by ethanol, the protection is dependent of the anti-oxidant action by bilirubin and CO. However, in the model of INDO gastropathy, we observe an anti-oxidant and anti-inflammatory action. The mechanism of gastro protective action of the CO, but not of the biliverdin, is dependent of the CO/ NOS/ GMPc pathway. / Objetivo: Avaliar o efeito protetor da via hemeoxigenase 1 (HO-1)/ biliverdina/ CO em modelos de gastropatia em camundongos e o papel da guanilato ciclase solÃvel (GCs) e da NOS constitutiva neste evento. MÃtodos: Protocolo 1: Camundongos foram prÃ-tratados hemina (indutor da HO-1; 1,3 ou 10mg/Kg, i.p.), biliverdina (produto da HO-1; 1,3 ou 10mg/Kg, i.p.), DMDC (doador de CO; 2,5, 7,5, 12,5 ou 25 μmol/Kg, i.p.) ou ZnPP I(inibidor da HO-1; 0,3, 1,0 ou 3,0 mg/kg. i.p.) uma hora antes da administraÃÃo por gavagem de etanol 50% (hemina, biliverdina, DMDC) ou 25% (ZnPP IX). Em outro grupo, os animais foram prÃ-tratados com ODQ (12,5 mg/kg, v.o) ou L-NAME (3 mg/Kg, v.o), trinta minutos antes dos tratamentos citados anteriormente. Depois de 1h, os camundongos foram sacrificados e os estÃmagos removidos para avaliaÃÃo das lesÃes gÃstricas (Image J). Protocolo 2: Camundongos foram prÃ-tratados hemina (3,0 mg/kg), biliverdina (3,0 mg/kg), DMDC (12,5 μmol/Kg) ou ZnPPIX (3,0 mg/Kg) uma hora antes da administraÃÃo de INDO 30 mg/Kg (hemina, biliverdina, DMDC) ou 10 mg/Kg (ZnPP IX). Em outro grupo os animais foram prÃ-tratados com ODQ (12,5 mg/kg, v.o) ou L-NAME (3 mg/Kg, v.o), trinta minutos antes dos tratamentos citados anteriormente. TrÃs horas depois, os camundongos foram sacrificados e os estÃmagos removidos para avaliaÃÃo das lesÃes gÃstrica, utilizando um paquÃmetro digital. Em todos os grupos experimentais, fragmentos da mucosa gÃstrica foram coletados para determinaÃÃo da concentraÃÃo de MDA, GSH e bilirrubina. Outra amostra de tecido foi retirada para analise microscÃpica e imunohistoquÃmica. A detecÃÃo das citocinas TNF-α, IL-1β e IL-10, bem como a atividade de MPO foram avaliados somente na gastropatia por INDO. Resultados: O etanol aumentou a expressÃo de enzima HO-1 e dos nÃveis de bilirrubina no tecido gÃstrico. Hemina, biliverdina ou DMDC reduziram a lesÃo gÃstrica, os nÃveis de MDA e o consumo de GSH induzido por etanol 50%. Os parÃmetros histolÃgicos, edema, hemorragia e perda de cÃlulas epiteliais, foram diminuÃdos na presenÃa de hemina, biliverdina ou DMDC. ZnPP IX amplificou o efeito do etanol 25%, aumentando a lesÃo gÃstrica, os nÃveis de MDA e o consumo de GSH. Os parÃmetros histolÃgicos tambÃm foram amplificados apÃs o tratamento com ZnPP IX. A concentraÃÃo de bilirrubina se mostrou elevada apenas na gastroproteÃÃo induzida por hemina e biliverdina, mas nÃo pelo DMDC. INDO aumentou a expressÃo da HO-1 e os nÃveis de bilirrubina na mucosa gÃstrica. Hemina, biliverdina ou DMDC reduziram a lesÃo gÃstrica, a atividade de MPO, os nÃveis de MDA e aumentaram a concentraÃÃo de GSH na gastropatia por INDO. Os parÃmetros histolÃgicos, edema, hemorragia, perda de cÃlulas epiteliais e a presenÃa de cÃlulas inflamatÃrias, foram inibidas pela hemina, biliverdina ou DMDC. ZnPP IX amplificou o efeito da INDO aumentando a lesÃo gÃstrica, a atividade de MPO, os nÃveis de MDA e o consumo de GSH. Os parÃmetros histolÃgicos tambÃm foram amplificados apÃs o tratamento com ZnPP IX. Bilirrubina se mostrou elevada apenas na gastroproteÃÃo induzida por hemina e biliverdina, mas nÃo pelo DMDC. Hemina, biliverdina e DMDC diminuÃram as concentraÃÃes de TNF-α e IL-1β e aumentaram a IL-10. ODQ e L-NAME reverteram o efeito protetor do DMDC, mas nÃo da biliverdina, na gastropatia induzida por etanol ou INDO. ConclusÃo: A via HO-1/biliverdina/CO participa do processo de defesa da mucosa gÃstrica contra lesÃes induzidas por etanol ou INDO. Na gastropatia por etanol, a proteÃÃo à dependente da aÃÃo antioxidante da bilirrubina e CO. Entretanto, no modelo de gastropatia por INDO, observamos uma aÃÃo antioxidante e antiinflamatÃria. Evidenciamos ainda que o mecanismo de aÃÃo gastroprotetor do CO, mas nÃo da biliverdina à dependente da via CO/GMPc/NOS.

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.