Diabetes is associated with increased psychological distress which, in turn, is associated with poorer diabetes outcomes. This study examined the impact of a nine-week Internet based cognitive-behavioral therapy intervention that targeted stress and mood in people with diabetes. It was hypothesized that the intervention would decrease psychological distress and improve diabetes outcomes and adherence to diabetes treatment regimens. Participants with type 1 and type 2 diabetes were randomly assigned to the intervention (n = 103) or a waiting-list control group (n = 74). ANCOVAs demonstrated significant group effects for the reduction of perceived generalized stress (F (1, 105) = 7.06, p = .01; d = .84), diabetes-related distress (F (1, 105) = 13.45, p < .01; d = .54), depression (F (1, 90) = 7.06, p < .01; d = .40), anxiety (F (1, 89) = 6.78, p = .01; d = .41), and negative affect (F (1, 103) = 13.02, p < .01; d = .56). There were also significant group effects for the reduction of psychological fatigue (F (1, 98) = 7.34, p = .01; d = .40), cognitive symptoms (F (1,95) = 6.40, p = .01; d = .48), hyperglycemic symptoms (F(1, 95) = 11.16, p <.01; d = .41) and hypoglycemic symptom (F(1, 98) = 6.16, p= .02; d= .53). Further, there were significant indirect effects of the intervention on the above diabetes symptoms, through psychological distress. There was no effect of the intervention on hemoglobin A1c (F(1.43) = 0.28, p= .60), though this analysis was underpowered. The intervention also had no effect on adherence to diabetes treatment regimen. This study provides evidence of a convenient and effective way to reduce psychological distress and improve symptoms in those with diabetes. It also provides evidence of reduced psychological distress as a mechanism for improving diabetes outcomes.

Malaria has consistently been rated as the worst parasitic disease in the world. This disease affects an estimated 5 billion households annually. Malaria has a high mortality rate leading to distorted socio-economic development of the world at large. The major challenge pertaining to malaria is its continuous and rapid spread together with the emergence of drug resistance in Plasmodium species (vector agent of the disease). For this reason, researchers throughout the world are following new leads for possible drug targets and therefore, investigating ways of curbing the spread of the disease. Cysteine proteases have emerged as potential antimalarial chemotherapeutic targets. These particular proteases are found in all living organisms, Plasmodium cysteine proteases are known to degrade host hemoglobin during the life cycle of the parasite within the human host. The main objective of this study was to use various in silico methods to analyze the hemoglobinase function of cysteine proteases in P. falciparum and P. vivax. Falcipain-2 (FP2) of P. falciparum is the best characterized of these enzymes, it is a validated drug target. Both the three-dimensional structures of FP2 and its close homologue falcipain-3 (FP3) have been solved by the experimental technique X-ray crystallography. However, the homologue falcipain-2 (FP2’)’ and orthologues from P.vivax vivapain-2 (VP2) and vivapain-3 (VP3) have yet to be elucidated by experimental techniques. In an effort to achieve the principal goal of the study, homology models of the protein structures not already elucidated by experimental methods (FP2’, VP2 and VP3) were calculated using the well known spatial restraint program MODELLER. The derived models, FP2 and FP3 were docked to hemoglobin (their natural substrate). The protein-protein docking was done using the unbound docking program ZDOCK. The substrate-enzyme interactions were analyzed and amino acids involved in binding were observed. It is anticipated that the results obtained from the study will help focus inhibitor design for potential drugs against malaria. The residues found in both the P. falciparum and P. vivax cysteine proteases involved in hemoglobin binding have been identified and some of these are proposed to be the main focus for the design of a peptidomimetric inhibitor.

La forte prévalence des hémoglobinopathies, notamment l'HbAS, l'HbC et l'α-thalassémie dans des zones d'endémie palustre est considérée comme la conséquence de la protection qu'elles procurent contre les formes létales du paludisme. De nombreux mécanismes ont été évoqués pour expliquer cette protection, ne sont pas cohérentes avec toutes les observations épidémio-cliniques disponibles. La première partie de ces travaux de thèse a abordé la réduction de la cytoadhérence des globules rouges (GR) parasités comme mécanisme potentiel commun aux trois hémoglobinopathies. Pour explorer plus en profondeur ce mécanisme, nous avons mené une étude comparative des phénotypes de cytoadhérence des isolats primaires de P. falciparum obtenus chez des patients HbAS et HbAC et HbAA. Cela avait pour but de déterminer si l'HbAS et l'HbAC jouaient un rôle dans la sélection et le maintien des souches virulentes de P. falciparum dans la nature. La deuxième partie de la thèse a concerné l'influence du trait drépanocytaire (HbAS) sur la déformabilité des GR non parasités d'une part et sur la rétention splénique des GR non parasités et parasités par les formes jeunes de P. falciparum d'autre part. Nous avons observé que l'adhésion des GR parasités aux cellules endothéliales micro-vasculaires humaines ainsi qu'aux monocytes était réduite avec les GR α-thalassémiques par rapport aux GR HbAA. Aucune différence statistiquement significative n’a en revanche été observée entre les profils de cytoadhérence des isolats primaires de P. falciparum issus de sujets HbAA, HbAS ou HbAC. L’étude de la déformabilité des GR a montré que les GR HbAS sont légèrement mais significativement moins déformables que les GR HbAA. En revanche, les GR HbAS parasités par les jeunes parasites de P. falciparum (anneaux) n’étaient pas plus retenus que leurs homologues HbAA dans la rate humaine isolé perfusée ex vivo, ou en microsphiltration, quelles que soient les conditions d’oxygénation. Nous n’avons observé aucune différence au niveau du taux de falciformation entre les GR parasités et non parasités que ce soit avec les GR HbAS ou avec les GR HbSS. En conclusion, nous proposons que la réduction de la cythoadhérence et la rétention splénique des GR contenant les formes matures de P. falciparum constituent un mécanisme commun à la protection des sujets HbAS, HbC et α-thalassémiques contre le paludisme. Ces deux phénomènes interconnectés peuvent rendre compte de l’ensemble des observations épidemio-cliniques disponibles sur la protection conférée par ces hémoglobinopathies. / The high prevalence of several inherited hemoglobin disorders, namely sickle cell trait (HbAS), HbAC and α-thalassemia, in malaria endemic areas is thought to be the consequence of their protective effects against malaria life-threatening manifestations. Numerous potential mechanisms have been proposed to explain this protective effect although many of them are not fully consistent with all available epidemiologic and clinical data. The first part of this thesis work explored the reduction of cytoadherence of infected RBC as a potential common mechanism for α-thalassemia-, HbAS- and HbAC-induced protection against malaria. To further explore this mechanism, and determine whether HbAS and HbAC select and maintain virulent P. falciparum parasite in nature, we compared the cytoadherence phenotype of P. falciparum isolates obtained from HbAS/HbAC and controls HbAA patients. The second part of the thesis work addressed the influence of HbAS on the deformability of uninfected RBC as well as the splenic retention of both uninfected RBCs and ring-infected RBCs. We observed a reduced adherence of α-thalassemic infected RBCs to human micro-vascular endothelial cells and monocytes compared to controls HbAA infected RBCs. The reduction was correlated to the number of non functional α- gene. Expression of PfEMP-1 on the surface of α- thalassemic infected RBCs was lower than on the surface of HbAA infected RBCs. There was no statistically significant difference between the cytoadherence of P. falciparum isolates obtained either from HbAS/HbAC or control HbAA malaria patients. The deformability of uninfected HbAS RBCs was slightly but significant lower than that of control uninfected HbAA RBCs. Retention rates of ring-infected HbAS and HbAA RBCs were similar either in human isolated spleen perfusion ex vivo and in microsphilters in vitro regardless of the oxygenation level. We did not observe any enhanced sickling of ring-infected RBCs compared to non infected RBCs, both in HbAS and HbSS samples. Based on these results along with available epidemiologic and previous experimental data, we propose a common malaria-protective mechanism of HbAS, HbAC and α-thalassemia whereby these hemoglobin disorders reduce the cytoadherence of mature P. falciparum-infected RBCs that stay in circulation where they are exposed to an enhanced splenic retention. These 2 mechanisms would act in conjunction to slower the rise of parasites loads in infected patients and protect them from sequestration-related complications of malaria.

Please read the abstract in the section 00front of this document / Dissertation (MA (Human Movement Science))--University of Pretoria, 2006. / Biokinetics, Sport and Leisure Sciences / unrestricted

Heme proteins carry out a diverse array of functions in vivo while maintaining a well-conserved 3-over-3 α-helical structure. Human hemoglobin (Hb) is well-known for its oxygen transport function. Type 1 non-symbiotic hemoglobins (nsHb1) in plants and bacterial flavohemoglobins (fHb) from a variety of bacterial species have been predicted to carry out a nitric oxide dioxygenase function. In nsHb1 and fHb this function has been linked to protection from nitrosative stress. Herein, I combine photoacoustic calorimetry (PAC), transient absorption spectroscopy (TA), and classical molecular dynamics (cMD) simulations to characterize molecular mechanism of diatomic ligand interactions with a hexa-coordinate globin from plant (rice hemoglobin), bacterial flavohemoglobins and human hemoglobin. In rice type 1 non-symbiotic hemoglobin (rHb1), the dynamics and energetics of structural changes associated with ligand photodissociation is strongly impacted by solvent and temperature, namely CO escape from the protein matrix is slower at pH = 6.0 compare to neutral pH (ns) due to the CD loop reorganization which forms a pathway for ligand escape. In human hemoglobin, exogenous allosteric effectors modulate energetics of conformational changes associated with the CO and O2 escape although the effectors impact on rate constants for ligand association is small. The conformational dynamics associated with ligand photorelease from fHbs from Cupriavidus necator (FHP) and Staphylococcus aureus (HMPSa) are strongly modulated by the presence of azole drugs indicating that drug association modulates structural properties of the heme binding pocket. In addition, we carried out a study of the formation of the DNA intercalated motif (i-motif). The formation of the structure is strongly favored at acidic pH; therefore, PAC was combined with a 2-nitrobenzaldehyde pH-jump to probe formation of the i-motif on fast timescales. i-Motif folding is two-step process with the initial protonation of cytosine residues being endothermic with ΔHfast=8.5 ± 7.0 kcal mol-1 and ΔVfast=10.4 ± 1.6 mL mol-1 and subsequent nucleation/i-motif folding (τ = 140 ns) with ΔHslow=-51.5 ± 4.8 kcal mol-1 and ΔVslow=-6.6 ± 0.9 mL mol-1. The above results indicate that PAC can be employed to study diverse biochemical reactions such as DNA folding, drug binding and ligand photorelease from proteins.

The spleen has been shown to contract in apneic situations in humans as well as in other diving mammals, expelling its stored red blood cell content into circulation. This natural blood boosting may increase the circulating hemoglobin concentration (Hb) by up to 10%, which would enhance the oxygen carrying capacity and likely increase performance. However, the triggers of this response in humans have not been fully clarified. Study I was therefore focused on the effect of hypoxia as a trigger of spleen contraction. It was found that 20 min of normobaric hypoxic breathing evoked a substantial reduction in spleen volume showing that hypoxia is an important trigger for spleen contraction. Knowing the role of hypoxia, Study II compared two different hypoxic situations – a 2 min apnea and 20 min normobaric hypoxic breathing – which resulted in the same level of arterial hemoglobin desaturation. Apnea evoked a twice as great spleen volume reduction, implying that variables other than hypoxia were likely involved in triggering spleen contraction. This may be hypercapnia which is present during apnea but not during normobaric hypoxic breathing. Study III therefore investigated the effects of breathing gas mixtures containing different proportions of CO2 prior to maximal apneas. Pre-breathing mixtures with higher percentages of CO2 resulted in greater spleen contraction, thus demonstrating hypercapnia's likely role as a trigger in addition to hypoxia. Study IV explored whether an all-or-nothing threshold stimulus for triggering spleen contraction existed, or if contraction was graded in relation to the magnitude of triggering stimuli. Exercise was therefore performed in an already hypoxic state during normobaria. Rest in hypoxia produced a moderate spleen volume reduction, with an enhanced spleen contraction resulting after hypoxic exercise, with a concomitant increase in Hb. This implies that spleen contraction is a graded response related to the magnitude of the stimuli. This could be beneficial in environments with varying oxygen content or work loads. Study V examined the possibility that spleen contraction is part of the acclimatization to altitude, during an expedition to summit Mt Everest. The long-term high altitude exposure, combined with physical work on the mountain, had no effects on resting spleen volume but resulted in a stronger spleen contraction, when provoked by apnea or exercise. This indicates that acclimatization to altitude may enhance the contractile capacity of the spleen, which may be beneficial for the climber. From these studies I concluded that hypoxia is an important trigger for spleen contraction but that hypercapnia also contributes in apneic situations. The spleen contraction likely provides a graded expulsion of erythrocytes in response to these stimuli, causing a temporary increase in gas storage capacity that may facilitate activities such as freediving and climbing. The storage of erythrocytes during rest serves to reduce blood viscosity, which would also be beneficial for the climber or diver. The human spleen contraction appears to become stronger with acclimatization, with beneficial effects at altitude. Such an upgraded response could be beneficial both in sports and diseases involving hypoxia.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T17:09:12Z (GMT). No. of bitstreams: 1 Santos_CamilaOrescodos_D.pdf: 28263366 bytes, checksum: 0bb038ac0bb395e9aed9a7089336b2d0 (MD5) Previous issue date: 2006 / Resumo: AHSP é uma proteína eritróide específica que apresenta afinidade de ligação com a-globinas, estabilizando essas moléculas e dessa forma, evitando a precipitação nos precursores eritróides e bloqueando danos celulares causados pela oxidação de cadeias globínicas. Camundongos portadores de P-talassemia com deleção do gene AHSP apresentaram maior precipitação de cadeias a-globinas nas hemácias e níveis acentuados de anemia. Estudos in vitro utilizando proteína recombinante demonstraram menor geração de espécies reativas de oxigênio quando a-globinas encontram-se estabilizadas pela AHSP. Com o objetivo de analisar a importância da proteína AHSP como modulador de gravidade clínica nas síndromes P-talassêmicas o gene AHSP foi analisado em amostras de DNA de pacientes com P-talassemia, atendidos pelo Hemocentro da Unicamp, e em amostras controles durante minha tese de mestrado. Durante estes estudos, três características do gene AHSP foram detectadas. Estas características do gene AHSP foram investigadas durante o desenvolvimento deste presente trabalho. Primeiro, um polimorfismo localizado no posição 12888 no gene AHSP, que leva a substituição de uma Asparagina na posição 75 por uma Isoleucina (N75I), foi identificado em uma paciente heterozigota para P-talassemia (Pj9/pA) com anemia grave e em processos transfusionais freqüentes. Outras duas famílias apresentaram o polimorfismo N75I. Entretanto, nestes casos a presença do polimorfismo no gene AHSP não se correlaciona claramente com a gravidade clínica. O sequênciamento de amostras de indivíduos controles de várias partes do mundo sugerirão uma baixa freqüência deste polimorfismo na população analisada. Estudos de indivíduos saudáveis positivos para o polimorfismo N75I demonstraram precipitação e oxidação de cadeias globínicas nas hemácias. Análises funcionais com proteína recombinante sugerem que a proteína AHSP N75I apresenta características de ligação com a-globínas normais, mas é menos eficiente em evitar geração de espécies reativas de oxigênio por estas cadeias globínicas. Estes efeitos, quando em conjunto com ?-talassemia poderiam resultar em anemia mais grave, demonstrando a proteína AHSP N75I como um modificador genético nas síndromes talassêmicas. Segundo, através de análises computacionais, nós identificamos uma estrutura secundária na região 3'-UTR do RNA mensageiro do gene AHSP. semelhante a um elemento responsivo a ferro (IRE), presente em todas as espécies que apresentam o gene AHSP. Várias evidências demonstraram que, mesmo não apresentando as características principais para a caracterização de um IRE, esta estrutura secundária do gene AHSP é reconhecido pelas proteínas reconhecedoras de IREs (IRPs) regulando a estabilidade da molécula de RNA mensageiro em resposta aos níveis de ferro: 1) Recuperação do RNA mensageiro do gene AHSP através da imunoprecipitacão das proteínas IRP1 e IRP2: 2) Níveis elevados de ferro desestabilizam o RNA mensageiro do gene AHSP: mutações neste IRE levam à desestabilização constitutiva do RNA mensageiro; 3) Níveis elevados de ferro desestabilizam o RNA mensageiro do gene AHSP em camundongos que apresentam excesso de ferro. Estes dados contribuem para o melhor entendimento de como IRE atípicos podem ainda ser funcionais e sugerem um mecanismo que poderia regular a estabilidade de cadeias globínicas de acordo com os níveis de ferro. Além disso, sugerem que o excesso de ferro, que ocorre em pacientes com (3-talassemia. podem ser determinante na gravidade da doença por, provavelmente, aumentar os níveis de a-globinas livres e precipitando nos precursores eritróides. E, em terceiro, através de busca de sítios de ligação de fatores de transcrição no gene AHSP, nós encontramos um elemento MARE localizado no final do segundo éxon. Estudos de imunoprecipitaçâo de cromatina demonstraram ligação dos fatores de transcrição Nrf2, Bachl e Maf ao elemento MARE do gene AHSP e regiões controle. Concluindo, estes resultados demonstraram pela primeira vez um polimorfismo no gene AHSP que produz uma proteína não completamente funcional que pode estar relacionada com gravidade à p-talassemia e, além disso, descrevem dois mecanismos inéditos da regulação da expressão do gene AHSP que podem ser importantes na regulação deste gene em outras doenças hematológicas e durante a eritropoiese / Abstract: Alpha-hemoglobin stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds the cx-chains of hemoglobin, preventing their precipitation and deleterious effects. Loss of AHSP exacerbates a-globin precipitation and anemia in a murine model for p-thalassemia. In vitro, recombinant AHSP inhibits the production of reactive oxygen species (ROS) by a-globin. To further define the role of AHSP as a modifier of P- thalassemia, we analyzed AHSP sequence for mutations in a large population of (3- thalassemic and control subjects. From this genomic screening three interesting features of the AHSP gene were found. First, a single nucleotide change that converts asparagine 75 to leucine (N75I) was identified in a patient who was heterozygous for P-thalassemia (p39/(3A). She presented with an unusually severe anemia that required regular blood transfusion. Another two families were positive detected for the SNP N75I. but the presence of other known genetic modifiers for thalassemia in these families made hard to correlate clinical severity with AHSP. Of the unrelated control subjects tested just one (0.35%) contained the SNP N75I. Analysis of red blood cells from this subject revealed normal hemoglobin indices but a small number of Heinz bodies, suggesting a non-fully functional AHSP. Analysis of the biochemical properties of the recombinant mutant protein showed that the binding affinity of AHSP N75I for a- hemoglobin is normal. Importantly, compared to wild type AHSP, the N75I mutant protein exhibited significantly reduced capacity to inhibit ROS production by a-hemoglobin. Hence, AHSP N75I may be less effective at conferring protection from oxidative-mediated damage by free a-hemoglobin in erythrocytes. These effects, when coupled with P-thalassemia, could result in more severe anemia, implicating AHSP N75I as a potential genetic modifier. Second, by computational algorithms, we identified IRE-like stem-loop structures in AHSP mRNA of multiple species, yet the primary sequences deviate significantly from canonical IRE consensus sequences determined by studies of classical IREs, such as Transferring receptor and Ferritin. Several lines of evidence now show that the AHSP IRE binds IRPs to regulate mRNA stability in an iron-dependent fashion: 1) AHSP mRNA co-immunoprecipitates with IRPs. 2) AHSP mRNA is destabilized by iron in both erythroid and heterologous cells; disruption of the IRE renders the mRNA constitutively unstable. To study how iron regulates AHSP expression in vivo, we treated mice with iron dextran for 10 days and then examined AHSP mRNA in Terll9+ erythroid progenitors by RT-PCR. We found that short-term iron overload reduced AHSP mRNA levels. Our findings indicate that AHSP mRNA stability is regulated by iron via an atypical 3'-UTR IRE. These findings extend the potential repertoire for functional IREs that do not conform as the previously defined canonical consensus sequences. In addition, they provide a potential mechanism by which erythroid cells can regulate globin stability according to iron status. As such, iron overload, which occurs in patients with p thalassemia, might aggravate the disease by further elevating the levels of toxic free a globin. And third, by computation analysis of transcriptional sites, we found a potential MARE element located at the end of the second exon. Experiments of chromatin imunoprecipitation assay determined the binding of the transcript factor Nrf2, Bachl and Maf to the MARE element of AHSP gene, as well to the positive controls. Overall, these results demonstrated for the first time a polymorphism on AHSP gene that produce a non fully functional protein that might be correlated with severity in p-thalassemia and two new mechanisms that control the AHSP gene expression with potential implications in another hematological diseases besides thalassemias and closely connecting AHSP with erythropoiesis / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Orientadores: Maria de Fatima Sonati, Munir Salomão Skaf / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T12:16:23Z (GMT). No. of bitstreams: 1 Jorge_SusanElisabethDominguesCosta_M.pdf: 12500595 bytes, checksum: 271d153e901aa04d248746dbcf6d90c6 (MD5) Previous issue date: 2009 / Resumo: A hemoglobina (Hb), pigmento respiratório de todos os organismos vertebrados, encontra-se em elevadas concentrações nas hemácias e é responsável pelo transporte de O2 dos pulmões para os tecidos. A Hemoglobina humana constitui-se de dois pares de cadeias polipeptídicas (globinas), cada qual envolvendo um grupo prostético heme, que se liga reversivelmente ao O2. Mais de 1000 variantes estruturais da Hb humana já foram descritas, parte delas relacionada a manifestações clinicas importantes. Algumas variantes apresentam alteração funcional, que se traduz em afinidade modificada pelo O2. Quando elevada, há uma produção compensatoriamente maior de hemácias, levando a policitemia; quando diminuída, resulta em cianose. O Laboratório de Hemoglobinopatias do Departamento de Patologia Clinica da FCM/UNICAMP e um dos laboratórios de referencia no pais, tendo identificado, ate o momento, 12 hemoglobinas novas e 51 variantes raras. Destas, as variantes novas Hb Hb Boa Esperanca (?16 Lys?Thr), Hb Itapira (?30 Glu?Val), Hb Bom Jesus da Lapa (?30 Glu?Ala), Hb Olinda [ß22 (b4) -25 (b7)], Hb Caruaru (ß 122 Phe?Ser) e Hb S-Sao Paulo (ß6 Glu?Val ; ß65 Lys?Glu) foram aqui caracterizadas funcionalmente, assim como as variantes raras Hb Iwata (a87 His??Arg), Hb Sunshine Seth (?94 Asp?His), Hb Deer Lodge (ß2 His?Arg), Hb Deer Lodge (ß2 His??Arg) + Hb S (ß6 Glu??Val), Hb G-Siriraj (ß7 Glu??Lys), Hb G-Siriraj (ß7 Glu??Lys) e Hb C (ß6 Glu??Lys) em associacao, Hb M-Saskatoon (ß63 His??Tyr), Hb Redondo (ß92 His? Asn), Hb Koln (ß98 Val??Met), Hb Coimbra (ß99 Asp??Glu) e Hb Dhonburi (ß126 Val?Gli)+ Btal. O metodo analitico empregado foi descrito por Rossi-Fanelli & Antonini (1958), e analisa espectrofotometricamente o comportamento da Hb frente a conhecidas pressões parciais de oxigênio (pO2), possibilitando a medida de afinidade (p50), da constante de Hill (n), que verifica o fenômeno de cooperatividade entre as cadeias globinicas para ligação do O2, o efeito Bohr (que indica facilidade na ligação Hb-O2 conforme e elevado o pH do meio), bem como a interação com o Inositol Hexafosfato (IHP), um fosfato polianion que dificulta a ligação entre a Hb e o O2. A exceção das Hbs Itapira e Bom Jesus da Lapa, em concentrações reduzidas nos hemolisados, todas as demais apresentam afinidade alterada pelo oxigênio. Como complementação ao estudo funcional, as Hbs Caruaru e São Paulo também foram submetidas a analise por dinâmica molecular, através dos programas VMD (Visual Molecular Dynamics), com dados inseridos nos softwares CHARMM (Chemistry at Harvard Molecular Mechanics) e NAMD (Nanoscale Molecular Dynamics). As demais variantes tiveram sua analise estrutural individualizada feita através da visualização de estruturas nativas depositadas no Protein Data Bank, para melhor compreensão da relação entre o aminoácido substituído e a função protéica alterada. Deste modo, foi possível estabelecer correlações entre a estrutura e a função e inferir sobre as manifestações clinicas resultantes da presença destas heme proteínas anômalas, ilustrando-se assim a importância desses estudos para a completa caracterização das variantes. / Abstract: The human hemoglobin (Hb) is the respiratory pigment of human organisms, found in high concentrations in the erythrocytes and is responsible for transporting O2 from the lungs to the peripheral tissues. This molecule is composed by two pairs of polypeptide chains (globin), each one involving a prosthetic group heme, which bind reversibly to the O2. More than 1000 structural variants had been described already; part of them related to important clinical manifestations. Some variants present functional alteration, with modified affinity for the O2. When it is increased, it has a higher compensatory production of the erythrocytes, causing policitemia; when decreased, it results on cyanosis. The Laboratory of Hemoglobinopaties of the Department of Clinical Pathology of the FCM/UNICAMP is one of the references laboratories in the country, and identified, until this moment, 12 new hemoglobins and 51 rare variants. The new variants Hb Hb Boa Esperanca (?16 Lys?Thr), Hb Itapira (?30 Glu?Val), Hb Bom Jesus da Lapa (?30 Glu?Ala), Hb Olinda [ß22 (b4) -25 (b7)], Hb Caruaru (ß 122 Phe?Ser) e Hb S-Sao Paulo (ß6 Glu?Val ; ß65 Lys?Glu) were, in the present study, functionally characterized, as well as the rare variants Hb Iwata (a87 His??Arg), Hb Sunshine Seth (?94 Asp?His), Hb Deer Lodge (ß2 His?Arg), Hb Deer Lodge (ß2 His??Arg) + Hb S (ß6 Glu??Val), Hb G-Siriraj (ß7 Glu??Lys), Hb G-Siriraj (ß7 Glu??Lys) e Hb C (ß6 Glu??Lys) em associacao, Hb M-Saskatoon (ß63 His??Tyr), Hb Redondo (ß92 His? Asn), Hb Koln (ß98 Val??Met), Hb Coimbra (ß99 Asp??Glu) e Hb Dhonburi (ß126 Val?Gli)+ Btal. The analytical method used was described for Rossi-Fanelli & Antonini (1958), which analyses, by spectrophotometer, the behavior of the Hb against known partial pressures of oxygen (pO2), making possible the measure of affinity (p50), the constant of Hill (n), that verifies the cooperativity, the Bohr effect (that it indicates the relation between Hb-O2 binding according to the pH), as well as the interaction with Inositol Hexaphosphoric acid (IHP), that difficult linkage between the Hb and the O2. Except from Hb Itapira and Hb Bom Jesus of the Lapa, in reduced concentrations at the total hemolysate, all the others presented modified affinity to the oxygen. As a complement of the functional study, the Hb Caruaru and Hb S-Sao Paulo were also submitted to previous molecular dynamics simulation. All the other variants were structurally analyzed by the study of the native structure deposited at the Protein Data Bank, to understand better the relation between the substituted residue and the modified one at the protein function. Therefore, it was possible to establish correlations between the structure and the function and to infer on the clinical manifestations because of the presence of these anomalous proteins, illustrating the importance of these studies for the complete characterization of the variants. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

A proteólise limitada de certas proteínas leva à liberação de peptídeos opióides endógenos. Vários relatos apontam que peptídeos derivados da hemoglobina como hemorfinas e hemopressinas têm efeito antinociceptivo, pela atividade de modulação de receptores acoplados a proteínas G. No presente estudo, um ensaio de captura do substrato (ECS) foi combinado com a marcação isotópica e LC-MS/MS para identificar e caracterizar um novo fragmento da hemoglobina que se liga à EP24.15. O peptídeo AGH, identificado neste trabalho, inibe respostas de hipernocicepção periféricas através de receptores opióides do tipo <font face=\"Symbol\">m . A persistência do peptídeo AGH no tecido nervoso perfundido sugere relevância fisiológica. Embora o AGH seja derivado de hemoglobina e tenha atividade opióide, falta-lhe a sequência chave das hemorfinas (YPWT), indicando que ele pode pertencer a uma nova classe de peptídeos derivados da hemoglobina. Adicionalmente, o AGH modula as interações entre as proteínas 14-3-3<font face=\"Symbol\">e e EP24.15 in vitro, podendo estar relacionado com a secreção não convencional da EP24.15. / Limited proteolysis of certain proteins leads to the release of endogenous opioid peptides. Several reports have shown that hemoglobin-derived peptides such as hemorphins and hemopressins have an antinociceptive effect by modulating GPCR activity. In the present study, a substrate capture assay (SCA) was combined with isotopic labeling and LC-MS/MS to identify and characterize a new bioactive hemoglobin fragment that binds to EP24.15. AGH, a new peptide identified in this work, inhibits peripheral hyperalgesic responses through <font face=\"Symbol\">m opioid receptors (MOR). The persistence of AGH peptide in perfused nervous tissue suggests its physiological relevance. Although AGH is derived from hemoglobin and it is a peptide with opioid activity, it lacks the key sequence of hemorphins (YPWT), indicating that it is part of a new class of peptides derived from hemoglobin. Additionally, the AGH modulates interactions between 14-3-3<font face=\"Symbol\">e and EP24.15 proteins in vitro and may be related to the unconventional EP24.15 secretion.

Introdução- A hiperglicemia (HG), durante o infarto agudo do miocárdio (IAM), está associada com aumento de mortalidade hospitalar em pacientes diabéticos e não diabéticos. Entretanto, não é conhecido o mecanismo responsável por esta associação. Assim estudou-se, simultaneamente, a correlação entre a glicemia e marcadores bioquímicos relacionados ao sistema neuro-humoral de estresse, metabolismo glicídico e lipídico, sistema de coagulação e inflamatório. Métodos- 80 pacientes foram incluídos consecutiva e prospectivamente. Foram realizadas duas coletas de sangue, a primeira com 24h a 48h do início dos sintomas do IAM (fase aguda) e a segunda após 3 meses do IAM (fase crônica), sempre com 12h de jejum. Foram analisados os seguintes parâmetros: glicose, cortisol, noradrenalina, hemoglobina glicada (HbA1c), insulina, LDL minimamente modificada eletronegativa, ácidos graxos livres (AGL), adiponectina, factor VII da coagulação, fibrinogênio, inibidor do ativação do plasminogênio tipo 1, proteína C reativa ultra-sensível (PCRus), colesterol total (c) e frações e triglicérides. Nas correlações univariadas entre glicemia e as variáveis contínuas empregou-se o teste de correlação de Pearson. As análises multivariadas foram feitas através de regressão logística (variáveis qualitativas) e modelo linear generalizado (quando as variáveis independentes incluídas foram quantitativas e nominais). Resultados- Na fase aguda, a glicemia correlacionou-se significativamente com HbA1c (r=0,75, p<0,001), insulina (r=0,25, p<0,001), AGL (r=0,3, p=0,01), adiponectina (r=-0,22, p=0,05), LDL-c (r=-0,25, p=0,03), VLDL-c (r=0,24, p=0,03) e triglicérides (r=0,27, p=0,01). No modelo multivariado, as variáveis correlacionadas de forma independente com a glicemia, na fase aguda, foram: HbA1c (p<0,001), insulina (p<0,001), e AGL (p=0,013). Para analisar uma variável de confusão, a história de diabetes mellitus (DM), incluiu-se esta variável num modelo, juntamente com as variáveis acima e todas mostraram associação significativas com glicose: HbA1c (p<0,001), insulina (p=0,001), AGL (p=0,013) e história de DM (p=0,027). Na fase crônica, glicose correlacionou-se com: cortisol (r=0,31, p=0,01), noradrenalina (r=0,54, p<0,001), HbA1c (r=0,78, p<0,001) e PCRus (r=0,46, p<0,001). Na análise multivariada, somente HbA1c (p<0,001) e noradrenalina (p<0,001) mantiveram correlação independente. Conclusão- A HbA1c foi a única variável que correlacionou-se de forma significativa e independente com a glicemia, tanto na fase aguda, quanto na crônica, mostrando que a hiperglicemia, durante o IAM, pode representar uma alteração crônica, sub-diagnosticada, do metabolismo glicídico. / Introduction- Hyperglycemia (HG) is an important prognostic factor in acute myocardial infarction (AMI). However, the pathophysiology is poorly understood. So we proposed a simultaneous correlation between glycemia and biochemical markers of stress, glucose and lipid metabolism, coagulation and inflammation system. Methods- Eighty AMI patients were included prospectively. Blood were collected between 24h and 48h from the pain (acute phase), and 3 months post AMI (chronic phase), with 12-h fasting. These parameters were analyzed: glucose, cortisol, norepinephrine, hemoglobin glycated (HbA1c), insulin, minimally modified electronegative LDL, free fatty acids (FFA), adiponectin, factor VII coagulant, fibrinogen, plasminogen activator inhibitor-1, high sensitive C reaction protein (hsCRP), total cholesterol (c) and fractions and triglyceride. The relationships between glucose and continuous variables were assessed by Pearsons correlation coefficient (r) and multivariate analysis with linear regression. Results- At acute phase, glucose correlated significantly with HbA1c (r=0.75, p<0.001), insulin (r=0.25, p<0.001), FFA (r=0.3, p=0.01), adiponectin (r=-0.22, p=0.05), LDL-c (r=-0.25, p=0.03), VLDL-c (r=0.24, p=0.03) and triglyceride (r=0.27, p=0.01). In a multivariate model, variables correlated were: HbA1c (p<0.001), insulin (p<0.001), and FFA (p=0.013). At the chronic phase, glucose correlated significantly with cortisol (r=0.31, p=0.01), norepinephrine (r=0.54, p<0.001), HbA1c (r=0.78, p<0.001) and hsCRP (r=0.46, p<0,001). By multivariable analysis, only HbA1c (p<0.001) and norepinephrine (p<0.001) remained correlated. Conclusion- HbA1c was the main variable that correlated significantly and independently with glycemia at acute and chronic phases, suggesting that HG during AMI can represent an exacerbation of abnormal glucose metabolism previously not diagnosed.