Background: Glycated hemoglobin is the indicator of long-term diabetes control and a value below 7 percent is recommended by the American Diabetes Association (ADA) to reduce cardiovascular complications. Diabetic patients have a two- to four-fold risk of cardiovascular disease and approximately two-thirds of diabetic patients die as a result of cardiovascular complications. Three large prospective randomized controlled long-term trials within the last decade reported no significant reduction in cardiovascular complications in type 2 diabetic patients by intensive glycemic control. To the author's knowledge, no known retrospective studies have examined the association between mean serial glycated hemoglobin and coronary atherosclerosis (CA) or acute coronary syndromes (ACS). Objective: This study was designed to determine the association between mean serial glycated hemoglobin with incident CA or ACS in type 2 diabetic patients after controlling for age, gender, hypertension, low density lipoprotein cholesterol (LDL-C), microalbuminuria, aspirin use, statin use, insulin use, tobacco use, and body mass index (BMI). Methods: The study was a retrospective cohort database analysis using the Austin Travis County CommUnityCare[trademark] clinics' electronic medical record for the time period between October 1, 2004 and September 30, 2009. The primary outcome of the study was the incidence of CA or ACS and the primary independent variable was glycated hemoglobin (<7% vs. [greater than or equal to]7%). The study subjects included type 2 diabetic patients aged 30 to 80 years with at least one glycated hemoglobin value per year for a minimum of two consecutive years. Study subjects were excluded if CA or ACS occurred within six months of the index date (i.e., first glycated hemoglobin). Logistic regression analysis was used to address the study objective. Results: Overall, 3069 subjects met the study inclusion criteria with a mean follow-up period of approximately two years. Two percent (N=62) of the subjects had incident CA or ACS. After controlling for age, gender, hypertension diagnosis, LDL-C, microalbuminuria, aspirin use, statin use, insulin use, tobacco use and BMI, there was no significant association (OR=1.026, 95% CI=0.589-1.785, p=0.9289) between mean serial glycated hemoglobin and the incident diagnosis of CA or ACS. Increasing age (OR=1.051, 95% CI=1.025-1.077, p<0.0001), male gender (OR=1.855, 95% CI=1.105-3.115, p=0.0195) and normal weight (normal or underweight compared to obese: OR=0.122, 95% CI=0.017-0.895, p=0.0438) were significantly associated with incident CA or ACS. Conclusions: Mean serial glycated hemoglobin (comparing [greater than or equal to]7% to <7%) was not significantly associated with CA or ACS over a mean follow-up period of approximately two years. Until more evidence becomes available, clinicians and diabetic patients should target glycated hemoglobin level below or close to 7 percent as recommended by the ADA soon after diagnosis while concomitantly controlling nonglycemic risk factors of cardiovascular disease (statin use, aspirin use, blood pressure control, smoking cessation and life style modification), to reduce their long-term risk of incident CA or ACS. / text

Οι αιμοσφαιρινοπάθειες, συμπεριλαμβανομένης της β-θαλασσαιμίας και της δρεπανοκυτταρικής αναιμίας, συγκαταλέγονται ανάμεσα στις πιο κοινές μονογονιδιακές αναταραχές παγκοσμίως, που χρήζουν αποτελεσματικής θεραπευτικής στρατηγικής. Σήμερα, η επανενεργοποίηση των γονιδίων της γ-σφαιρίνης φαίνεται να είναι μια ενδιαφέρουσα θεραπευτική προσέγγιση για τους ασθενείς που πάσχουν από β-τύπου αιμοσφαρινοπάθειες. / Hemoglobinopathies are amongst the most common single gene disorders worldwide, including beta-thalassemia and sicle cell disease (SCD), seeking for therapeutic intervention. The reactivation of the human gamma-globin genes is considered to be a therapeutic strategy for beta-type hemoglobinopathies patients.

Hemoglobin biosynthesis in erythrocyte precursors involves several steps. The correct ratios and concentrations of normal alpha (α) and beta β) globin proteins must be expressed, apoproteins must be folded correctly, heme must be synthesized and incorporated into these globins, and the resulting α and β subunits must be rapidly and correctly assembled into heterotetramers. These events occur on a large scale in vivo, and dysregulation causes serious clinical disorders such as thalassemia syndromes. Recent work has implicated a conserved erythroid protein known as Alpha-Hemoglobin Stabilizing Protein (AHSP) as a participant in these events. Current evidence suggests that AHSP enhances α subunit stability and diminishes its participation in harmful redox chemistry. There is also evidence that AHSP facilitates one or more early-stage post-translational hemoglobin biosynthetic events. In this work, the rate constants associated with AHSP binding to and dissociation from native ferric and ferrous human α subunits have been determined, along with the binding and dissociation equilibrium constants. Also, several mutant AHSP proteins were used to better define the cis-trans peptidyl-prolyl isomerization events that AHSP is known to undergo, and several naturally occurring human a subunit missense mutants were used to probe AHSP function. Additionally, several post-binding events regarding AHSP:α-subunit interactions were investigated, such as autooxidation, heme uptake, hemin loss, effects on ligand binding, and secondary structure acquisition. Finally, AHSP was co-expressed with α and β subunits in transgenic Escherichia coli as a way of probing the effects of AHSP on hemoglobin production. Collectively, these data support the model that AHSP rapidly binds α subunits, stabilizes them, and then is displaced by β subunits during hemoglobin production.

Das akute Lungenversagen (Adult Respiratory Distress Syndrome: ARDS) stellt ein in der Intensivmedizin häufig auftretendes Krankheitsbild dar und weist trotz intensiver Bemühungen nach wie vor eine hohe Letalität auf. Ein wichtiger pathophysiologischer Faktor bei der mit dem ARDS verbundenen schweren Gasaustauschstörung ist der intrapulmonale Rechts-links-Shunt mit daraus resultierender Abnahme des Oxygenierungsindex. Um therapeutisch eine Verbesserung der Oxygenierung zu erreichen, wurde unter anderem von Gallart et al. ein Konzept entwickelt, bei dem durch den kombinierten Einsatz eines intravenös gegebenen pulmonalen Vasokonstriktors (Almitrine) und eines inhalativ gegebenen pulmonalen Vasodilatators (inhalatives Stickstoffmonoxid: iNO) die Shuntfraktion verkleinert wird (Gallart et al. 1998). Im Rahmen dieser Arbeit wurde die Kombination von iNO mit der intravenösen Gabe von zellfreiem Hämoglobin (Hemoglobin Based Oxygen Carrier, HBOC) als Vasokonstriktor am Tiermodell des akuten Lungenversagens hinsichtlich hämodynamischer Parameter und der arteriellen Oxygenierung untersucht. Die Ergebnisse wurden verglichen mit einer zweiten Versuchsgruppe, in der die Tiere lediglich iNO und Hydroxyethylstärke als Kontrollsubstanz erhielten. In Pilotversuchen wurden dafür ein Tiermodell des akuten Lungenversagens etabliert und die Auswirkungen der Gabe von HBOC bei vorbestehendem Lungenschaden untersucht. Anhand der durchgeführten Versuche konnte gezeigt werden, dass sich durch die HBOC-Gabe sowohl der systemische als auch der pulmonalarterielle Blutdruck signifikant erhöhte. Durch die zusätzliche Gabe von iNO ließ sich dieser Effekt antagonisieren. Ein additiver Effekt auf die arterielle Oxygenierung durch den kombinierten Einsatz von HBOC und iNO -im Vergleich zur alleinigen Gabe von iNO- war nachweisbar, die Unterschiede waren jedoch nicht signifikant. Weitere Untersuchungen werden zeigen müssen, ob sich dieser Effekt bei größerer Fallzahl als signifikant erweist, oder ob dieser tatsächlich nicht vorhanden sein sollte.

Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.

Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.

Thesis (M.S)--State University of New York at Binghamton, Decker School of Nursing, 2007. / Includes bibliographical references.

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 104-117.

Submitted by CLAUDEMIR OLIVEIRA SOUZA null (clau_souza10@hotmail.com) on 2018-04-05T00:34:55Z No. of bitstreams: 1 Dissertação Claudemir final.pdf: 2598018 bytes, checksum: 6a02fac38da72d304450321a6bebe674 (MD5) / Approved for entry into archive by Ana Carolina Gonçalves Bet null (abet@iq.unesp.br) on 2018-04-05T20:46:17Z (GMT) No. of bitstreams: 1 souza_co_me_araiq_int.pdf: 2522583 bytes, checksum: 763389bef408c84daca17f39d0af0cd5 (MD5) / Made available in DSpace on 2018-04-05T20:46:17Z (GMT). No. of bitstreams: 1 souza_co_me_araiq_int.pdf: 2522583 bytes, checksum: 763389bef408c84daca17f39d0af0cd5 (MD5) Previous issue date: 2018-03-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As hemoproteínas desempenham um papel vital nos organismos. Dentre elas, as hemoglobinas extracelulares gigantes (Hbs) se destacam por sua alta cooperatividade, alta estabilidade oligomérica, grande afinidade para ligação com oxigênio e resistência à oxidação. Devido estas propriedades, as Hbs apresentam um grande potencial biotecnológico, como é o caso de estudos buscando protótipos de substituição sanguínea e elaboração de biossensores. Porém, o uso das hemoglobinas gigantes em biossensores para detecção de metais pesados ainda não é encontrado na literatura, este tipo de biossensor agiliza e diminui o custo das análises de empresas e agências de fiscalização ambiental, obtendo o resultado in loco. Diante do exposto, o objetivo deste trabalho foi verificar a sensibilidade da hemoglobina de Amynthas gracilis (HbAg) com os metais cobre e cádmio em diferentes valores de pH e imobilizá-la em substrato sólido com o auxílio dos seguintes polímeros: o catiônico polietilenoimina (PEI) e o aniônico hidrocloreto de polialilamina (PAH). Os estudos por absorção ótica, espalhamento de luz (LSI), dicroísmo circular (CD) e fluorescência da interação da HbAg em função da concentração do metal pesado cobre no pH 7,0 mostraram que a concentração de 30 μmol.L-1 é uma concentração crítica onde observa-se uma maior oxidação do grupo heme, agregação e dissociação da proteína, além de alteração em suas α- hélices. No pH 5,0 foi observada oxidação do grupo heme mas sem alterações nas estruturas secundárias da HbAg e formação de agregados. As análises dos espectros de absorção ótica, CD, fluorescência e LSI da HbAg na presença de diferentes concentrações do metal pesado cadmio não apresentaram mudanças significativas quando comparadas com o espectro da HbAg nativa. A imobilização da HbAg em PEI no pH 7,0 foi monitorada por absorção ótica e voltametria cíclica, entretanto a HbAg não foi possível imobilizar a HbAg no polímero PAH no mesmo valor de pH, sugerindo que a interação da HbAg com os biopolímeros se deve por interações eletrostáticas. A imobilização da HbAg em PEI manteve as características da hemoglobina em pH 7,0. A hemoglobina apresentou respostas ao cobre nas medidas de voltametria cíclica realizadas em concentrações de até 20 μmol.L-1 do metal, evidenciando o grande potencial de biossensoriamento do sensor fabricado. Assim, nestes estudos foi possível verificar que a HbAg na presença de cobre em pH 7,0 se oxida, agrega e dissocia, enquanto que em pH 5,0 ela apresenta apenas oxidação. Na presença de cádmio, a HbAg não apresentou nenhuma alteração através das técnicas avaliadas. Com isso, sugere-se que a HbAg pode ser utilizada como um biossensor de indicação de presença de concentrações micromolares do metal pesado cobre. / Hemoproteins are a group of proteins that play a vital role in organisms. Among them, giant extracellular hemoglobins (Hbs) stand out for their high cooperativity, high oligomeric stability, high affinity for oxygen binding and resistance to oxidation. Due to these properties, the Hbs show a great biotechnological potential, as is the case of studies searching for prototypes of blood replacement and preparation of biosensors. However, the use of giant hemoglobins in biosensors for heavy metals detection is not yet reported on the literature, this kind of biosensors speeds up and lowers costs of analysis of companies and environmental inspection agencies, obtaining on the spot result. Thus, the aim of this work was verified the sensibility of HbAg with the metals copper and cadmium on different pH values and it's immobilization in solid surfaces with the polymers: the cationic polietilenimine (PEI) and anionic polyallylamine hydrochloride (PAH). Studies of optical absorption (UVVis), light scattering (LSI), circular dichroism (CD) and fluorescence of HbAg interaction with copper at pH 7.0 shown that 30 μmol.L-1 it’s a critical concentration where observed a great oxidation of group heme, aggregation and protein dissociation as well as alteration on your α-hélices. In pH 5.0 was observed oxidation of heme group but without alterations in HbAg secondary structure and no aggregates formation. UV-Vis spectra analysis, CD, fluorescence and LSI of HbAg at different concentrations of cadmium doesn’t shown significant changes on native HbAg spectra. The HbAg immobilization on PEI pH 7,0 has monitored by optical absorption and cyclic voltammetry, however it was not possible to immobilize the HbAg in the PAH polymer at the same pH value, suggesting that HbAg interaction with biopolymers is due to electrostatic ligations. The HbAg Immobilization in PEI maintained the hemoglobin characteristics at pH 7.0. The Hemoglobin shown responses to copper at concentrations of up to 20 μmol.L-1 of the metal in cyclic voltammetry measurements performed, demonstrated the great potential of the fabricated biosensor. Thus, in these studies it was possible to verify that HbAg on copper presence at pH 7.0 oxidizes, to aggregates and dissociates whereas at pH 5.0 it presents only oxidation. In the presence of cadmium, the HbAg showed no change through the techniques evaluated. Thereby, it is suggested that HbAg can be used as a biosensor indicating the presence of micromolar concentrations of copper heavy metal.

Orientador: Patricia Soares Santiago / Banca: Marli Leite de Moraes / Banca: Valquiria da Cruz Rodrigues Barioto / Resumo: As hemoproteínas desempenham um papel vital nos organismos. Dentre elas, as hemoglobinas extracelulares gigantes (Hbs) se destacam por sua alta cooperatividade, alta estabilidade oligomérica, grande afinidade para ligação com oxigênio e resistência à oxidação. Devido estas propriedades, as Hbs apresentam um grande potencial biotecnológico, como é o caso de estudos buscando protótipos de substituição sanguínea e elaboração de biossensores. Porém, o uso das hemoglobinas gigantes em biossensores para detecção de metais pesados ainda não é encontrado na literatura, este tipo de biossensor agiliza e diminui o custo das análises de empresas e agências de fiscalização ambiental, obtendo o resultado in loco. Diante do exposto, o objetivo deste trabalho foi verificar a sensibilidade da hemoglobina de Amynthas gracilis (HbAg) com os metais cobre e cádmio em diferentes valores de pH e imobilizá-la em substrato sólido com o auxílio dos seguintes polímeros: o catiônico polietilenoimina (PEI) e o aniônico hidrocloreto de polialilamina (PAH). Os estudos por absorção ótica, espalhamento de luz (LSI), dicroísmo circular (CD) e fluorescência da interação da HbAg em função da concentração do metal pesado cobre no pH 7,0 mostraram que a concentração de 30 μmol.L-1 é uma concentração crítica onde observa-se uma maior oxidação do grupo heme, agregação e dissociação da proteína, além de alteração em suas α- hélices. No pH 5,0 foi observada oxidação do grupo heme mas sem alterações nas estruturas se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hemoproteins are a group of proteins that play a vital role in organisms. Among them, giant extracellular hemoglobins (Hbs) stand out for their high cooperativity, high oligomeric stability, high affinity for oxygen binding and resistance to oxidation. Due to these properties, the Hbs show a great biotechnological potential, as is the case of studies searching for prototypes of blood replacement and preparation of biosensors. However, the use of giant hemoglobins in biosensors for heavy metals detection is not yet reported on the literature, this kind of biosensors speeds up and lowers costs of analysis of companies and environmental inspection agencies, obtaining on the spot result. Thus, the aim of this work was verified the sensibility of HbAg with the metals copper and cadmium on different pH values and it's immobilization in solid surfaces with the polymers: the cationic polietilenimine (PEI) and anionic polyallylamine hydrochloride (PAH). Studies of optical absorption (UVVis), light scattering (LSI), circular dichroism (CD) and fluorescence of HbAg interaction with copper at pH 7.0 shown that 30 μmol.L-1 it's a critical concentration where observed a great oxidation of group heme, aggregation and protein dissociation as well as alteration on your α-hélices. In pH 5.0 was observed oxidation of heme group but without alterations in HbAg secondary structure and no aggregates formation. UV-Vis spectra analysis, CD, fluorescence and LSI of HbAg at different concentrations of... (Complete abstract click electronic access below) / Mestre