Particle analysis using multiwavelength UV-visible spectroscopy provides the potential for extracting quantitative red blood cell information, such as hemoglobin concentration, cell size, and cell count. However, if there is a significant presence of hypochromism as a result of the concentrated hemoglobin (physiological value of 33%), successful quantification of red cell values would require a correction. Hypochromism has been traditionally defined as a decrease in absorption relative to the values expected from the Beer-Lambert Law due to electronic interactions of chromophores residing in close proximity of one another. This phenomenon has been suggested to be present in macroscopic systems composed of strong chromophores such as nucleic acids, chlorophyll, and hemoglobin. The study presented in this dissertation examines the presence of hypochromism in red blood cells as a part of a larger goal to qualitatively and quantatively characterize red blood cells using multiwavelength UV-visible spectroscopy. The strategy of the study was three-fold: 1) to determine the instrumental configuration that would provide the most complete information in the acquired spectra, 2) to develop an experimental model system in which the hemoglobin content in red blood cells could be modified to various concentrations, and 3) to implement an interpretation model based on light scattering theory (which accounts for both the scattering and absorption components of the optical density spectrum) to provide quantitative information for the experimental system. By this process, hypochromicity was redefined into two categories with molecular hypochromicity representing the traditional definition and macroscopic hypochromicity being an attenuation of the absorption component due to a scattering-related effect. Successful simulations of experimental red cell spectra containing various amounts of hemoglobin were obtained using the theoretical model. Furthermore, successful quantitative interpretation of the red blood cell spectra was achieved in the context of corpuscular hemoglobin concentration, corpuscular volume, and cell count solely by accounting for the scattering and absorption effects of the particle, indicating that molecular hypochromicity was insignificant in this macroscopic system.

A limited understanding of iron uptake mechanisms is available for Streptococcus pyogenes, a hemolytic human pathogen capable of using a variety of hemoproteins in addition to ferric and ferrous iron. This study characterizes the transporters of iron-complexes siuADBG (for streptococcal iron uptake) and siaABC (for streptococcal iron acquisition). These ABC-type transporters are encoded by iron regulated operons and their protein products are homologous to components of heme and siderophore transporters found in both Gram-positive and Gram-negative bacteria. Mutants of the membrane permeases siuG and siaB were constructed and characterized. Mutations in both transporters demonstrated growth reduction in comparison to the parent strain when grown in complex medium containing iron in the form of hemoglobin. The addition of heme to the growth medium inhibited ferric uptake by the wild-type while the addition of protoporphyrin IX did not, suggesting that heme utilization as an iron source is responsible for the inhibition of ferric accumulation. Inactivation of siuG reduced the ability of heme to inhibit ferric incorporation by the cells. Inactivation of siaB in addition to siuG had a cumulative effect, indicating that both siu and sia transporters are involved in heme utilization. We also demonstrated that purified rSiaA, the surface receptor of SiaABC, binds heme and hemoglobin in vitro, and we propose a mechanism of heme binding by SiaA. Studies in a zebrafish infection model revealed that the siuG mutant was attenuated in producing disease. While the siaB mutant also presented virulence attenuation, infection by this mutant was characterized by an increase in the host inflammatory response. These observations show that iron acquisition is important for S. pyogenes virulence. We propose that the SiaABC and SiuADBG, together with the multi-metal transporter MtsABC, are involved in iron acquisition from different iron sources present in the human body, thus contributing to the survival and pathogenesis of S. pyogenes.

INTRODUCCIÓ: L'estandardització de mesures d’hemoglobina glicada A(1c) (HbA(1c)) ha estat una de les principals preocupacions en el camp de la bioquímica clínica des de la publicació dels estudis DCCT i UKPDS. Des del desenvolupament del mètode de referència per a mesurar específicament hemoglobina glicada A(1c) en sang humana, s’han publicat equacions per a la transformació de resultats traçables al sistema de referència de la IFCC a resultats traçables als sistemes d’estandardització japonès (JDS/JSCC) i nord-americà (NGSP). Existeixen alguns inconvenients en l’ús d’equacions com el baix nombre de mostres utilitzat en el seu desenvolupament. L’objectiu principal d’aquest estudi fou elucidar dues equacions per a la transformació de resultats traçables al sistema de referència IFCC a resultats traçables als esquemes japonès i nord-americà. Estudiar les implicacions clíniques dels resultats obtinguts, la relació entre, por una banda, de la concentració basal de glucosa en sang amb la concentració de glucosa mitjana estimada a partir del resultat de HbA(1c) i, per altra, de la relació entre la mitjana de les concentracions basals de glucosa i el valor de HbA(1c) i, per últim, l’estudi de la capacitat diagnòstica de la concentració basal de glucosa respecte la HbA(1c). MATERIAL I MÈTODES: Es mesurà el percentatge d’hemoglobina glicada A(1c) en 3000 mostres de sang total per al desenvolupament de cadascuna de les equacions (2000 per a l’elucidació de cada equació i 1000 per a estudiar les diferències entre diferents valors d’hemoglobina A(1c) obtinguts) en dos analitzadors, un calibrat traçable al sistema de referència de la JDS/JSCC o al sistema de referència de la NGSP i un altre calibrat de forma traçable al mètode de referència de la IFCC. S’obtingueren les dues equacions mitjançant mètodes de regressió lineal simple. RESULTATS: Les equacions obtingudes foren: JDS/JSCC(%) = 1,0545*IFCC(%) + 1,129(%) (R^2 = 0,9972) i NGSP(%) = 0,9293*IFCC(%) + 2,1054(%) (R^2 = 0,9979). S’observaren diferències estadísticament significatives entre els resultats obtinguts mitjançant l'utilizació tan de les equacions anteriorment publicades como de les desenvolupades en el present treball i els resultats obtinguts directament de l’analizador. En quant a l’estudi de les seves implicacions clíniques, no s’observà relació lineal entre la concentració basal de glucosa en sang i el valor de la concentració mitjana de glucosa en sang estimada a partir dels valors de HbA(1c), així com tampoc s’observà cap tipus de relació lineal entre el valor de la concentració basal mitjana de glucosa en sang i el valor d’HbA(1c). No s’observaren millors resultats en els paràmetres diagnòstics en l’ús de la concentració basal de glucosa respecte de la mesura d’HbA(1c). CONCLUSIONS: Es possible realitzar la transformació de resultats d’HbA(1c) traçables al sistema de referència de la IFCC a valors traçables als esquemes d’estandardització japonès i nord-americà mitjançant les equacions desenvolupades en el present estudi. L’elevat nombre de mostres utilitzat en el desenvolupament de les equacions proporciona una notable robustesa a les mateixes, garantint-se la qualitat dels resultats obtinguts mitjançant la seva utilització. No s’observà cap tipus de relació lineal entre la concentració de glucosa basal en sang i la glucosa mitjana estimada a partir de l’HbA(1c), ni entre la mitjana de les concentracions basals de glucosa i el valor d’hemoglobina glicada, pel que no es poden obtenir equacions lineals per a l’estimació d’aquestes variables. La HbA(1c) segueix essent la millor prova de laboratori per a el diagnòstic de diabetis mellitus i l’avaluació del control metabòlic dels pacients diabètics. / INTRODUCTION: HbA(1c) measurements are the cornerstone in diabetic patient’s control. Master equations have been published to transform values from the reference method to values traceable to national standardization systems. There are diverse inconveniences in these equations like low sample number use and heterogeneity of methods used to develop equations. The aim of this study was to obtain two equations for reference method values transformation to values traceable to the JDS/JSCC and the NGSP standardization schemes and to compare it with the already published equations. A secondary objective was to study the clinical implications of the obtained results and the relationship between fasting plasma glucose and average estimated glucose concentrations derived from HbA(1c) values and, on the other hand, the relationship between mean values of fasting plasma glucose and HbA(1c) values. MATERIAL AND METHODS: HbA(1c) was measured in 3000 blood samples in two analyzers calibrated traceably to the JDS/JSCC and the NGSP schemes and to the reference method. Two equations for value transformation were developed by simple linear regression methods with use of 2000 paired values and compared with the already published equation using 1000 HbA(1c) values. RESULTS: The equations obtained were JDS/JSCC(%) = 1,0545*IFCC(%) + 1,129(%) (R^2 = 0,9972) i NGSP(%) = 0,9293*IFCC(%) + 2,1054(%) (R^2 = 0,9979). Statistically significant differences were found between the results obtained using these equations, the results obtained with the published equations and those obtained directly from the analyzer. No linear relationship were found neither between fasting plasma glucose and average estimated glucose concentrations derived from glycohemoglobin values nor between mean values of fasting plasma glucose and glycohemoglobin values. Fasting plasma glucose does not provide better diagnostic parameters than HbA(1c) CONCLUSIONS: These new developed master equations are useful tools to transform reference method values to the JDS/JSCC and NGSP schemes values. The elevated nunber of values used in its development provides high robustness to the equations. HbA(1c) remains as the best laboratory test for the evaluation of diabetic patients.

This dissertation presents the development and use of a novel optical imaging system capable of monitoring changes in blood flow, oxygenated hemoglobin, deoxygenated hemoglobin, and absolute pO₂ in the brain. There are several imaging modalities capable of monitoring these parameters separately. Laser speckle contrast imaging (LSCI) and multi-spectral reflectance imaging (MSRI) have been used to monitor relative blood flow and hemoglobin changes respectively. Phosphorescence quenching, while not typically used for imaging, is capable of noninvasive measurements of pO₂. Combining these three techniques has led to the development of an imaging system that could ultimately lead to a better understanding of brain physiology. By combining techniques such as LSCI and MSRI, it becomes possible to estimate the cerebral metabolic rate of oxygen (CMRO₂), an important indicator of neuronal function. It is equally important to understand absolute pO₂ levels so that oxygen metabolism can be examined in context. Integrating phosphorescence quenching and a spatial light modulator into the imaging system allowed absolute pO₂ to be simultaneously measured in distinct regions. This new combined system was used to investigate pathophysiological conditions such as cortical spreading depression (CSD) and ischemia. The observed hemodynamic changes associated with these events were largely dictated by baseline oxygen levels and varied significantly in different regions. This finding highlighted the importance of having a system capable of monitoring hemodynamic changes and absolute pO₂ simultaneously while maintaining enough spatial resolution to distinguish the changes in different regions. It was found that animals with low baseline pO₂ were unable to deliver enough oxygen to the brain during events like CSD because of the high metabolic demand. In order for this technique to become more prevalent among researchers, it is essential to make it cost effective and simple to use. This was accomplished by replacing the expensive excitation sources with cheaper light emitting diodes (LEDs) and redesigning the software interface so that it was easier to control the entire device. The final system shows the potential to become a key tool for researchers studying the role of absolute pO₂ and other hemodynamic parameters during pathophysiological conditions such as CSD and ischemia. / text

No description available.

No description available.

Motivation Hypertension and type 2 diabetes mellitus are common in the black population of South Africa. The literature also shows that elevated blood glucose concentrations can lead to an increase in blood pressure and a blunted decline in nocturnal blood pressure. Therefore, the motivation for this study was to determine if blood glucose may play a role regarding the blunted nocturnal decline in blood pressure in African and Caucasian men. Aim The aim of this study was to investigate the relationship between a blunted nocturnal decline in blood pressure and blood glucose in African and Caucasian men. Methodology A comparative population study was preformed that consisted of 202 school teachers (101 African and 101 Caucasian) between the ages of 25–60 years from the North West Province, South Africa. Subjects were excluded if their body temperature was elevated, had a dependence or abuse of psychotropic substances, were regular blood donors and/or vaccinated in the previous three months. Ambulatory systolic (SBP) and diastolic blood pressure (DBP) were measured. Blood samples from the antebrachial vein were collected in sodium fluoride tubes to determine the serum glucose level and glycosylated hemoglobin A1c (HbA1c) percentage. Estimated average glucose (eAG) was determined from the percentage HbA1c by means of a regression formula. Means and proportions were compared by standard t–test and the chi–square test, respectively. Pearson correlations were used to determine unadjusted associations and multiple regression analysis to determine adjusted associations between variables. Results and Conclusion African men had an elevated HbA1c (p<0.001), eAG (p<0.001), nighttime SBP (p<0.001) and DBP (p<0.001). These results remained similar when non–dipping African and Caucasian men were compared. The Africans also smoked more (p=0.012), consumed more alcohol (p=0.049), had a higher percentage of non–dippers (p=0.054), HIV infected subjects (p<0.001) and a larger number of subjects that used anti–hypertensive medication (p=0.049). The unadjusted analysis showed positive correlations between all the blood pressure measurements and serum glucose, HbA1c and eAG in the African non–dipper men. While in the non–dipper Caucasian men, only daytime SBP and nighttime SBP (22:00–06:00) correlated positively with serum glucose, HbA1c and eAG. Furthermore, when viewing the relationship between carotid intima–media thickness (CIMT) and the blood pressure measurements in the African population, only nighttime (00:00– 04:00) SBP (r=0.581, p<0.001) and DBP (r=0.566, p<0.001) showed positive associations. After adjustments were made for age and body mass index the associations between the various blood pressure measurements and blood glucose disappeared in the non–dipper Caucasian men. However, in the non–dipper African men both nighttime (22:00–06:00) SBP and (00:00– 04:00) SBP showed positive correlations with serum glucose, HbA1c and eAG. After full adjustments (age, BMI, smoking, alcohol intake, physical activity, C–reactive protein and baroreceptor sensitivity) were made, nighttime (00:00–04:00) SBP was the only measure of blood pressure that correlated positively with HbA1c (p=0.069) and eAG (p<0.001) in the nondipper African men. No significant relationships were found for Caucasian men. Furthermore, to determine if the association between nighttime (00:00–04:00) SBP and eAG were independent of CIMT, we adjusted for CIMT. By doing so the positive association between SBP and eAG remained significant in the non–dipper African men (R2=0.617; =0.438; p=0.008) and nonsignificant in the non–dipper Caucasian men (R2=0.423; =0.169; p=0.33). However, the relationship between CIMT and eAG disappeared when we adjusted for SBP, suggesting that the SBP and eAG relationship drives CIMT. In conclusion, the association between the early morning SBP (00:00–04:00) and the blood glucose in non–dipping African men suggests that the blunted decline in nocturnal blood pressure during the early morning hours is associated with chronically elevated blood glucose. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2011.

Motivation Hypertension and type 2 diabetes mellitus are common in the black population of South Africa. The literature also shows that elevated blood glucose concentrations can lead to an increase in blood pressure and a blunted decline in nocturnal blood pressure. Therefore, the motivation for this study was to determine if blood glucose may play a role regarding the blunted nocturnal decline in blood pressure in African and Caucasian men. Aim The aim of this study was to investigate the relationship between a blunted nocturnal decline in blood pressure and blood glucose in African and Caucasian men. Methodology A comparative population study was preformed that consisted of 202 school teachers (101 African and 101 Caucasian) between the ages of 25–60 years from the North West Province, South Africa. Subjects were excluded if their body temperature was elevated, had a dependence or abuse of psychotropic substances, were regular blood donors and/or vaccinated in the previous three months. Ambulatory systolic (SBP) and diastolic blood pressure (DBP) were measured. Blood samples from the antebrachial vein were collected in sodium fluoride tubes to determine the serum glucose level and glycosylated hemoglobin A1c (HbA1c) percentage. Estimated average glucose (eAG) was determined from the percentage HbA1c by means of a regression formula. Means and proportions were compared by standard t–test and the chi–square test, respectively. Pearson correlations were used to determine unadjusted associations and multiple regression analysis to determine adjusted associations between variables. Results and Conclusion African men had an elevated HbA1c (p<0.001), eAG (p<0.001), nighttime SBP (p<0.001) and DBP (p<0.001). These results remained similar when non–dipping African and Caucasian men were compared. The Africans also smoked more (p=0.012), consumed more alcohol (p=0.049), had a higher percentage of non–dippers (p=0.054), HIV infected subjects (p<0.001) and a larger number of subjects that used anti–hypertensive medication (p=0.049). The unadjusted analysis showed positive correlations between all the blood pressure measurements and serum glucose, HbA1c and eAG in the African non–dipper men. While in the non–dipper Caucasian men, only daytime SBP and nighttime SBP (22:00–06:00) correlated positively with serum glucose, HbA1c and eAG. Furthermore, when viewing the relationship between carotid intima–media thickness (CIMT) and the blood pressure measurements in the African population, only nighttime (00:00– 04:00) SBP (r=0.581, p<0.001) and DBP (r=0.566, p<0.001) showed positive associations. After adjustments were made for age and body mass index the associations between the various blood pressure measurements and blood glucose disappeared in the non–dipper Caucasian men. However, in the non–dipper African men both nighttime (22:00–06:00) SBP and (00:00– 04:00) SBP showed positive correlations with serum glucose, HbA1c and eAG. After full adjustments (age, BMI, smoking, alcohol intake, physical activity, C–reactive protein and baroreceptor sensitivity) were made, nighttime (00:00–04:00) SBP was the only measure of blood pressure that correlated positively with HbA1c (p=0.069) and eAG (p<0.001) in the nondipper African men. No significant relationships were found for Caucasian men. Furthermore, to determine if the association between nighttime (00:00–04:00) SBP and eAG were independent of CIMT, we adjusted for CIMT. By doing so the positive association between SBP and eAG remained significant in the non–dipper African men (R2=0.617; =0.438; p=0.008) and nonsignificant in the non–dipper Caucasian men (R2=0.423; =0.169; p=0.33). However, the relationship between CIMT and eAG disappeared when we adjusted for SBP, suggesting that the SBP and eAG relationship drives CIMT. In conclusion, the association between the early morning SBP (00:00–04:00) and the blood glucose in non–dipping African men suggests that the blunted decline in nocturnal blood pressure during the early morning hours is associated with chronically elevated blood glucose. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2011.

Chronic kidney disease (CKD) is highly prevalent, with increasing numbers of patients affected by the disease world-wide, and anemia is a common finding in patients with CKD. Anemia impacts negatively on cardiovascular disease, exercise capacity and quality of life, resulting in a significant mortality and morbidity. The aim of this study was to evaluate the levels of ischemia-modified albumin and lactate in patients with established anemia associated to CKD and its correlations with hemoglobin levels. Hematocrit, hemoglobin, iron, ferritin, albumin, creatinine, lactate and IMA were measured in 17 patients with established anemia associated to CKD and 19 controls by standard methods. The results of hematocrit, hemoglobin, iron and albumin were lower in anemia group than control group. Ferritin, creatinine and lactate levels were higher in anemia of CKD group than control group. IMA increases in anemia group (0.8115 ± 0.1304) ABSU compared to control (0.4951 ± 0.0393) ABSU. Significant correlations between IMA and lactate, IMA and hemoglobin, IMA and creatinine, and hemoglobin and lactate were observed. IMA and lactate increase during anemia and this elevation could be associated to hypoxia due to low hemoglobin levels. However, our data suggest that lactate is more sensitive to detect hypoxia in anemia compared to IMA. / A doença renal crônica (DRC) é altamente prevalente, com um aumento do número de pacientes no mundo inteiro, e a anemia é muito comum nestes pacientes. A anemia tem impacto negativo sobre a doença cardiovascular, capacidade de executar exercícios e a qualidade de vida, resultando numa significativa morbidade e mortalidade. O objetivo deste estudo foi avaliar os níveis de albumina modificada pela isquemia (IMA) e lactato em pacientes com anemia associada à DRC e sua correlação com os níveis de hemoglobina. Hematócrito, hemoglobina, ferro, ferritina, albumina, creatinina, lactato e IMA foram avaliados em 17 pacientes com anemia associada à DRC e em 19 pacientes do grupo controle, por métodos padronizados. Os resultados do hematócrito, hemoglobina, ferro e albumina foram menores no grupo de anêmicos do que no grupo controle. Ferritina, creatinina e lactato foram maiores no grupo de anêmicos do que no grupo controle. A IMA aumentou no grupo de anêmicos (0,8115 ± 0,1304) ABSU se comparado ao grupo controle (0,4951 ± 0,0393) ABSU. Foram observadas significativas correlações entre IMA e lactato, IMA e hemoglobina, IMA e creatinina e hemoglobina e lactato. A IMA e lactato aumentaram durante a anemia e esta elevação poderia ser associada com hipóxia devido aos baixos níveis de hemoglobina. Entretanto, nossos dados sugerem que o lactato possui maior sensibilidade na detecção da hipóxia na anemia, quando comparado com a IMA.

A hemoglobina possuí um grande potencial antimicrobiano, sendo os peptídeos antimicrobianos (AMPs) derivados de proteínas contêm o grupamento heme denominados hemocidinas. O carrapato bovino Rhipicephalus (Boophilus) microplus é capaz de adquirir e processar grandes quantidades de sangue para seu metabolismo. Neste trabalho, purificamos hemocidinas produzidas no tubo digestório do carrapato. Identificamos 10 fragmentos da hemoglobina bovina que possuem atividade contra Candida albicans (Hb 1-94, 3-94, 1-87, 93-141, 102-141, 103-141, 107-141, 104-141 e 98-114 e Hb\"beta\" 127-145). Determinamos que todos os peptídeos apresentam um alto conteúdo de aminoácidos básicos, além de uma estrutura secundária em \"alfa\"-hélice, características encontradas em outros AMPs. Verificamos também que as duas principais proteases que atuam na produção das hemocidinas são uma cisteíno- e uma aspártico-proteinase. A identificação de diversas hemocidinas, no tubo digestório de R. (Boophilus) microplus, indica uma possível participação das hemocidinas na proteção contra microorganismos / The hemoglobin posses a great potential as an antimicrobial, being denominated hemocidins the antimicrobial peptides (AMPs) derived from heme containing proteins. The cattle tick Rhipicephalus (Boophilus) microplus is capable of processing large amounts of blood to its own metabolism. In this work we purified the hemocidins that are produced in the tick gut. We identified 10 fragments from bovine hemoglobin that presents activity towards Candida albicans (Hb 1-94, 3-94, 1-87, 93-141, 102-141, 103-141, 107-141, 104-141 e 98-114 e Hb\"beta\" 127-145). We determined that all the peptides presents a high content of basic amino acids, besides a secondary structure in \"alfa\"-helix conformation, characteristics found in other AMPs. We also verified that a cistein- and an aspartic-protease are the two main proteases involved on the production of these hemocids. The identification of several hemocidins in the gut of R. (Boophilus) microplus, suggests that these AMPs may play a role in the defense of the tick against microorganisms