True Blood – Understanding the impact of reduced oxygen carrying capacity using a physiological, haematological and metabolomics perspective.

Oxygen carrying capacity has a positive association with endurance performance. As such, techniques such as altitude training can increase red cell mass (Hbmass) and have been shown to improve sea level performance. In addition to haematological changes (i.e. Hbmass), hypoxia can have a metabolic influence (e.g. increased oxidative and anaerobic enzyme concentration) resulting in changes to metabolites, which reflect systems level adaption to low oxygen carrying capacity environments. The use of metabolomics; therefore, represents an ideal analytical method to examine the physiological adaptations to hypoxia as this technique provides the ability to simultaneously analyse large number of metabolites present in human biological samples. The purpose of this thesis was to examine the acute (14 d) and chronic (42 d) influence of low oxygen carrying capacity on performance (Chapter Four), haematology (Chapters Three, Four and Five) and the metabolic profile (Chapter Three and Five). Chapter Three was conducted as an observational study to examine metabolic changes associated with moderate altitude exposure (~3000 m; 196.4 ± 25.6 h) during a 14 d Live High Train Low altitude camp in 10 endurance runners (six males, four females; 29 ± 7 y). Resting whole blood samples were collected before altitude (baseline), 3 d and 14 d of altitude camp and analysed using mass spectrometry. From this data, acute metabolic profiles to altitude exposure were developed, which demonstrated significant separation between measured time points. Specifically, through mass spectrometry 36 metabolites were identified from metabolite classes relating to amino acids, glycolysis, and purine metabolism and indicate a shift in substrate utilisation (i.e. greater carbohydrate use) during acute hypoxic exposure. From canonical variate analysis, trajectories of the 36 identified metabolites were identified, with two different trajectories observed during the acute moderate hypoxic exposure. Importantly, principal components analysis highlighted greater variance in measured metabolites between-person when compared to within-person. This finding is consistent with previous literature and indicates individual variability during the adaptive response to altitude exposure. During Chapter Four and Five, Australian Red Cross blood donation (~470 mL) was used to reduce the oxygen carrying capacity of 13 male participants, who were then monitored over a 42 d period. Measures of haemoglobin concentration and haematocrit % were obtained before blood removal (Baseline) and at seven time points (24 h, 7 d, 14 d, 21 d, 28 d, 35 d and 42 d) following blood removal. Additionally, exercise tests were conducted to assess fitness (V̇ O2max) and performance (4-minute self-paced cycling time trial [4MMP]) (Chapter Four and Five) and 4 mL blood samples were obtained for metabolomic analysis (Chapter Five). The purpose of Chapter Four was to examine the impact of reduced oxygen carrying capacity on V̇ O2max and both the performance and pacing during a 4MMP. During this study, only participants that were trained cyclists (n = 7) were used for the analyses. Blood removal resulted in a maximal decrease of 5.4 % in V̇ O2max (24 h), while the average power output during the 4MMP decreased significantly at 24 h (7 ± 6%), 7 d (6 ± 8%) and 21 d (4 ± 6%) when compared with baseline values. Furthermore, the aerobic contribution during the 4MMP was significantly reduced, when compared with baseline, by 5 ± 4%, 4 ± 5% and 4 ± 10% at 24 h, 7 d and 21 d, respectively. The rate of decline in power output upon commencement of the 4MMP was significantly attenuated and was 76 ± 20%, 72 ± 24% and 75 ± 35% lower than baseline at 24 h, 21 d and 42 d, respectively. These changes were unrelated to differences in haemoglobin concentration. Findings from this study indicated that reducing oxygen carrying capacity can influence pacing and the performance of middle-distance endurance events. Specifically, it appears that changes in pacing and performance during middle-distance endurance events are related to the ability to contribute power from aerobic metabolism, as an increase in anaerobic contribution was not observed. The focus of Chapter Five was to investigate, in addition to measures obtained in Chapter Four, the acute and chronic changes to the metabolic profile of individuals after blood donation. Untargeted metabolomic analyses was used on 4 mL whole blood samples obtained from 13 participants at baseline and at multiple time points over 42 d following the removal of some 470 mL of whole blood. Similar to findings in Chapter Four, whole blood removal resulted in a maximal decrease in V̇ O2max (-6%) and 4MMP (-7%), with both occurring at 7 d. However, with the inclusion of the additional six “untrained” participants, significant reductions in oxygen delivery (V̇ O2) only persisted for 24 h compared to 21 d in the subset of trained males (Chapter Four). The accelerated return is likely associated with participant fitness (i.e. V̇ O2max: 53.0 ± 10.9 mL・kg-1・min-1; (Chapter Five) vs. V̇ O2max: 60.7 ± 5.5 mL・kg-1・min-1 (Chapter Four)) with the addition of lesser trained males seemingly diminishing the influence of oxygen delivery in the later time points. The metabolomics analysis revealed multi-factorial changes with 40 metabolites deemed significant post blood removal. Through hierarchical cluster analysis, consistent with acute findings during Chapter Three, purine metabolites were significantly elevated immediately following blood removal (acute) and remained elevated throughout the 42 d monitoring period. This finding indicates a chronic adaptive response, which may occur to enhance oxygen delivery to the periphery. Overall, the series of studies have shown separation in plasma metabolites during acute moderate altitude exposure, which is likely linked to substrate utilisation and purine metabolism. Furthermore, decreasing oxygen carrying capacity does not appear to influence the anaerobic contribution to high-intensity middle-distance endurance exercise even with observed reductions in aerobic contribution (Chapter Four). The use of metabolomics throughout this thesis (Chapters Three and Five) has identified purine metabolism as an important marker of adaptation to hypoxia. Further research should focus on purine metabolism as a possible robust marker of hypoxic exposure and work to identify the unknown metabolites which were shown to be significant in the multivariate models

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Dielectrophoretic characterization of particles and erythrocytes

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE\u3edielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at \u3e95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE\u3edielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE\u3edielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE\u3emicrodevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE\u3emicrochannel system to be integrated with the DC- class=SpellE\u3eiDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error

Pulmonary disease, its risk factors and necessity for long-term follow-up care in childhood cancer survivors.

Treatment for childhood cancer puts the young patients at risk to develop adverse health outcomes. These adverse health outcomes can develop acute, already during treatment, or slowly over years to decades after completion of treatment. They can be of transient nature or long-lasting and chronic. Adverse health outcomes can potentially affect every organ system, including the lung. The term late effects is used to describe this heterogeneity of adverse health outcomes. Pulmonary late effects contribute to a higher morbidity and mortality in childhood cancer survivors compared to siblings or the general population

Examining the regional physiology of the transplant kidney during normothermic machine perfusion.

Ischaemia-reperfusion injury (IRI) is an unavoidable consequence of deceased-donor kidney transplantation that has a profound effect upon both immediate and long-term graft function. Disruption to microvascular perfusion (MVP) and tissue oxygenation (PtO2) are central to the development of IRI, but detailed regional pathophysiology remains unresolved in the context of renal transplantation. Normothermic machine perfusion (NMP) is a novel preservation technology that aims to improve pre-implantation kidney quality, but may also be used experimentally to investigate transplant reperfusion. My PhD set out to determine the optimal perfusion conditions to improve kidney quality, and to investigate the underlying heterogeneity in pathophysiology within regions of the kidney which may impact kidney quality. Firstly, we used a porcine model of donor kidney retrieval, NMP, and simulated reperfusion to test the hypothesis that reducing current NMP perfusate oxygenation (PPO2) from superoxic levels would improve renal function and reduce reperfusion injury. In kidneys exposed to either short or long cold ischaemic times, reducing PPO2 from the clinical standard to normoxic or hypoxic PPO2 altered oxygen kinetics during NMP but did not influence tubular function, clearance, urine output, or biomarkers of renal injury during simulated reperfusion. Secondly, we used porcine and human models of transplant reperfusion to test the hypothesis that the renal medulla would be disproportionately affected by tissue hypoperfusion, hypoxia and acute inflammation. In a porcine reperfusion series, PtO2 and MVP were significantly altered following IRI when compared to pre-ischaemic baselines, with greater variation and heterogeneity seen in the medulla than in the cortex. In a human reperfusion series, there was widespread initial microcirculatory disruption, persistent lower medullary PtO2 and a distinct medullary inflammatory environment. In summary we have described novel porcine and human renal medullary physiology and inflammation during transplant reperfusion that highlight the need for medulla-specific strategies to ameliorate IRI. We have further determined changes to renal physiology and injury in response to perfusate oxygenation in a novel preservation technology that may guide clinical implementation

Protein Structure and Interactions Studied by Electrospray Mass Spectrometry

Since the emergence of electrospray ionization (ESI) mass spectrometry (MS) as a tool for protein structural studies, this area has experienced tremendous growth. ESI-MS is highly sensitive, and it allows the analysis of biological systems ranging in size from a few atoms to large multi-protein complexes. This work aims to solve questions in protein structural biology by using ESI-MS in conjunction with other techniques. We initially apply ESI-MS for studying the monomeric protein cytochrome c (Chapter 2). The physical reasons underlying the irreversible thermal denaturation of this protein remain controversial. By utilizing deconvoluted charge state distributions, oxidative modifications were found to be the major reason underlying the observed behavior. The positions of individual oxidation sites were identified by LC-MS/MS-based tryptic peptide mapping. Chapter 3 and 4 focus on noncovalent protein complexes. ESI allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these assemblies by MS. It remains somewhat unclear, however, in how far this approach is suitable for measuring binding affinities. We demonstrate that the settings used for rf-only quadrupoles in the ion path are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful Kd measurements. Overall, our data support the viability of the direct ESI-MS approach for determining binding affinities of protein–protein complexes in solution. Having established suitable conditions for the analysis of noncovalent protein complexes, ESI-MS is applied for monitoring the folding and assembly of hemoglobin (Hb). The native structure of this protein comprises four heme-bound subunits. Hb represents an important model system for exploring coupled folding/binding reactions, an area that remains difficult to tackle experimentally. We demonstrate that efficient Hb refolding depends on the heme ligation status. Only under properly optimized conditions is it possible to return denatured Hb to its tetrameric native state with high yield. ESI-MS allows the observation of on-pathway and off-pathway intermediates that become populated during this highly complex self-assembly process. In summary, this work demonstrates that ESI-MS is a highly versatile tool for addressing questions at the interface of chemistry and structural biology

Life Course Socioeconomic Position and C-Reactive Protein: Mediating Role of Health-Risk Behaviors and Metabolic Alterations. The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil)

Background: Chronic inflammation has been postulated to be one mediating mechanism explaining the association between low socioeconomic position (SEP) and cardiovascular disease (CVD). We sought to examine the association between life course SEP and C-reactive protein (CRP) levels in adulthood, and to evaluate the extent to which health-risk behaviors and metabolic alterations mediate this association. Additionally, we explored the possible modifying influence of gender. Methods and Findings: Our analytical sample comprised 13,371 participants from ELSA-Brasil baseline, a multicenter prospective cohort study of civil servants. SEP during childhood, young adulthood, and adulthood were considered. The potential mediators between life course SEP and CRP included clusters of health-risk behaviors (smoking, low leisure time physical activity, excessive alcohol consumption), and metabolic alterations (obesity, hypertension, low HDL, hypertriglyceridemia, and diabetes). Linear regression models were performed and structural equation modeling was used to evaluate mediation. Although lower childhood SEP was associated with higher levels of CRP in adult life, this association was not independent of adulthood SEP. However, CRP increased linearly with increasing number of unfavorable social circumstances during the life course (p trend <0.001). The metabolic alterations were the most important mediator between cumulative SEP and CRP. This mediation path accounted for 49.5% of the total effect of cumulative SEP on CRP among women, but only 20.2% among men. In consequence, the portion of the total effect of cumulative SEP on CRP that was mediated by risk behaviors and metabolic alterations was higher among women (55.4%) than among men (36.8%). Conclusions: Cumulative SEP across life span was associated with elevated systemic inflammation in adulthood. Although health-risk behaviors and metabolic alterations were important mediators of this association, a sizable fraction of this association was not mediated by these factors, suggesting that other pathways might play a role, especially among men

Doctor of Philosophy

dissertationDue to the tremendous progress of modern medicine, more people are surviving cancer. A cancer diagnosis no longer connotes the end of life, but instead, a change in life. Recently, middle age and older adults with hematological cancer have become eligible for treatment with allogeneic hematopoietic cell transplantation (allo HCT), enabling them to survive their underlying cancer diagnosis. While some individuals fully recover from allo HCT, up to two thirds of allo HCT recipients develop new-onset diabetes. While research has been conducted on the physiological effect diabetes has on HCT outcomes, there is a knowledge gap regarding middle age and older adults' psychosocial response to the condition. The objective of this qualitative study was to explore the psychosocial experience of developing new-onset diabetes after allo HCT. Nineteen participants above 50 years of age were interviewed. Qualitative data generated through interviews were analyzed using constructivist grounded theory methods. The result was the mid-range theory of dealing with new-onset diabetes as a long-term effect of allo HCT. This theory had 4 stages; 1) finding out about diabetes, 2) formulating an understanding of diabetes in relation to cancer, 3) formulating a diabetes identity, and 4) dealing with diabetes after allo HCT. Three distinct patterns of movement through these stages emerged, depending on how participants recovered from their allo HCT. The first pattern occurred in the group of participants with no or minimal after allo HCT complications. The second pattern was seen in the group with episodic complications, iv and the third in those with ongoing complications. Two primary factors were responsible for these differences of moving through the stages, and ultimately, whether participants adapted to new-onset diabetes: the amount of treatment-related work and the perceptions of diabetes. The group with minimal complications was able to understand, identify, and integrate diabetes into their lives, while those with ongoing complications experienced barriers to socially constructing and identifying with their type of diabetes, and were subsequently unable to integrate diabetes into their lives. This mid-range theory provides a working framework for the development of clinical and educational interventions specific to this patient population

Quantum chemistry and conformational sampling meet together : a powerful approach to study and design metalloprotein reactivity

Metalloprotein are proteins containing metal ion cofactors. Compared to chemical catalysts, metalloproteins have a well-defined configuration around the active site which ensures higher specificity, selectivity and reaction rates. Metalloproteins are soluble in water, their function can be optimized genetically by modifying an host (e.g. a bacteria) and are biodegradable. Therefore, they are ideal templates for the creation of novel green catalysts and therapeutics. Nonetheless, metalloproteins, as found in nature, are usually not ready for industrial scale-up and may need to be re-designed. Molecular simulations can guide the search for new metalloprotein functionality, cutting the costs of the experimental work. Modeling metalloproteins' function requires the sampling of both the electronic and nuclear degrees of freedom to exhaustively describe their chemical reactivity. The combination of conformational sampling and quantum chemical technique allows to model how they catalyze a reaction or covalently bind a ligand, without missing information about the dynamics of the whole protein. In this thesis, these computational techniques are systematically employed to study and guide present and future design efforts of laccases and hemoglobin. Laccases are copper-containing oxidoreductases that can oxidize a large variety of substrates at expenses of oxygen, which is reduced to water. Therefore, their interest in green chemistry applications: they work with air and produce water as sole by-product. So far, most efforts made to enlarge the chemical space of laccases have focused on increasing the redox potential of the first copper-based electron acceptor, a choice that yielded limited success. Here, it is proposed to focus on the desired substrate, modeling the active site of laccases to better fit and oxidize it. To do so, a computational protocol was developed, based on Monte Carlo sampling of the enzyme-substrate conformational space, followed by quick quantum chemical calculations to score oxidation. The protocol was first validated against experimental data, proving its capability to reproduce data and provide a rationale to laccases functioning. This new tool was then used to improve the oxidation of aniline by a laccase by simulating the effect of certain mutations (amino acid substitutions) which were tested in the lab by our collaborators. As a result, the design mutations significantly improved the oxidation of aniline (which leads to the formation of polyaniline, an organic semiconductor). Another design was carried out which lead to a significant improvement in activity of another laccase toward three different substrates. Therefore, the methodology developed proved to be capable of reproducing and rationalizing experimental results and rendering a la carte design of laccases toward a given (class of) substrates possible. A similar protocol, which uses an empirical computational method instead of quantum chemical techniques, was used to selectively attach a photosensitizer (a molecule that produces a chemical change in another molecule in a photochemical process) to the surface of a laccase. Hemoglobin, a heme-containing protein that carries oxygen from the lungs to the tissue in the body, is a candidate for blood substituent design. However, its design is rendered difficult by the limited knowledge of its functioning. Here, a mixed Monte Carlo-quantum chemical approach was used to support a theory about hemoglobin's allosteric mechanism and structurally characterize the tertiary end-states of the allosteric transition for the first time. These calculations, which were benchmarked against available experimental data, disclosed the role of the amino acids next to the oxygen binding site. This information was used in a subsequent molecular dynamics study which showed how the four subunits of hemoglobin give rise the allosteric response, highlighting the signalling paths and their hierarchyLas metaloproteínas son proteínas que contienen iones metálicos como cofactores. En comparación con los catalizadores químicos, las metaloproteínas tienen una configuración bien definida alrededor del sitio activo que asegura mejor especificidad, selectividad y velocidad de reacción. Las metaloproteínas son solubles en agua, su función puede ser optimizada genéticamente mediante la modificación de un huésped y son biodegradables. Por lo tanto, son ideales para la creación de nuevos catalizadores verdes y terapéuticos. No obstante, las metaloproteínas, como se encuentran en la naturaleza, no suelen estar listas para la industria y pueden necesitar ser re-diseñadas. Las simulaciones moleculares pueden orientar el diseño de mejores metaloproteınas, reduciendo el trabajo experimental. Modelar la función de las metaloproteınas necesita el muestreo de los grados de libertad electrónicos y nucleares para describir exhaustivamente su reactividad química. La combinación de muestreo conformacional y técnicas de química cuántica permite modelar la catálisis de una reacción o como un ligando se une covalentemente a una metaloproteína, sin omitir información sobre la dinámica de la proteína. En esta tesis, estas técnicas computacionales se utilizan sistemáticamente para estudiar y orientar presentes y futuros esfuerzos de diseño de lacasas y hemoglobina. Las lacasas son oxidorreductasas que pueden oxidar una gran variedad de sustratos y reducir oxígeno a agua. Por lo tanto, tienen interés en las aplicaciones de la química verde: funcionan con aire y producen agua como subproducto. Hasta ahora, la mayoría de los esfuerzos realizados para ampliar el espacio químico de lacasas se han centrado en aumentar el potencial redox del primer aceptor de electrones a base de cobre, una elección que obtuvo un éxito limitado. En este trabajo, se propone centrarse en el sustrato deseado, diseñando el sitio activo de las lacasas para mejor enlace y oxidación. Para ello, un protocolo de cálculo fue desarrollado, basado en el muestreo del espacio conformacional de la enzima-sustrato con técnicas Monte Carlo, seguido de cálculos de química cuántica para cuantificar la oxidación. El protocolo fue validado por primera vez contra datos experimentales, lo que probó su capacidad para proporcionar una justificación de las lacasas de funcionar. A continuación se utilizó esta nueva herramienta para mejorar la oxidación de la anilina por una lacasa, simulando el efecto de ciertas mutaciones que se probaron en el laboratorio por nuestros colaboradores. Como resultado, las mutaciones mejoraron significativamente la oxidación de anilina. Otro diseño produjo una mejora significativa en la actividad de otro lacasa hacía tres sustratos diferentes. Por lo tanto, la metodología desarrollada demostró ser útil para diseñar las lacasas hacia un dado sustrato. Un protocolo similar, que utiliza un método de cálculo empírico en lugar de técnicas de química cuántica, se utilizó para unir selectivamente un fotosensibilizador a la superficie de una lacasa. La hemoglobina, una proteína que contiene cuatro grupos hemo para transportar el oxígeno de los pulmones a los tejidos en el cuerpo, es un candidato para el diseño sustituyente de la sangre. Sin embargo, su diseño se ve dificultado por el limitado conocimiento de su funcionamiento. Aquí, la combinación de técnicas Monte Carlo y química cuántica se utilizó para apoyar una teoría sobre el mecanismo alostérico de la hemoglobina y caracterizar estructuralmente los estados finales de la transición alostérica terciaria por primera vez. Estos cálculos, que fueron comparados con los datos experimentales disponibles, permitieron conocer el papel de los aminoácidos próximos al sitio de unión de oxígeno. Esta información se utilizó en un estudio posterior de la dinámica molecular que mostró cómo las cuatro subunidades de la hemoglobina dan lugar a la respuesta alostérica, destacando las rutas de señalización y su jerarquía