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Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / The geometric tortoise is one of the world‟s rarest terrestrial tortoises and is endemic to the Southwestern Cape, South Africa. There has been cause for conservation concern for Psammobates geometricus, yet as is common for many species, quantitative physiological research has been lacking. Considering the important role of red blood cells in oxygen circulation, and the role of white blood cells in immune resistance, blood profiles have been used across taxa as a reliable indicator of health status and physiological processes. Forming part of a larger chelonian conservation programme in South Africa, I studied the haematological changes in P. geometricus, to better understand their physiological responses to changes in climatic conditions. Sampled peripheral blood from males, females and juveniles of the largest known wild geometric tortoise population over four seasons (spring, summer, autumn and winter) from August 2000 to June 2001. Blood samples were used to make smears and determine red cell count (RCC), packed cell volume (PCV), haemoglobi concentration (Hb), red cell indices and differential white cell counts. Digital imaging analysis was used for the histological evaluation of stained blood smears, including descriptions of red and white blood cell morphologies, as well as erythrocyte developmental stages. In the cooler periods, geometric tortoises showed low Hb and mean cell haemoglobin concentration values. Erythrocytes were larger and rounder in winter and spring, which were likely due to hydration states. In addition, increased numbers of immature erythrocytes in circulation suggested an erythropoietic response in winter and spring. This regenerative response is common in reptiles emerging from periods of limited activity and is associated with increasing primary production following rainfall events. In the following summer and autumn, increased mean cell haemoglobin concentrations suggested elevated metabolic rates influenced by rising temperatures. This would seem pertinent to meet the extra physical demands associated with foraging effort in the season characterised with limited water and food supply, and mating behaviour, which occurs in the summer. Low body conditions across all cohorts provided evidence for nutrition stress, while erythrocyte size, shape and degenerative responses indicated dehydration stress. Physiological responses to seasonal influences are specific to growth or reproductive demands and differed for each cohort. Males experienced increased Hb, PCV, RCC, and erythrocyte sizes in summer and autumn, which relate to the erythropoieticstimulating effects of androgens. Female erythropoietic cycles in spring accommodate the increased metabolic demands of increased foraging needed for a larger body size and egg production, and again in autumn again for vitellogenesis. Juvenile tortoises showed minimal differences, and could indicate species-specific responses to environmental changes. A spring-related erythropoiesis was observed in juveniles while during summer and autumn, juveniles showed less evidence for dehydration stress than in adults. No haemoparasites were observed in peripheral blood. Seven leukocyte types were identified and included heterophils, eosinophils, basophils, lymphocytes, plasma cells, monocytes and azurophils, in addition to thrombocytes. Heterophils were the most abundant leukocyte, followed by lymphocytes and eosinophils while monocytes and basophils were equally low; plasma cells and azurophils were rare. Heterophil counts were higher in spring than in summer and autumn, and in summer, were more abundant in females than in juveniles. Eosinophil counts were low in spring for all cohorts, and additionally, female and juvenile counts were low in summer. Eosinophils in juveniles were significantly lower than in adults in winter and spring. Lymphocyte numbers increased in autumn for all cohorts, while summer counts were higher in juveniles than in adults. Basophils and monocytes showed minimal seasonal changes, although basophil counts in females in winter tended to be high. Thrombocytes were lowest in spring for all cohorts. Understanding the physiological responses associated with seasonal changes and for each cohort is critical for effective chelonian conservation management. Results obtained from this study indicate a clinically healthy population of Psammobates geometricus and represented the first of this kind to establish baseline haematological reference data for this Critically Endangered tortoise species.

The purpose of this thesis is to design a physical microchannel model for micro-Couette blood flow that provides constant and controlled conditions to study and analyze Red Blood Cell (RBC) aggregation. The innovation of this work is that the Couette blood flow is created by the motion of a second fluid with different properties, thereby entraining the blood. The experimental work is coupled with three-dimensional numerical simulations performed using a research Computational Fluid Dynamic (CFD) Solver, Nek5000, based on the spectral element method, while the experiments are conducted using a micro Particle Image Velocimetry (μPIV) system with a double frame CCD camera and an inverted laser imaging microscope. The design of the channel (150 × 33 μm and 170 × 64 μm microchannels) is based on several parameters determined numerically, such as the velocity and viscosity ratios and the degree of miscibility between the fluids, and the resulting configurations are fabricated in the laboratory using standard photolithography methods. The microchannel designed numerically is then tested experimentally, first, with a Newtonian fluid (glycerol), then with RBC suspensions to be compared to the simulations results. It was found that, numerically, using a velocity ratio of 4 between the two fluids, a third of the channel thickness corresponds to the blood layer. Within that range, it can be concluded, that the velocity profile of the blood layer is approximately linear as confirmed by experimental tests, resulting in the desired profile to study RBC aggregation in controlled conditions. The effect of several parameters, such as the hematocrit and the shear rate, on the RBC aggregates and the velocity profile is investigated, through experiments on the RBC suspensions. The final goal of this research is to ensure the compatibility of the results between the experiments and the Newtonian numerical model for several ranges of shear rate with the future intention of finding an accurate method to be able to quantitatively analyze aggregates and determine the number of RBC in each aggregate depending on the flow conditions (the shear rate).

Purpose: To investigate the influence of intensity versus total energy expenditure on neutrophilia and blood oxidative stress to acute exercise. Methods: Nine males (18-30 yr) completed one maximal (Max) and three submaximal exercise sessions: 1) 45 min at 10% above (LT+) lactate threshold (LT), 2) 45 min at 10% below (LT-) LT, and 3) 10% below LT until caloric expenditure equaled the 10%+ trial (LT-kcal). Blood was sampled before (PRE), immediately (POST), 1 h, and 2 h after exercise to measure neutrophils, myeloperoxidase, superoxide (O2-), neutrophil activation (O2-/neutrophils), ascorbic acid, uric acid, malondialdehyde, and lipid hydroperoxides. Results: Intensity-dependent neutrophilia occurred POST exercise with significant increases (P ≤ 0.05) after Max and LT+. A second neutrophilia wave occurred 2 h postexercise. Superoxide was elevated POST (Max) and 2 h post (Max and LT+). In contrast, O2-/neutrophils was increased at 2 h only (Max and LT +). These data indicate that immediately postexercise, total neutrophil number rather than activation best represents neutrophil-generated reactive species within blood. POST Max, ascorbic acid and uric acid were decreased indicating a blood oxidative stress occurred. Alternately, total energy expenditure was not related to any marker of neutrophilia or oxidative stress. Conclusion: Exercise intensity plays a major role in postexercise blood oxidative stress, whereas total exercise energy expenditure does not. Further, neutrophils recruited into circulation during exercise may impose a threshold dependent oxidative stress in blood plasma after exercise.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18626号 / 生博第317号 / 新制||生||42(附属図書館) / 31526 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 西田 栄介, 教授 James Hejna / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

Close to one million red blood cell (RBC) units are transfused annually in Canadian hospitals, with surgical inpatients accounting for up to 44% of transfusions. There is evidence of significant variation in transfusion practice in the operating room (i.e., intraoperative). Although variation is expected based on disease severity and patient preference, inappropriate clinical care due to either under- or over-transfusion likely also contributes to significant variation. Indeed, estimates of unwarranted intraoperative RBC transfusions in the literature range from 19% to 49%, owing partly to a lack of evidence-based consensus on RBC transfusion practice in the OR. Our two systematic reviews have highlighted this gap, demonstrating a lack of evidence from trials or actionable clinical practice guidelines to inform decisions in the OR. Perhaps more importantly, avoidance of blood product exposure is an important patient-prioritized outcome that has yet to be studied empirically in the OR. As such, the observed variation in transfusion practice suggests that transfusion decision-making during surgery represents a clear and important knowledge and evidence gap. Transfusion decision-making in the OR is a complex and dynamic process that we cannot begin to improve without first understanding it. It is influenced by 1) physiologic parameters such as acute blood loss, the effects of general anesthesia, and surgical manipulation. Decision-making is also likely heavily influenced by 2) behavioural factors in the OR (heuristics, team dynamics, institutional culture), for which very little empirical work has been conducted. Finally, the importance of 3) patient input in influencing transfusion decisions is inadequately studied, given the documented disconnect between patient priorities and outcomes used in the medical literature and by clinicians. In this context, the aim of my thesis was to develop an empirical understanding of transfusion decision-making in the OR based on stakeholder perceptions and priorities, informed by an integrated patient engagement process. With this work, I address an important knowledge gap in intraoperative blood transfusion, thereby contributing to efforts to reduce variation in blood transfusion practice in surgery. It is my hope that this work will be influential in informing actionable perioperative tools to optimize blood management including providing both evidence and knowledge gaps for future research.

No description available.

Cette thèse traite de la dynamique de cellules sanguines dans la microcirculation. Cette appellation regroupe les deux thématiques de mon travail. La première est l'étude du mouvement de globules rouges soumis à un écoulement de cisaillement. Prenant la suite des travaux réalisés par Manouk Abkarian, Magalie Faivre et Annie Viallat, nous avons étudié le mouvement de cellules dans un flux oscillant et mis en évidence l'apparition de chaos (Dupire J. et al, PRL 104,168101 (2010)). Nous avons ensuite repris l'étude sous écoulement constant pour comprendre les régimes de mouvement encore non étudiés (article accepté à PNAS). Tous ces travaux se basent sur un modèle à forme ellipsoïdale constante (type Keller & Skalak) auquel a été rajouté un terme tenant compte de l'élasticité de la membrane. Pour mieux modéliser la mémoire de forme, nous avons recalculé les équations du modèle en tenant compte d'une nouvelle forme non contrainte du cytosquelette élastique. Elle nous permet entre autres d'ajuster le modèle aux données expérimentales en utilisant des valeurs de viscosité et de module élastique de cisaillement compatibles avec la littérature. Le deuxième partie traite de l'étude du mouvement de globules blancs dans un réseau de canaux microfluidiques. Ce réseau est régulier et possède des dimensions biomimétiques. Nous étudions comment la rhéologie des cellules influe sur leur mouvement à travers le dispositif. Nous montrons que l'entrée des cellules, et donc leur première déformation, peut être utilisée pour obtenir des informations sur leur rhéologie (viscosité, élasticité, tension). / This thesis deals with dynamics of blood cells in microflow. This title regroups two aspects of my work. The first one studies the movement of red blood cells (RBC) under flow. Continuing the work done by M. Abkarian, M. Faivre and A. Viallat, we looked at RBCs in an oscillating shear flow and showed the presence of chaos in the motion (Dupire J. et al, PRL 104,168101 (2010) ). Then we continued the study of RBC under constant flow to understand the regime of motion that were still to elucidate (PNAS, accepted for publication). These works use a ellipsoidal fixed shape model (based on Keller and Skalak's) to which we add an elastic membrane term. To take into account the shape memory, we calculated again the equations of motion considering a new stress-free shape of the elastic cytoskeleton. It allows us to fit the model on the experimental data using viscosity and elasticity coefficient compatible with the litterature. The second part deals with the motion of white blood cell (WBC) in a microfluidic channel network. The device has a regular geometry and has biomimetic shape characteristics matching the human lung mean values. We aim to study how the cell's rheology is related to their motion through the device. We show how the entry of the cell, and thus their first deformation, can be used to obtain information about a single cell rheology (viscosity, elasticity, tension). The motion is then decomposed in 2 phases : a transient regime right after the entrance and a final stationary regime. We study these regimes in terms of cellular deformation and wall friction.

Nous avons étudié les marqueurs hémorhéologiques, hématologiques et biochimiques chez des sujets drépanocytaires homozygotes SS (HbS/HbS) et hétérozygotes composites SC (HbS/HbC) dans deux cohortes, pédiatriques et adultes, de patients drépanocytaires, et ce, à travers 7 complications récurrentes de la drépanocytose : 2 appartenant au profil hémolytique (l’ulcère de jambes et la glomérulopathie) et 5 appartenant au phénotype visqueux/vaso-occlusif (l’hypertension artérielle, le syndrome thoracique aigu (STA), la crise vaso-occlusive (CVO), la rétinopathie et l’ostéonécrose). Nous avons montré que : 1) une viscosité sanguine et une déformabilité érythrocytaire élevées sont des facteurs de risques de CVO chez les enfants homozygotes ; 2) Une viscosité sanguine élevée est associée à une hypertension artérielle systémique relative chez des adultes SS ; 3) les enfants SC présente une fonction vasculaire mieux préservée que les enfants SS pour faire face à une augmentation de la viscosité sanguine ; 4) les patients adultes SS avec une ostéonécrose présentent une déformabilité érythrocytaire plus élevée que les patients sans ostéonécrose ; 5) une viscosité sanguine élevée est associée à la présence d’une rétinopathie chez les adultes SC mais pas chez les SS ; 6) les patients adultes SS présentant une glomérulopathie ont un taux d’hémolyse élevé, une déformabilité érythrocytaire réduite et des agrégats érythrocytaires très robustes ; 7) les patients adultes SS avec des ulcères de jambes récurrents ont un taux d’hémolyse accru et une déformabilité érythrocytaire réduite. De plus, nos travaux confirment que l’-thalassémie module les propriétés de déformabilité érythrocytaire, mais montrent pour la première fois qu’elle module aussi les propriétés d’agrégation érythrocytaire, et notamment la force des agrégats érythrocytaires. En conclusion, ces travaux permettent de préciser le rôle de la rhéologie sanguine dans un certain nombre de complications de la drépanocytose et d’enrichir le modèle préexistant divisant les complications de la drépanocytose selon 2 phénotypes : hémolytique versus visqueux/vaso-occlusif. Nous montrons pour la première fois que le phénotype hémolytique est caractérisé aussi par des anomalies de la rhéologie du globule rouge : rigidité accrue et agrégats érythrocytaire robustes. / Hemorheological, hemathological, and biochemical marquers of patients with sickle cell anemia (SS) and patients with sickle cell SC disease (SC) were studied in 2 cohorts: children and adults. We focused on 7 recurrent complications: 5 belonging to the viscosity/vaso-occlusion phenotype (systemic hypertension, acute chest syndrome (ACS), vaso-occlusive crisis (VOC), retinopathy and osteonecrosis) and 2 belonging to the hemolytic phenotype (leg ulcer and glomerulopathy). Our results show that 1) high viscosity is associated with increased risk for VOC in SS children; 2) blood viscosity is increased in SS adults with systemic relative hypertension; 3) SC children have preserved vascular function compared to SS children; 4) SS adults with osteonecrosis are characterized by higher red blood cell (RBC) deformability than SS adults without osteonecrosis; 5) high blood viscosity is associated with retinopathy in SC adults but not in SS adults; 6) SS adults affected by glomerulopathy have high hemolytic rate, low RBC deformability and increased RBC aggregates strenght; 7) SS adults with recurrent leg ulcers have high hemolytic rate and reduced RBC deformability. Moreover, our studies shows that alpha-thalassemia modulate RBC deformability and RBC aggregation properties. In conclusion, this work shows for the first time that the hemolytic phenotype is characterized by an abnormal RBC rheology which may play a role in several sickle cell complications.