Дисертації з теми "Blood cell imaging"

Fragility fractures represent a socio-economic burden, yet effective systematic treatments for the prevention of osteoporotic fractures are still lacking. Bone is a dynamic and highly vascularised tissue. Evidence suggests that with ageing, low bone mass and disruption of the microstructure and mechanical properties could be driven by reduced vascular supply and blood vessel attracting signals. However, it remains unclear whether alterations in the intracortical vasculature occur with age and if preservation of the cortical vasculature could prevent bone fractures. The main objective of this PhD was to develop and utilise high resolution micro-computed tomography (μCT) imaging ex-vivo of cortical bone microstructure to test the hypothesis that bone vascular networks are altered with age. The following aims have been addressed i) To develop an image processing and analysis framework that allows for systematic measurement of the 3D architecture of cortical porosity comprising vascular canal networks and osteocyte lacunae in murine cortical bone ii) Application and validation of developed methodology to assess the effects of age on vascular canal phenotype iii) Extension of methodology to a unique transgenic mouse strain were osteoblast-derived vascular endothelial growth factor (VEGF) has been deleted iv) Application of phase-contrast enhanced X-ray tomography imaging to visualise soft tissue within vascular canal networks. Using both desktop μCT (1.7 μm) and synchrotron X-ray tomography (0.65 μm), cortical bone microstructure was assessed at a sufficient spatial resolution to detect and extract cortical porosity. Extracted porosity measurements from synchrotron X-ray tomography were classified into vascular canals and osteocyte lacunae and 3D spatial relationships computed. First, the tibiofibular junction from 15-week and 10-month-old female C57BL/6J mice (n=6) was selected and vascular networks compared. It was found that the posterior region of the tibiofibular junction had a higher vascular canal volume than the anterior, lateral and medial regions (+127.62%, 693.08% and 659.64% respectively, p < 0.05) at 15 weeks of age. By 10 months, bone cortices were thinner (-13.04%, p < 0.01) and reduction in vascular density was evident in the posterior region (-46.54%, p < 0.01) providing the first evidence for location of the intracortical vasculature impacting age related effects on bone porosity. To explore the effect of osteoblast-derived VEGF on the intracortical microstructure, VEGF was knocked out (KO) in mature osteocalcin (Ocn) expressing osteoblasts. Again, the tibiofibular junctions of aged (1 year) wildtype (WT) and transgenic (VEGF Ocn KO) female mice (n=5) were imaged, analysed and compared. Results revealed that the lack of osteoblast-derived VEGF increased total porosity (vascular networks and osteocyte lacunae combined) in the tibiofibular junction (+39.92%, p < 0.01) with changes evident in the anterior and posterior compartments. Attempts to extend the use of the developed methodology to separate osteocyte lacunae from vascular canals was unsuccessful in the VEGF Ocn KO model due to low mineralised matrix in the VEGF Ocn KO bones. Finally, an approach that allows the 3D visualisation and assessment of the soft tissues in calcified bone using phase contrast-enhanced X-ray tomography has been reported. Using this technique, vascular structures were detected within 95.77% of the intracortical canals of the murine tibiofibular junction, supporting the theory that the intracortical network is the living space of the bone vasculature. This project has developed novel methodology which has allowed demonstration of an age-related reduction in the intracortical vasculature associated with reduced cortical bone thickness and deleterious changes in bone porosity due to the lack of VEGF, thus supporting further investigations into targeting the blood supply to treat age-related bone disease.

Peripheral artery disease (PAD) affects more than 200 million people worldwide. PAD refers to illness due to a reduction or complete occlusion of blood flow in the artery, especially to the extremities in disease conditions, such as atherosclerosis or diabetes. Critical limb ischemia (CLI) is a severe form of PAD associated with high morbidity and mortality. Currently, no effective and permanent treatments are available for this disease. The current endovascular medications (e.g., angioplasty or stents) only relieve the clinical symptoms while the surgical therapies (e.g., bypass or endarterectomy) require grafting vessels from a healthy organ to the diseased limb of the patient. However, even with these therapeutic techniques, 30% of patients still undergo limb amputation within a year. Thus, understanding of disease mechanism and development of new therapeutic approaches are in urgent needs. Meis1 (myeloid ecotropic viral integration site 1) gene belongs to the three-amino-acid loop extension subclass of homeobox gene families, and it is a highly conserved transcription factor in all eukaryotes. Up to date, little is known about the role of Meis1 in regulating vascular remodeling under ischemic condition. In this study, we aim to investigate the role and underlying mechanism of Meis1 in the regulation of arteriogenesis and angiogenesis using hindlimb ischemia model of transgenic neonatal mice. The long-term goal is to develop a new treatment for patients with PAD. Three separate but related studies were planned to complete the proposed research aims. To better understand the role of Meis1, we reviewed, in the first chapter, all literature relevant to the recent advances of the Meis1 in normal hematopoiesis, vasculogenesis, and heart developments, which were mostly studied in zebrafish and mouse. Briefly, Meis1 is found to be highly expressed in the brain and retina in zebrafish and additional in the heart, nose, and limb in mouse during the very early developmental stage, and remains at a low level quickly after birth. Meis1 is necessary for both primitive and definitive hematopoiesis and required for posterior erythroid differentiation. The absence of Meis1 results in a severe reduction of the number of mature erythrocytes and weakens the heart beats in zebrafish. Meis1 deficiency mouse is dead as early as E11.5 due to the severe internal hemorrhage. In addition, Meis1 is essential in heart development. Knock-down of Meis1 can promote angiotensin II-induced cardiomyocytes (CMs) hypertrophy or CMs proliferation, which can be repressed by a transcription factor Tbx20. Meis1 appears to play a complicated role in the blood vessels. Although the major blood vessels are still normal when global deletion of Meis1, the intersegmental vessel cannot be formed in Meis1 morphants in the zebrafish, and the small vessels are either too narrow or form larger sinuses in Meis1 deficient mouse. The effects of Meis1 on the vascular network under normal and disease (ischemia) condition remain largely unknown, and the existing data in this field is limited. In the second chapter, we developed a method protocol to identify mice of all ages, especially neonates that we faced methodological difficulties to easily and permanently label prior to our major experiments. In this study, single- or 2-color tattooing (ear, tail, or toe or combinations) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day (PND) 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than three weeks. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. Thus, tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals, which shows a new approach than any existing method to identify mouse at all ages, especially the neonatal pups used in the present study (Chapter 4). Various formation of hindlimb ischemia with ligations of femoral artery or vein or both have been reported in the literature. The ischemic severity varies dependent on mouse strains and ligation methods. Due to the tiny body size of our experimental neonatal mice (PND2), it is technically challenging to separate the femoral artery from femoral vein without potential bleeding. In the third chapter, we aimed to explore a suitable surgical approach that can apply to neonatal mice. To this end, we compared the effects of femoral artery/vein (FAV) excision vs. femoral artery (FA) excision on hindlimb model using adult CD-1 mice. We showed during the 4-week period of blood reperfusion, no statistically significant differences were found between FAV and FA excision-induced ischemia regarding the reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell size. We conclude that FAV and FA excision in CD-1 mice generate a comparable severity of hindlimb ischemia. In other words, FAV ligation is no more severe than FA ligation. These findings provide valuable information for researchers when selecting ligation methods for their neonate hindlimb models. Based on these findings, we selected FAV ligation of hindlimb ischemia approach to study the function of Meis1 in vascular remodeling of neonatal mice. In the fourth chapter (the main part of my dissertation), we investigated the roles of Meis1 in regulating arteriogenesis and angiogenesis of neonatal mouse under the ischemic condition. To this end, endothelial cell-specific deletion of Meis1 was generated by cross-breeding Meis1flox/flox mice with Tie2-Cre mice. Wild-type (WT, Meis1f/f) and endothelial cell-specific knock-out (KO, Meis1ec-/-Tie2-Cre+) C57BL/6 mice at the age of PND2 were used. Under the anesthesia, the pups were subject to hindlimb ischemia by excising FAV. Laser Doppler Imager was used to measure the blood flow pre- and post-surgery up to 28 days. Toe necrosis, skeletal regeneration, and vascular distributions were examined at the end of experiments (PND28 post-ischemia). Surprisingly, during 4-week periods after ischemia, the blood flow ratios (ischemic vs. control limb) in KO mice significantly increased compared to WT on PND14 and PND28, suggesting the inhibitory effects of Meis1 on blood flow recovery under ischemic condition. Meanwhile, WT mice showed more severe necrotic limb (lower ratio of limb length and area, and higher necrotic scores at PND7) than those in the KO mice. Furthermore, significant increases in diameters of Dil-stained arterioles of the skin vessel and the vessels on the ligation site were observed in KO mice, indicating the enhanced arteriogenesis in KO mice. To investigate the underlying mechanism, RNA from the ischemia and control limb was extracted and q-PCR was used to study the potential genes involved in the mechanism. Casp3 and Casp8 were found downregulated showing less apoptosis in the KO mice. On the other hand, endothelial cells (ECs) were isolated from the lungs of 3-5 WT and KO neonates using CD31 Microbeads. CD31+ cells were plated and treated with 0, 0.5, and 1μM doxorubicin for 24 hours and analyzed with various assays. Meis1-KO ECs demonstrated higher cell viability and formed a higher number of vascular tubes than those in WT ECs following 0.5μM Dox treatment, presenting the potential ability of angiogenesis in KO-ECs. Furthermore, the increased viability in KO ECs may be due to the decreased expression or activities of Casp8 and Casp3. In conclusion, my present studies have developed a new methodology to easily and permanently identify all mice at any ages. The insignificant differences between FAV and FA ligations suggest that a relative-easy surgical approach could be used to generate hindlimb ischemic model, which potentially reduces the cost, decreases the surgical time and prevents damage of femoral nerve from surgical tools. More importantly, by using transgenic mice, we found that Meis1-KO dramatically increased blood flow and protected the ischemic hindlimb through vascular remodeling. Obviously, the molecular and cellular mechanisms underlying the above beneficial effects appear complicated and likely to involve multiple cellular remodeling processes and molecular signaling pathways to enhance arteriogenesis and angiogenesis and/or reduce cellular apoptosis through Meis1-mediated pathways. Our study demonstrated that under ischemic condition, knockout of Meis1 increases expression of Hif1a, which then activates Agt or VEGF, thus enhances arteriogenesis or angiogenesis; In addition, knockout of Meis1 activates Ccnd1, which subsequently promotes regeneration of skeletal muscle, and reduces expression of Casp8 and Casp3, thus preventing limb tissue from ischemia-induced apoptosis. Our innovative findings offer great potential to ultimately lead to new drug discovery or therapeutic approaches for prevention or treatment of PAD.
PHD

Les techniques quantitatives ultrasonores basées sur la paramétrisation du coefficient de rétrodiffusion (BSC) supposent généralement que le tissu étudié est homogène et isotrope. Cependant, certains tissus tels que les agrégats des globules rouges, le myocarde ou les muscles du biceps présentent des propriétés acoustiques dépendantes de l'angle (BSC et/ou coefficient d'atténuation). L'objectif de cette thèse était d'intégrer l'anisotropie dans l'analyse de la rétrodiffusion tissulaire. Dans un premier temps, un modèle de rétrodiffusion ultrasonore est proposé et évalué numériquement pour caractériser les structures anisotropes des agrégats des globules rouges. Ensuite, une procédure expérimentale a été évaluée pour mesurer l'anisotropie du BSC dans des fantômes des tissus. Plus précisément, une comparaison des performances est effectuée entre une sonde à transducteur ultrasonore micro-usiné capacitif et une sonde piézoélectrique commerciale pour mesurer l'anisotropie de rétrodiffusion en utilisant la stratégie d'imagerie par orientation du faisceau focalisé
Quantitative ultrasound techniques based on the parameterization of the backscatter coefficient (BSC) generally assume that the tissue under investigation is homogeneous and isotropic. However, some tissues such as flowing red blood cell (RBC) aggregates, myocardium or bicep muscles exhibit angle-dependent acoustic properties (BSC and/or attenuation coefficient). The objective of this thesis was to incorporate anisotropy in tissue backscatter analysis. First, an ultrasonic backscattering model is proposed and evaluated numerically to characterize the anisotropic structures of RBC aggregates. Then, an experimental procedure was evaluated to measure BSC anisotropy in tissue-mimicking phantoms. More specifically, a performance comparison is carried out between a capacitive micromachined ultrasonic transducer probe and a commercial piezoelectric probe to measure backscatter anisotropy by using the focused beam steering imaging strategy

I am reporting on lensless imaging of human red blood cell using coherent x-ray scattering (CXS) technique. The successful microfabrication of a sample-mask structure using focused ion beam (FIB) milling was the key element in this imaging technique. The sample-mask structure is 600 - 800 nm gold films deposited using sputtering or electron beam evaporation on Si₃N₄ membrane windows. We used commercially available 100 nm thick Si₃N₄ membranes held by 3 mm diameter silicon frames that are designed for use in transmission electron microscopy. The red blood cell (RBC) sample was mounted in front of a 3 μm hole milled through both gold and Si₃N₄ layers on the opposite side. Three smaller reference apertures with diameters 300, 250 and 200 nm on the gold side were milled all the way through both layers at a distance of 9 am center-to center from the sample aperture. These holes are used for holographic lensless x-ray imaging. It was found that a gold surface roughens during ion milling due to a sputter instability and which produces cup-like features with a characteristic length up to few hundred nm. We found apertures milled through gold films deposited by sputtering show good circularity and sidewall roughness of 20 nm. We present result on CXS measurements in transmission geometry near Fe L₃₋ and C K-absorption edges on a single RBC. We captured high resolution images of the sample by simple Fourier inversion of the recorded far-field scattered intensity. We found 8.5 % reduction in the transmission intensity near Fe L₃₋edge due to presence of Fe in the form of hemoglobin molecules inside RBC. This absorption agrees with estimated agrees well with the estimated value of 9 % within experimental uncertainty. From limited data measured below C K-edge we measured a presence of at least 300 nm thick carbon inside RBC which lies in the range of the estimated value of 1.8 μm. The resolution of our lensless imaging technique is about 55 nm near Fe L₃₋edge and 78 nm near C K-edge.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

L'encéphalopathie associée au sepsis (EAS) est définie comme un dysfonctionnement cérébral diffus induit par une réponse systémique à une infection. Chez les patients septiques, l'imagerie par résonance magnétique (IRM) a indiqué à la fois des anomalies de la substance grise (SG) et blanche (SB) associées à des troubles cognitifs graves, y compris le delirium. Pour améliorer notre compréhension des changements hémodynamiques, métaboliques et structuraux associés au sepsis, différentes séquences d'IRM ont été réalisées chez des rats ayant subi une injection ip de solution saline ou de lipopolysaccharide bactérien (LPS) 2,5h plus tôt ou une ligature et ponction caecale 24h plus tôt. Après ip LPS, l'IRM de contraste de phase a été réalisée pour étudier le flux des artères cérébrales antérieures et moyennes et le marquage des spins artériels (ASL) pour étudier la perfusion des structures cérébrales de la SB et SG. Des séquences d'imagerie par diffusion pondérée (DWI) ont été utilisées pour évaluer les changements structurels. Après la chirurgie CLP, ASL a été utilisé pour étudier les changements de la microcirculation. L'imagerie pondérée en T2, l'imagerie du tenseur de diffusion (DTI) et les statistiques spatiales basées sur les faisceaux (TBSS) ont été réalisées pour caractériser les événements structurels dans différentes structures cérébrales. Après imagerie, les animaux ont été sacrifiés et leur cerveau a été traité pour l'histologie afin de détecter l'enzyme synthétisant les prostaglandines vasoactives cyclooxygénase-2 (COX-2) et le canal hydrique astrocytaire aquaporin-4 (AQP4) dont l'expression peut être régulée à la hausse, évaluer la présence d'immunoglobulines périvasculaires (Ig) indiquant une rupture de la barrière hémato-encéphalique (BHE) et étudier la morphologie des glies puisque la microglie et l’astroglie changent de morphologie lors des conditions inflammatoires. L'IRM n'a indiqué aucun changement hémodynamique dans la substance grise après l'administration de ip LPS, alors qu'une perfusion cérébrale accrue a été montrée au niveau du corps calleux comme indiqué par l'ASL. DTI a indiqué une augmentation de la diffusion des molécules d’eau parallèlement aux fibres du corps calleux. Ces changements étaient accompagnés d'une dégradation de BHE dans la SB ainsi que la substance grise corticale et striatale adjacente tel est indiqué par la présence périvasculaire d'IgG, sans aucun changement majeur de COX-2 vasculaire ou de morphologie des glies du coprs calleux. Le dysfonctionnement du SNC induit par le sepsis a résulté en une augmentation du contraste pondéré en T2 dans le cortex, le striatum et la base du cerveau, une diminution de la perfusion sanguine dans le cortex et une augmentation de la diffusion hydrique du corps calleux et du striatum ventral. Ces changements ont été associés dans la SB à des modifications de la morphologie des glies et dans la substance grise à une expression constitutive de COX-2 et AQP4 plus faible dans le cortex cérébral. La comparaison entre CLP ayant subit ou non une IRM sous anesthésie à l'isoflurane a montré une réponse inflammatoire réduite tel est indiqué par l'expression de COX- 2, une activation réduite des glies ainsi qu’une lésion réduite de la BHE dans le CLP subissant une IRM sous anesthésie. Collectivement, nos résultats suggèrent que des changements hémodynamiques peuvent survenir en l'absence de flux altéré dans les artères irriguant le cerveau antérieur. Ensuite, l'altération de la structure de la SB est une étape précoce de la pathogenèse de l’EAS qui peut résulter soit de la dégradation de la BHE, soit de l'activation des glies. Cette étude sous-tend l'effet délétère d'une seule exposition à l'anesthésie à l'isoflurane qui peut être atténuée par une seconde exposition chez les rats ayant subi une laparotomie ainsi que les effets de l'inflammation systémique induite par le CLP sur les glies pouvant être atténués par imagerie sous anesthésie à l'isoflurane
Sepsis-associated encephalopathy (SAE) refers to central nervous system dysfunction during the systemic inflammatory response to infection. In septic patients with encephalopathy MRI has indicated both gray and white matter abnormalities that were associated with worse cognitive outcome including delirium. To improve our understanding of sepsis-associated hemodynamic, metabolic, and structural changes, different MRI sequences were performed in rats that either underwent an i.p injection of saline or bacterial lipopolysaccharide (LPS) 2.5h earlier or cecal ligation and puncture (CLP) 24h earlier. After ip LPS, phase contrast MRI was performed to study anterior and middle cerebral arteries flow and Arterial Spin Labeling (ASL) to study perfusion of white and grey matter brain structures. Diffusion Weighted Imaging (DWI) sequences was used to assess structural changes. After CLP surgery, ASL was used to study microcirculation changes. T2-Weighted Imaging, Diffusion Tensor Imaging (DTI) and tract-based spatial statistics (TBSS) were performed to characterize structural events in different brain structures. After imaging, animals were sacrificed and their brains processed for histology to detect the vasoactive prostaglandin-synthesizing enzyme cyclooxygenase-2 (COX-2) and the astrocytic aquaporin-4 water channel (AQP4) the expression of which can be upregulated during inflammation, to assess the presence of perivascular immunoglobulins (Ig) indicating blood-brain barrier (BBB) leakage and to study glia cell morphology as both microglia and astrocytes are known to change their morphology in inflammatory conditions. Magnetic resonance rat brain imaging indicated no hemodynamic changes in the grey matter after ip LPS administration while an increased CBF was shown in corpus callosum white matter as indicated by ASL. DTI indicated increased water diffusion parallel to fibers of the corpus callosum white matter. These changes were accompanied by BBB breakdown in the white matter and adjacent cortical and striatal grey matter as indicated by the perivascular presence of IgG, but no major changes in vascular COX-2 or white matter glia cell morphology. CLP induced sepsis-associated CNS dysfunction resulted in higher T2-weighted contrast intensities in the cortex, striatum and base of the brain, decreased blood perfusion distribution to the cortex and increased water diffusion in the corpus callosum and ventral striatum compared to sham surgery. These changes were associated in the white matter with modifications in glia cells morphology and in the grey matter with lower expression of constitutive COX-2 expression and AQP4 in the cerebral cortex. The comparison between CLP that underwent or not MRI under isoflurane anesthesia indicated reduced inflammatory response as indicated by COX-2 expression, reduced glia activation and reduced BBB damage in CLP that underwent MRI under isoflurane anesthesia. Collectively, our results suggest that hemodynamic changes may occur in the absence of altered flow in forebrain irrigating arteries. Then, altered white matter structure is an early step in SAE pathogenesis that may result either from BBB breakdown or glial cells activation. This study underlies the deleterious effects of a single exposure to isoflurane anesthesia that may be mitigated by a second exposure in sham-operated rats and the effects of CLP-induced systemic inflammation on glial cells that can be attenuated by imaging under isoflurane anesthesia

A contagem de células vermelhas do sangue desempenha um papel importante no diagnóstico de animais silvestres e domésticos. Apesar da existência de muitas tecnologias em diferentes contadores automatizados para análise de sangue, quando se trata do sangue de animais silvestres ainda é difícil encontrar uma solução simples e economicamente viável para múltiplas espécies. O objetivo deste estudo é desenvolver um contador automático de células vermelhas. Amostras de sangue (1 jaguatirica - Leopardus pardalis, 1 macaco - Cebus apella, 1 quati - Nasua nasua, 62 cães - Canis familiaris e 5 cavalos - Equus caballus) foram analisadas usando três métodos: 1-contagem manual, 2-contagem automática por imagem e 3-contagem semiautomática por imagem; as amostras de cães e cavalos foram analisadas por um quarto método: 4-contagem automática por impedância. As contagens dos métodos 2 e 3 foram obtidas usando o contador de células vermelhas proposto. Os resultados foram comparados usando a correlação de Pearson e gráficos com diferentes métodos como valor de referência. As contagens dos métodos 1, 2 e 3 correlacionaram muito bem com as contagens do método 4 (r ≥ 0.94). As contagens produzidas pelo método 2 apresentaram alta correlação com o método 3 (r = 0.998). Os resultados indicam que o contador proposto pode ser usado como um método de contagem automática ou semiautomática em clínicas que usam o método manual para contagem de células vermelhas do sangue de animais.
A RBC count plays an important role in the diagnostic of wild and domestic animals. Despite the many technologies available in different automated hematology analyzers, when it comes to blood of wild animals it is still difficult to find an easy and affordable solution for multiple species. This study aims to develop an automatic red blood cell counter. Blood samples (1 ocelot - Leopardus pardalis, 1 monkey - Cebus apella, 1 coati - Nasua nasua, 62 dogs - Canis familiaris and 5 horses - Equus caballus) were analyzed using three methods: 1-manual count, 2automatic count by image and 3-semi-automatic count by image; blood from dogs and horses were also analyzed by a fourth method: 4-automatic count by impedance. The counts of methods 2 and 3 were produced by the proposed red blood cell counter. Results were compared using Pearson’s correlation and plots with different methods as the criterion standard. RBC counts of methods 1, 2 and 3 correlated very well with those on the method 4 (r ≥ 0.94). RBC counts produced by method 2 were highly correlated with method 3 (r = 0.998). The results indicate that the proposed method can be used as an automatic or semi-automatic counting method in clinics that are currently using the manual method for RBC assessment.

During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.

Cardiovascular disease is the leading cause of death in the United States, and coronary artery disease (CAD) kills over 370,000 people annually. There are available therapies that offer a temporary solution; however, only a heart transplant can fully resolve heart failure, and donor organ shortages severely limit this therapy. C-kit+ human cardiac stem cells (hCSCs) offers a viable alternative therapy to treat cardiovascular disease by replacing damaged cardiac tissue; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation has limited the efficacy of stem cell therapy. Tissue engineering solutions offer potential tools to overcome current limitations of stem cell therapy. Materials derived from natural sources such as keratin from human hair offers innate cellular compatibility, bioactivity, and low immunogenicity. Keratin proteins extracted using oxidative chemistry known as keratose (KOS) have shown therapeutic potential in a wide range of applications including cardiac regeneration. My studies utilize KOS hydrogels to modulate c-kit+ hCSC differentiation, and explore the capability of differentiated cells to regenerate vascular tissue. In the first Chapter, we reviewed literature relevant to keratin-based biomaterials and their biomedical applications, the use of stem cells in cardiovascular research, and the differentiation of vascular smooth muscle cells (VSMCs). The section on biomedical applications of keratin biomaterials focuses on the oxidized form of keratin known as keratose (KOS), because this was the material used for our research. Since we planned to use this material in conjunction with c-kit+ hCSCs, we also briefly explored the use of stem cells in cardiovascular research. Additionally, we examined some key signaling pathways, developmental origins, and the cell phenotype of VSMCs for reasons that will become clear after observing results from chapters 2 and 3. Based upon our review of the literature, KOS biomaterials and c-kit+ hCSCs were determined to be promising as a combined therapeutic for the regeneration of cardiac tissue. Research in Chapter 2 focused on characterizing the effects of KOS hydrogel on c-kit+ hCSC cell viability, proliferation, morphology, and differentiation. Results demonstrated that KOS hydrogels could maintain hCSC viability without any observable toxic effects, but it modulated cell size, proliferation, and differentiation compared to standard tissue culture polystyrene cell culture (TCPS). KOS hydrogel produced gene and protein expression consistent with a VSMC phenotype. Further, we also observed novel "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. Results from this study lead us to question what signaling pathways might be responsible for the apparent VSMC differentiation pattern we observed on KOS hydrogels. Research in Chapter 3 explored the time course of VSMC differentiation, cell contractility, inhibition of VSMC differentiation, and measured protein expression of transforming growth factor beta 1 (TGF-β1) and its associated peptides for hCSCs cultured on KOS hydrogels, tissue culture polystyrene, and collagen hydrogels. A review of VSMC differentiation signaling pathways informed our decision to investigate the role of TGF-β1 in VSMC differentiation. Results demonstrated that KOS hydrogel differentiated hCSCs significantly increased expression for all three vascular smooth muscle (VSM) markers compared to TCPS differentiated cells. Additionally, KOS differentiated hCSCs were significantly more contractile than cells differentiated on TCPS. Recombinant human (rh) TGF-β1 was able to induce VSM differentiation on TCPS. VSM differentiation was successfully inhibited using TGF-β NABs and A83-01. Enzyme-Linked Immunosorbent Assay (ELISA) analysis revealed that both TCPS and KOS hydrogel differentiated cells produced TGF-β1, with higher levels being measured at early time points on TCPS and later time points on KOS hydrogels. Results from supplementing rhTGF-β1 to TCPS and KOS hydrogels revealed that KOS seems to interact with TGF-β to a greater extent than TCPS. Western blot results revealed that latency TGFβ binding protein (LTBP-1) and latency associated peptide (LAP) had elevated levels early during differentiation. Further, the levels of LTBP-1 and LAP were higher on KOS differentiated hCSCs than TCPS hCSCs. This study reaffirms previous results of a VSM phenotype observed on KOS hydrogels, and provides convincing evidence for TGF-β1 inducing VSM differentiation on KOS hydrogels. Additionally, results from ELISA and western blot provide evidence that KOS plays a direct role in this pathway via interactions with TGF-β]1 and its associated proteins LTBP-1 and LAP. Results from chapter 2 and 3 offered significant evidence that our cells exhibited a VSMC phenotype, and that TGF-β1 signaling was a key contributor for the observed phenotype, but we still needed an animal model to explore the therapeutic potential of our putative VSMCs. Research in Chapter 4 investigated a disease model to test the ability of KOS hydrogel differentiated cells to regenerate vascular tissue. To measure vascular regenerative capability, we selected a murine model of critical limb ischemia (CLI). CLI was induced in 3 groups (n=15/group) of adult mixed gender NSG mice by excising the femoral artery and vein, and then treated the mice with either PBS (termed as PBS-treated), Cells differentiated on TCPS (termed as Cells from TCPS), or KOS hydrogel-derived VSMCs (termed as Cells from KOS). Blood perfusion of the hind limbs was measured immediately before and after surgery, then 14, and 28 days after surgery using Laser Doppler analysis. Tissue vascularization, cell engraftment, and skeletal muscle regeneration were measured using immunohistochemistry, 1,1'-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiL) vessel painting, and hematoxylin and eosin (HandE) pathohistological staining. During the 4-week period, both cell treatment groups showed significant increases in blood perfusion compared to the PBS-treated control, and at day 28 the Cells from KOS group had significantly better blood flow than the Cells from TCPS group. Additionally, the Cells from KOS group demonstrated a significant increase in the ratio of DiL positive vessels, capillary density, and a greater density of small diameter arterioles compared to the PBS-treated group. Further, both cell-treated groups had similar levels of engraftment into the host tissue. We conclude that Cells from KOS therapy increases blood perfusion in an NSG model of CLI, but does not lead to increased cell engraftment compared to other cell based therapies. Overall, the results from this dissertation demonstrated that KOS hydrogels produce VSMC differentiation from c-kit+ hCSCs mediated by TGF-β1 signaling, and that the differentiated cells are able to increase blood perfusion in a CLI model by increasing capillary density, suggesting enhanced angiogenesis. Future studies should explore potential protein-protein interactions between KOS, TGF-β1 and its associated proteins. Additionally, we should plan animal studies that examine the efficacy of our cells to regenerate cardiac tissue following an acute myocardial infarction (AMI).
PHD

Traumatic brain injury (TBI) is a leading cause of disability worldwide. Annually, 150 to 200/1,000,000 people become disabled as a result of brain trauma. Axonal degeneration is a critical, early event following TBI of all severities but whether axon degeneration is a driver of TBI remains unclear. Molecular pathways underlying the pathology of TBI have not been defined and there is no efficacious treatment for TBI. Despite this significant societal impact, surprisingly little is known about the molecular mechanisms that actively drive axon degeneration in any context and particularly following TBI. Although severe brain injury may cause immediate disruption of axons (primary axotomy), it is now recognized that the most frequent form of traumatic axonal injury (TAI) is mediated by a cascade of events that ultimately result in secondary axonal disconnection (secondary axotomy) within hours to days. Proposed mechanisms include immediate post-traumatic cytoskeletal destabilization as a direct result of mechanical breakage of microtubules, as well as catastrophic local calcium dysregulation resulting in microtubule depolymerization, impaired axonal transport, unmitigated accumulation of cargoes, local axonal swelling, and finally disconnection. The portion of the axon that is distal to the axotomy site remains initially morphologically intact. However, it undergoes sudden rapid fragmentation along its full distal length ~72 h after the original axotomy, a process termed Wallerian degeneration. Remarkably, mice mutant for the Wallerian degeneration slow (Wlds) protein exhibit ~tenfold (for 2–3 weeks) suppressed Wallerian degeneration. Yet, pharmacological replication of the Wlds mechanism has proven difficult. Further, no one has studied whether Wlds protects from TAI. Lastly, owing to Wlds presumed gain-of-function and its absence in wild-type animals, direct evidence in support of a putative endogenous axon death signaling pathway is lacking, which is critical to identify original treatment targets and the development of viable therapeutic approaches. Novel insight into the pathophysiology of Wallerian degeneration was gained by the discovery that mutant Drosophila flies lacking dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously recapitulated the Wlds phenotype. The pro-degenerative function of the dSarm gene (and its mouse homolog Sarm1) is widespread in mammals as shown by in vitro protection of superior cervical ganglion, dorsal root ganglion, and cortical neuron axons, as well as remarkable in-vivo long-term survival (>2 weeks) of transected sciatic mouse Sarm1 null axons. Although the molecular mechanism of function remains to be clarified, its discovery provides direct evidence that Sarm1 is the first endogenous gene required for Wallerian degeneration, driving a highly conserved genetic axon death program. The central goals of this thesis were to determine (1) whether post-traumatic axonal integrity is preserved in mice lacking Sarm1, and (2) whether loss of Sarm1 is associated with improved functional outcome after TBI. I show that mice lacking the mouse Toll receptor adaptor Sarm1 gene demonstrate multiple improved TBI-associated phenotypes after injury in a closed-head mild TBI model. Sarm1-/- mice developed fewer beta amyloid precursor protein (βAPP) aggregates in axons of the corpus callosum after TBI as compared to Sarm1+/+ mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phosphorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after TBI. Strikingly, whereas wild type mice exhibited a number of behavioral deficits after TBI, I observed a strong, early preservation of neurological function in Sarm1-/- animals. Finally, using in vivo proton magnetic resonance spectroscopy, I found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1-/- mice compared to controls immediately following TBI. My results indicate that the Sarm1-mediated prodegenerative pathway promotes pathogenesis in TBI and suggest that anti-Sarm1 therapeutics are a viable approach for preserving neurological function after TBI.

’arthropathie dégénérative est une maladie ayant des répercussions socio-économiques majeures chez l’homme et le cheval. Il n’existe pour l’heure aucun traitement curatif de cette maladie, le cartilage articulaire étant dépourvu de pouvoir de cicatrisation spontané. De nombreux espoirs reposent sur l’utilisation de cellules souches mésenchymateuses (CSM), pour leur potentiel pro-régénératif et anti-inflammatoire. Le premier objectif de cette étude était d’évaluer la tolérance des CSM de sang de cordon ombilical (SCO) et de moelle osseuse (MO) dans des articulations saines. L’étude contrôlée en aveugle menée sur 12 chevaux expérimentaux a démontré que l’injection de CSM-MO provoquait significativement plus de signes de réaction inflammatoire que l’injection de CSM-SCO, et que l’injection des CSM, quelle que soit leur origine, provoquait une réaction inflammatoire discrète à modérée, supérieure à celle d’une injection de placébo, avec une grande variabilité individuelle de sensibilité à une même lignée de cellules. Le second objectif était d’évaluer l’efficacité des CSM-MO et -SCO dans un modèle d’arthropathie induite. L’étude contrôlée en aveugle menée sur 8 chevaux expérimentaux a mis en évidence une réduction significative de la progression des signes indicateurs d’arthropathie à l’imagerie après injection de CSM-MO allogéniques par rapport à l’injection du placébo. Ces résultats encourageants, à considérer à la lumière des limites des études menées, indiquent un effet bénéfique des CSM-MO allogéniques dans la prise en charge de l’arthrose chez le cheval. Ils soulignent la nécessité de poursuivre les recherches afin de confirmer ces résultats, et d’optimiser les effets des CSM à travers leur combinaison à un vecteur ou par une approche acellulaire avec administration des nanovésicules qu’elles sécrètent, et considérées être à l’origine de leurs effets thérapeutiques
Osteoarthritis is a common cause of pain and economic loss in both humans and horses. There is currently no curative treatment for osteoarthritis, because of the lack of spontaneous regenerative capacity of the articular cartilage. Mesenchymal stem cells (MSC) based regenerative medicine comes across as a promising strategy given their pro-regenerative and anti-inflammatory potential. The first objective of this study was to evaluate the safety of umbilical cord blood (UCB) and bone marrow (BM) derived MSC in healthy joints. The blind controlled study conducted on 12 experimental horses showed that the injection of BM-MSC caused significantly more signs of inflammatory reaction than the injection of UCB-MSC, and that the injection of MSC, regardless of their origin, caused a discrete to moderate inflammatory reaction, greater than that of the placebo, with great individual variability in sensitivity to the same cell line. The second objective was to evaluate the efficacy of BM-MSC and UCB-MSC in a model of induced osteoarthritis. The blind controlled study conducted on 8 experimental horses showed a significant reduction in the progression of osteoarthritis associated signs with imaging techniques after injection of allogeneic BM-MSC compared to placebo. These promising results, to be considered in light of the limitations of the studies, indicate a beneficial effect of allogeneic BM-MSC in the management of osteoarthritis in horses. They underline the need for further research to confirm these results, and to optimize the effects of MSC through their combination with a vector or through an acellular approach with administration of the nanovesicles they secrete that ared considered to be responsible for their therapeutic effects

Point-of-care Diagnostics (POCD) is one of the rapidly growing areas of health-care sector that avails to the needs of the patient at the point-of-care. An ideal POCD device is required to be compact, portable, offer quick results, require no or minimal sample preparation, and inexpensive with a low cost per test. Micro fluidics has a potential to cater to these needs, thereby leading to a growing research interest to develop micro uidic POCD (POCD) devices. POCD devices can be broadly categorized into cellular diagnostics and non-cellular diagnostics. Micro fluidic flow cytometers (imaging and laser based) are the emerging diagnostics tools for biological cell identi cation, categorization, and counting. Despite the advances in this area these flow cytometers have not yet been turned into POCD devices. This thesis focuses on finding solutions for the major problems associated with these micro fluidic flow cytometers towards becoming POCD devices. However, the developed techniques are quite general and can be equally well applied for non-cellular diagnostics as well. More specfi cally this thesis presents techniques for focusing of cells in flow, in- flow decantation, and pumping along with the experimental demonstration of these techniques in the context of deformability estimation of cells in ow, blood cell counting, and quantitative microscopic urinalysis. Focusing of the cells while in flow is at the heart of the operation of flow cytometers. The developed technique reduces the complexity of fabrication and offers its applicability for a wide range of flow rates, thereby decreasing the cost per device and simultaneously offering the flexibility of its use in both imaging and laser-based flow cytometers. The in- flow decantation technique adds an extra dimension to the possibility of realizing sheath-free ow focusing, by separating the particle-free fluid from the sample itself. The simplicity of the design and the applicability of the technique for wide varieties of ow rates, particle concentrations, and sizes while having the ability to offer 100 % purity at high yields, offers its potential applications into the realization of POCD that can operate on plasma separated from whole blood for several biochemical assays, rare cell enrichment in cancer diagnostics, immunodiagnostics etc. Pumping is an unavoidable task to cause ow of either sample or sheath fluids into the micro fluidic device. The developed pump uses the mechanical energy from the fingers and stores inside an elastic block to cause pumping action when released subsequently. The pumping mechanism is very general and is independent of the type of elastomeric block involved. However, the low rate quantity and stability depend upon the nature of stress-strain curve and the stiffness of the elastomeric block used inside the pump. This pump is inexpensive (< 4 USD), compact, portable, and reusable (> 500 times) making it as an ideal choice for the POCDs. Using the developed ow focusing device, deformability and the associated elastic moduli of RBCs (that are obtained from healthy and diabetic subjects) and cancer cells (with and without Emodine Anticancer drug treatment) were obtained and the results were found to be in agreement with literature. To further demonstrate the applicability of the ow focusing device for blood cell imaging and counting, blood cells (Red Blood Cells - RBCs and White Blood Cells - WBCs) in ow were imaged and the counts were compared with standard hematological analyzer counts. Similar experiments were performed on urine samples to demonstrate the technique's applicability for quantitative microscopic urinalysis. Wide variety of cells that can be found in Urine such as RBCs, WBCs, Epithelial cells, and Casts were imaged, and a quantitative analysis was performed to infer the diagnosis and the observations were compared with clinical results. All these results indicate the robustness of the developed techniques and their excellent applicability for a wide range of POCDs.

This thesis reports the characterization of Red Blood Cell (RBC) morphology in a non-contact and label-free manner using a flexible, low-cost continuous imaging system. Detection of abnormalities in red blood cell properties, including shape, size, and number, can reveal a range of pathologies. A usual laboratory hematologic diagnosis consists of a complete blood count (CBC) and a peripheral blood smear (PBS) review. An Automated Hematology Analyzer reports the complete blood count, which includes Hematocrit, Hemoglobin content in RBCs and Total Count, Mean Cell Volume, Distribution width for each blood cell (RBC, WBC, Platelets). But they fail to provide any information about the cell shape, which is an essential property in determining cell morphology. A peripheral blood smear analysis involves imaging RBCs under a microscope and determining cell shape, although cell volume cannot be found as cell thickness remains unknown. Since cells are imaged in dry form and distributed non-uniformly, finding cell count using a PBS review is not possible. So, there is a need for a different technique that can be used to study red blood cell morphology effectively. Today one of the rapidly growing research fields in studying cell morphology and cell dynamics is Quantitative Phase Imaging (QPI). It combines advancements in optics, imaging theory, and computational methods to image phase information of the sample quantitatively. In this work, determination of RBC total count, MCV, and RDWusing a single, fast, portable, and cost-effective optical setup has been proposed. It involves quantifying the phase delay introduced by the red blood cells using a QPI method called the Transport of Intensity Equation (TIE). The application of TIE as a QPI method does not require complex setups or expensive components, unlike other QPI techniques. A partially coherent light beam from a conventional LED is used to illuminate a diluted blood sample loaded in a microfluidic channel. Through-focus intensity images of RBCs arranged in a monolayer are acquired using a low-cost continuous imaging system. Intensity images are processed using a Fast Fourier Transform (FFT) based Poisson solver to find a solution to the transport of intensity equation. The solution to TIE is the phase distribution of light at the focus. From phase, the thickness profile of each cell can be calculated, hence the cell volume. RBC total count is calculated by counting the cells in the given field of view. Cell shape is determined from the focal image. So the proposed system provides complete information about the red blood cells morphology at a much lower cost than hematology analyzers and peripheral blood smear analysis. The overall aim of this research project is to explore the potential of our imaging system combined with the TIE algorithm as a reliable tool for characterizing red blood cells morphology

Stored red blood cell (sRBC) morphology is currently scored manually by technicians in a slow labor intensive process prone to error. This project proposes a way to simplify, automate, and expedite the morphology scoring process by using a novel microfluidic device that I designed to facilitate the flow of a single layer of red blood cells (RBCs). The appearance of this flow allows for the capture of a series of high clarity images captured via digital camera coupled to a microscope that are ideally suited for image analysis algorithm-based morphological scoring. During storage, RBCs heterogeneously shift from the form of discocyte to the reversibly altered form of discoechinocyte as storage lesion progresses. Beyond this level of degradation, the cell assumes the form of a spheroechinocyte or spherocyte and becomes irreparably damaged. The microfluidic device and image analysis algorithm developed in this research classified the individual morphology of 5000 RBCs taken from storage into the physiologically relevant category of either “discocyte,” “reversibly changed,” or “irreversibly changed.” This process took only 15 minutes. The accuracy in classification was verified as 92.6% in a separate trial when compared against classification of the same sample images via manual inspection. The morphological distribution of the RBC population remained consistent in both cases. The findings of this project suggest that microfluidic device assisted automated image analysis can provide a quick and effective way to quantitatively estimate the viability of a sRBC population and the extent of storage lesion endured. This technology could provide augmented RBC storage and transfusion research capabilities and have clinical applications, such as the ability to conveniently differentiate between the transfusion qualities of two sRBC units of the same age.
acase@tulane.edu

Plusieurs études ont démontré une association entre l’agrégation érythrocytaire du milieu sanguin et plusieurs anomalies hémorhéologiques. Cette agrégation peut être quantifiée à l'aide du coefficient de rétrodiffusion ultrasonore. Pour décrire l’interaction entre l'onde ultrasonore et les tissus biologiques, on se sert de modèles. Ainsi, le modèle de facteur de structure (MFS) est utilisé pour évaluer le coefficient de rétrodiffusion des globules rouges agrégés. Toutefois, ce modèle numérique ne permet pas des mesures en temps réel du niveau d’agrégation et n'informe pas sur la structure du milieu sanguin comme par exemple la taille de l’agrégat. Pour pallier à ces difficultés, nous proposons un modèle où la théorie du milieu effectif est combinée au modèle de facteur de structure. Tout en permettant une mesure en temps réel de l’agrégation, ce modèle nommé TMEMFS fournit en plus deux indices structuraux de l’agrégation: le rayon de l’agrégat ainsi que sa compacité. Par le biais de simulations numériques en 3D, on a comparé le coefficient de rétrodiffusion suivant les modèles MFS et TMEMFS. Ceci dans le but de vérifier que dans la solution du problème direct, les propriétés acoustiques des globules rouges et les propriétés structurales du milieu agrégeant correspondaient à la réalité. Pour simuler des agrégats de globules rouges, une disposition hexagonale compacte a été utilisée. Les effets du rayon et de la compacité sur le coefficient de rétrodiffusion ont été étudiés. Basé sur la microstructure sanguine considérée, les résultats obtenus avec le modèle TMEMFS sont semblables à ceux du modèle MFS. Ce travail constitue un support théorique pour une mesure quantitative in vivo de l’agrégation érythrocytaire à des fins diagnostics.
Many studies have reported that an enhanced level of red blood cell aggregation is associated with the presence of hemorheological disorders. Pathological aggregation has been characterized by quantitative ultrasound based on the backscattering coefficient. In order to describe the interaction between the incident ultrasound and the interrogated biological tissues, mathematical models are used. Mathematical modeling is known to be the optimal way to describe the interaction occurring between ultrasound and tissues at the cellular level. The structure factor model (SFM), considered as the exact scattering model has been developed to predict the backscattering coefficient from blood. However, the numerical SFM cannot be applied in real time for practical measurements and does not provide aggregate size to assess the level of aggregation. Therefore, we come up with a new model based on the effective medium theory in order to tackle this difficulty. The effective medium theory combined with the structure factor model (EMTSFM) can be applied in real time and contrary to the SFM provides two indices of the aggregate state in vivo: aggregate size and compactness. Based on a 3D simulation study, the backscattering coefficients (BSCs) predicted by the effective medium theory combined with the Structure Factor Model (EMTSFM) are compared to the BSCs computed with SFM. Our aim here is to assess the accuracy of the EMTSFM against the SFM by comparing their BSC in the framework of a forward problem, i.e., the calculation of the BSC from the known acoustic and structure aggregate parameters. This was done in order to validate the proposed model. To simulate aggregates, RBCs are stacked following a hexagonal close packing scheme. The influences of the aggregate radius and compactness on the BSC are studied as well. The results showed good agreement between the SFM and the EMTSFM based on our simulated microstructure of RBC aggregates. Our work provides thus the theoretical background to assess locally the aggregation level for diagnosis purposes.
Le travail a été réalisé en collaboration avec le laboratoire de mécanique acoustique de Marseille, France. Les simulations ont été menées avec les langages Matlab et C. Ce projet s'inscrit dans le champ de recherche dénommé caractérisation tissulaire par ultrasons.

Chez les patients atteints de la sclérose en plaques (SEP), les lymphocytes T autoréactifs utilisent des molécules d'adhérence (CAM) pour traverser la barrière hémo-encéphalique (BHE), pénétrer dans le système nerveux central (SNC) et médier la détérioration de la myéline. Les lymphocytes T régulateurs (Treg) constituent l’un des éléments clés de la tolérance immunitaire, protégeant contre les réactions auto-immunes. Cependant, l'entrée et la fonction des Treg dans le SNC restent largement inconnues. Notre laboratoire a démontré la contribution de plusieurs CAM, dont la molécule melanoma cell adhesion molecule (MCAM), dans la migration des lymphocytes pathogéniques à travers la BHE. L'objectif de cette étude est de déterminer si les Treg migrent dans le SNC en utilisant MCAM et s’ils exercent des fonctions anti-inflammatoires qui pourraient atténuer l'inflammation du SNC. L'expression de MCAM, des marqueurs fonctionnels de Treg (CTLA-4, CCR6, CCR5), ainsi que leur sécrétion de cytokines (IL-10, GrzmB, TGF-ß, IFN-γ, TNF α, GM-CSF, IL-17a), ont été étudiées sur des Treg du sang périphérique, du liquide céphalo-rachidien (LCR) et de la culture in vitro, provenant de patients atteints de SEP et d’individus sains (HC), par cytométrie de flux, en corroboration avec qPCR et ELISA. De plus, la présence de MCAM+ Treg dans le SNC a été évaluée par immunohistofluorescence (CD4, CD25, Foxp3, MCAM, noyaux) sur des souris atteintes d'encéphalomyélite auto-immune expérimentale (EAE). Nos données ont montré une augmentation de l'expression de MCAM sur les Treg de patients atteints de la forme cyclique de SEP (RRMS) par rapport aux HC. Nous avons observé une tendance vers une fréquence plus élevée de MCAM+ Treg dans le LCR par rapport au sang périphérique des patients atteints de SEP, ce qui suggère que MCAM pourrait jouer un rôle important dans la migration des Treg. Ces cellules MCAM+ Treg semblent avoir un phénotype plus fonctionnel et anti-inflammatoire que leurs contreparties MCAM-. De plus, nous avons trouvé des niveaux plus élevés de MCAM+ Treg dans les périodes de rémission de l'EAE, ce qui souligne leur implication durant cette phase de la maladie. Dans l'ensemble, nos données montrent que MCAM est une CAM essentielle pour la migration des Treg vers le SNC.
In multiple sclerosis (MS), autoreactive T cells upregulate cellular adhesion molecules (CAMs) to cross the blood brain barrier (BBB), enter the central nervous system (CNS) and mediate damage to myelin. Regulatory T cells (Treg) are one of the key components of immune tolerance, protecting against autoimmune reactions. However, Treg's entry and function in the CNS remains largely unknown. Our lab has demonstrated the contribution of several CAMs, including melanoma cell adhesion molecule (MCAM), in the migration of pathogenic lymphocytes across the BBB. The goal of this study is to determine whether Treg migrate into the inflamed CNS using MCAM and exert anti-inflammatory functions, possibly dampening CNS inflammation. The expression of MCAM and Treg functional markers and chemokine receptors (CTLA-4, CCR6, CCR5,), as well as cytokine secretion (IL-10, GrzmB, TGF-ß, IFN-γ, TNF α, GM-CSF, IL-17a), were studied on MS patients and healthy individuals (HC) Treg from the peripheral blood, cerebrospinal fluid (CSF), and in vitro culture, by flow cytometry, in corroboration with qPCR and ELISA. Moreover, the presence of MCAM+ Treg in the CNS was assessed by immunohistofluorescence (CD4, CD25, Foxp3, MCAM, nuclei) on experimental autoimmune encephalomyelitis (EAE) affected mice. Our data showed an increase in the expression of MCAM on Treg during relapse-remitting MS patients (RRMS) compared to HC. We observed a trend for a higher frequency of MCAM+ Treg cells in the CSF versus the peripheral blood of MS patients, suggesting that MCAM might play an important role in the migration of Treg. These MCAM+ Treg seem to have a more functional and anti-inflammatory phenotype than their MCAM- counterparts. Moreover, we found higher levels of MCAM+ Treg in periods of EAE remission, underlining their involvement during this disease phase. Overall, our data depicts MCAM as an essential CAM for Treg homing to the CNS.

Medical devices are used widely at every stage of disease diagnosis and treatment. To eradicate certain infectious diseases, the development of highly sensitive diagnostic tools and techniques is essential. The work reported in this thesis presents a novel approach, which can be used for the diagnosis of various diseases in the field of clinical cytology. The central theme of this approach was to develop a simple, holistic and completely automated system for point-of-care (POC) diagnostics. This is realized through the Development of an Absorption Flow-Cytometer with Synergistic Integration of Microfluidic, Optics and simple Electronics. Quantitative diagnosis of malaria has been taken as test case for the characterization and validation of the developed technology. Malaria is a life-threatening disease widely prevalent in developing countries. Approximately half the world population undergoes a test of malaria and it kills close to half a million people every year. Early detection and treatment will reduce the number of fatalities and also decrease its transmission rate. In the recent past, several diagnostic tools have been developed to detect malaria but there are varied demands on diagnostic instruments in healthcare settings and endemic contexts. The objective of this thesis is to develop an instrument capable of identifying malaria-infected red blood cells (i-RBCs) from a given few micro-liters of whole blood. The optical absorption properties of blood cells were measured at a single-cell level to diagnose malaria. The proof-of-concept for the instrument was established in four stages, after which a prototype was also developed and validated. In the first stage, a system capable of simultaneously imaging cells and also measuring their optical absorbance properties was developed. The developed system was employed to characterize absorption properties of red blood cells (malaria-infected and healthy ones) on blood-smear. A custom-made bright-field transmission microscope in combination with a pair of laser diode and photo-detector was used to simultaneously image and measure transmittance of infected and uninfected RBCs. In the second stage, the technique was extended to enable high-throughput measurements with the use of microfluidic sample handling and synchronous data acquisition. Using this technique, the optical absorbance and morphology of infected and healthy RBCs have been characterized in statistically significant numbers. The correlation between cell morphology (from images) and single-cell optical absorbance level helped to establish the thresholds for differentiating healthy and infected cells. In the third stage, a portable prototype capable of assessing optical absorbance levels of single cells was fabricated. The developed prototype is capable of assessing cells at throughputs of about 1800 cells/ second. It was initially validated with sample suspensions containing infected and healthy RBCs obtained from malaria cultures. For the device to be usable at the field-level, it has to function in the presence of all other cellular components of whole blood. The optical absorbance of other cellular components of blood like white blood cells and platelets, were characterized. The device was finally tested with blood samples spiked with malaria-infected RBCs validating the overall proof-of-concept and the developed prototype. The deployment of such cost-effective, automated POC system would enable malaria diagnosis at remote locations and play a crucial role in the ongoing efforts to eradicate malaria. In future, the presented technology can be extended to develop POC diagnostic tool for other diseases as well. As it enables quantitative estimation of malaria, the present optical absorption flow analyzer would also find application in disease prognosis monitoring, anti-malarial drug development and other studies requiring measurements on a single-cell basis. The hyper-imaging system can be used to characterize and validate the threshold information, and can be incorporated in the prototype. Thus, it is a continuous process to characterization and implementation in the prototype. The optofluidic absorption flow analyzer will help enable affordable clinical diagnostic testing in resource limited settings. This approach will be extended to diagnose other diseases, using differences in optical absorption as criteria for differentiating healthy and infected cells.

Medical devices are used widely at every stage of disease diagnosis and treatment. To eradicate certain infectious diseases, the development of highly sensitive diagnostic tools and techniques is essential. The work reported in this thesis presents a novel approach, which can be used for the diagnosis of various diseases in the field of clinical cytology. The central theme of this approach was to develop a simple, holistic and completely automated system for point-of-care (POC) diagnostics. This is realized through the Development of an Absorption Flow-Cytometer with Synergistic Integration of Microfluidic, Optics and simple Electronics. Quantitative diagnosis of malaria has been taken as test case for the characterization and validation of the developed technology. Malaria is a life-threatening disease widely prevalent in developing countries. Approximately half the world population undergoes a test of malaria and it kills close to half a million people every year. Early detection and treatment will reduce the number of fatalities and also decrease its transmission rate. In the recent past, several diagnostic tools have been developed to detect malaria but there are varied demands on diagnostic instruments in healthcare settings and endemic contexts. The objective of this thesis is to develop an instrument capable of identifying malaria-infected red blood cells (i-RBCs) from a given few micro-liters of whole blood. The optical absorption properties of blood cells were measured at a single-cell level to diagnose malaria. The proof-of-concept for the instrument was established in four stages, after which a prototype was also developed and validated. In the first stage, a system capable of simultaneously imaging cells and also measuring their optical absorbance properties was developed. The developed system was employed to characterize absorption properties of red blood cells (malaria-infected and healthy ones) on blood-smear. A custom-made bright-field transmission microscope in combination with a pair of laser diode and photo-detector was used to simultaneously image and measure transmittance of infected and uninfected RBCs. In the second stage, the technique was extended to enable high-throughput measurements with the use of microfluidic sample handling and synchronous data acquisition. Using this technique, the optical absorbance and morphology of infected and healthy RBCs have been characterized in statistically significant numbers. The correlation between cell morphology (from images) and single-cell optical absorbance level helped to establish the thresholds for differentiating healthy and infected cells. In the third stage, a portable prototype capable of assessing optical absorbance levels of single cells was fabricated. The developed prototype is capable of assessing cells at throughputs of about 1800 cells/ second. It was initially validated with sample suspensions containing infected and healthy RBCs obtained from malaria cultures. For the device to be usable at the field-level, it has to function in the presence of all other cellular components of whole blood. The optical absorbance of other cellular components of blood like white blood cells and platelets, were characterized. The device was finally tested with blood samples spiked with malaria-infected RBCs validating the overall proof-of-concept and the developed prototype. The deployment of such cost-effective, automated POC system would enable malaria diagnosis at remote locations and play a crucial role in the ongoing efforts to eradicate malaria. In future, the presented technology can be extended to develop POC diagnostic tool for other diseases as well. As it enables quantitative estimation of malaria, the present optical absorption flow analyzer would also find application in disease prognosis monitoring, anti-malarial drug development and other studies requiring measurements on a single-cell basis. The hyper-imaging system can be used to characterize and validate the threshold information, and can be incorporated in the prototype. Thus, it is a continuous process to characterization and implementation in the prototype. The optofluidic absorption flow analyzer will help enable affordable clinical diagnostic testing in resource limited settings. This approach will be extended to diagnose other diseases, using differences in optical absorption as criteria for differentiating healthy and infected cells.