Megakaryocytes (MK) are responsible for platelet biogenesis, which is thought to occur canonically in the adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may significantly contribute to circulating platelet mass. While a diversity of cells specific to the BM are known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single cell RNA sequencing (scRNA-Seq) coupled with histological, ploidy and flow cytometric analyses to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed towards roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. In addition to MKs, the lung houses many resident hematopoietic cells, including hematopoietic stem and progenitor cells (HSPCs). The existence of lung HSPCs suggests that the differentiation and development of lung resident hematopoietic cells may occur in-situ. To investigate the potential role the lung has in instructing site specific hematopoiesis, we employed explant cultures of murine and human fetal lungs. This displayed adherent endothelial cells transitioning into floating hematopoietic cells, suggesting that the fetal lung is a source of hemogenic endothelial cells that have the functional capacity to undergo endothelial to hematopoietic transition (EHT) to produce HSPCs. Flow cytometric and functional assessment of fetal lung explants showed the production of HSPCs that expressed key EHT and pre-HSPC markers. Expression profiles revealed by scRNA-Seq and small molecule modulation demonstrated that fetal lung EHT is reliant on canonical EHT signaling pathways. These findings suggest that functional HECs are present in the fetal lung, thus establishing this location as a potential extramedullary site of de-novo hematopoiesis. Overall, these findings suggest that the lung may have a greater role in instructing tissue specific hematopoiesis and/or overall hematopoietic development.

No description available.

Obesity and insulin resistance are characterized by elevated pro-inflammatory proteins in the blood and immune cell accumulation in the visceral adipose tissue. Resident leukocytes release tumor necrosis factor α (TNFα) and other inflammatory cytokines which stimulate adipocyte lipolysis, recruit leukocytes to adipose tissue, promote pro-inflammatory immune cell polarization, facilitate oxidative stress, and activate intracellular kinases which dull insulin signaling cascades in metabolic tissues. Immune cell mediated dysregulation of stromal and parenchymal cells has raised suspicion that insulin resistance is an immune disorder initiated by activated white blood cells with over-nutrition. Efforts to improve pathological metabolism by reducing inflammation have yielded mixed results in humans and animal models. The role of inflammation and immune cell accumulation in the visceral fat (VF) in the progression of insulin resistance remains presently debated. There is, however, a consensus that identifying the triggers for obesity and impaired insulin signaling is of the utmost importance. The goal of this report is to identify dietary fat absorption as a key initiator of inflammatory action and insulin desensitization which may be dampened by reducing immune cell accumulation in adipose tissue. To explore how lean, healthy organisms become obese and insulin resistant, we examined the inflammatory consequences of isocaloric but variable macronutrient loads in the VF of lean mice. Mice were administered single liquid meals composed of low-fat (10% fat) or high-fat (60% fat) diet and observed by intravital microscopy to quantify leukocyte-endothelium interactions in mesenteric postcapillary venules (MPCV) 1, 2, 3, and 4 hours after oral gavage. Leukocyte rolling and leukocyte adhesion were transiently elevated within 1 hour after feeding and returned to baseline levels 4 hours later. Endothelial cell surface expression of P-selectin (Psel), a rapidly activated cell adhesion molecule (CAM), confirmed that high-fat feeding induced Psel dependent leukocyte rolling through the VF microcirculation. Furthermore, leukocyte accumulation in the VF was modestly increased by a single high-fat meal (HFM). Repetitive high-fat diet (HFD) consumption for 24 hours prolonged elevated leukocyte-endothelium interactions and promoted neutrophil accumulation in the VF. The neutrophilic enzyme myeloperoxidase (MPO), a producer of the chlorinating agent hypochlorous acid, increased in abundance and activity in the VF of HFM fed mice. Elevated leukocyte-endothelium interactions, leukocyte infiltration, and MPO activity in VF were not observed in Psel deficient (Psel-/-) mice following lipid overload. To ascertain if MPO is required for sustained endothelial activation, leukocyte-endothelium interactions and leukocyte infiltration were monitored in high-fat fed MPO deficient (MPO-/-) mice. Similar to the Psel-/- mice, MPO-/- mice were protected from the inflammatory effects of high-fat feeding. Our data supports postprandial hyperlipemia as an inducer of transient and Psel dependent inflammatory reactions that are sustained by prolonged HFD consumption. To study whether early phase inflammatory interventions granted late phase metabolic improvements, wild-type (WT), Psel deficient (Psel-/-), and MPO deficient (MPO-/-) C57BL/6 mice were given ad libitum access to LFD (10% fat) or HFD (60% fat) for 12-16 weeks. All mouse groups given HFD became obese. Prolonged HFD consumption sustained elevated leukocyte-endothelium interactions in MPCVs and was accompanied by increased local and systemic TNFα in WT mice. High-fat fed WT mice were hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant compared to LFD fed controls. Psel-/- mice were protected from leukocyte-endothelium interactions as well as local and systemic TNFα accumulation despite extended HFD consumption. Surprisingly, high-fat fed Psel-/- mice were equally hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant as the inflamed, high-fat fed WT mice. MPO-/- mice were also protected from elevated systemic TNFα and gained slightly less weight than the other high-fat fed groups. While MPO-/- mice were hyperglycemic and glucose intolerant, they did have improved insulin stimulated glucose clearance. The data presented in this report demonstrates the pro-inflammatory nature of postprandial hyperlipemia and the insulin desensitizing nature of prolonged HFD consumption. Ablation of VF immune cell accumulation by Psel deletion is not sufficient for improving insulin signaling or glycemic control, which is consistent with prior reports. Deletion of MPO, however, did result in slightly less obesity and marginally improved insulin signaling. We conclude that while immune cell accumulation in the VF contributes to the progression of insulin resistance, it is not a prerequisite for metabolic pathology development. / Physiology

In a 1982 paper, Gould and Vrba argue that a conflation of the two components of adaptation of a feature, historical development of the feature and present utility, has caused evolutionists to overlook a missing term in the science of form, which they call 'exaptation'. In the present project, I show that evolutionary biology still contains a confusion in the use of 'adaptation' due to an inappropriate perception of the interaction between the two components of adaptation. Because of the confusion, evolutionists have missed another term in the science of form. Evolutionary theory, specifically the treatment of adaptation, would profit from the introduction of a term referring to features that have a selective history which causes them to appear overly well adapted to their present function. I suggest we refer to these features as hyperadaptations, since they appear to be hyperbolized adaptations. By introducing hyperadaptation into the conceptual framework of adaptation, we can sharpen our understanding of related concepts (adaptation to function, exaptation, maladaptation, etc.) and remove or reduce some confusion regarding the interplay between analysis of historical pathways and ascriptions of (current) function in the diagnosis of adaptation. Furthermore, the improved framework should allow evolutionists to more adequately explain biological phenomena. / Master of Arts

La radiothérapie est utilisée seule ou en association avec la chimiothérapie dans le traitement de plus de 50% des cancers. En dépit des progrès techniques dans la balistique, l'irradiation des tissus sains entourant la tumeur et les effets secondaires qui lui sont associés sont une limite à la dose d'irradiation utilisée. Ces effets secondaires, lorsqu'ils concernent le tube digestif, ont un retentissement important sur la qualité de vie des patients et peuvent même engager leur pronostic vital. La compréhension des mécanismes impliqués dans le développement de ces lésions est donc un enjeu majeur dans l'identification de cibles thérapeutiques permettant leur prévention et leur traitement. Durant ma thèse nous avons étudié le rôle des mastocytes dans le développement de la rectite radique in vivo et dans la réponse endothéliale à l'irradiation in vitro. Nos résultats suggèrent un rôle délétère des mastocytes dans le développement de la rectite radique humaine et murine, notamment par l'influence de certains de leurs médiateurs tels que l'histamine et les protéases sur le phénotype des cellules musculaires lisses de la muscularis propria. Le ciblage de certains médiateurs mastocytaires pourrait représenter une nouvelle stratégie thérapeutique pour prévenir et/ou limiter les atteintes radiques digestives. D'autre part, nos travaux montrent que des médiateurs mastocytaires comme l'histamine peuvent exacerber la réponse inflammatoire de l'endothélium à l'irradiation par des mécanismes de signalisation impliquant l'activation de la voie p38 MAPKinase et du facteur de transcription NF-B. L'étude approfondie des voies de signalisation activées lors du développement des lésions radiques pourrait offrir de nouvelles possibilités thérapeutiques dans la gestion des dommages radiques aux tissus sains. / Radiation therapy is used alone or in combination with chemotherapy in more than 50% of cancer treatments. Despite recent advances in treatment delivery such as dose-sculpting techniques, irradiation of healthy tissues surrounding the tumor and the associated side effects limit the radiation amount used. Those side effects when concerning the gastrointestinal tract, have profound repercussions on patient's quality of life and may even engage their vital prognosis. The comprehension of the mechanisms implicated in the development of these lesions is thus a major stake in the identification of therapeutic targets allowing their prevention and treatment. During my PhD, we studied the role of mast cells in the development of radiation proctitis in vivo and in the endothelial response to irradiation in vitro. Our results suggest that mast cells have a deleterious role in the development of human and murine radiation proctitis, in particular by the influence of some of its mediators such as histamine and proteases on the phenotype of the smooth muscle cells of the muscularis propria. Targeting mast cells'mediators may represent new therapeutic tools to prevent and/or limit digestive radiation damage. Other shares our work shows that mast cells mediators such as histamine can exacerbate the endothelial inflammatory response to irradiation by mechanisms involving the activation of the p38MAPKinase pathway and the transcription factor NF-B. The study of intracellular signaling pathways activated during radiation damage development may offer new therapeutic possibilities in the management of healthy tissues radiation damage.

Xue, Hongmei. / "December 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 165-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Liu, Limei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-156). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Liu, Jian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 127-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Zhang, Yang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-169). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Leung, Pan Cheung Catherine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 105-120). / Abstracts in English and Chinese. / DECLARATION --- p.II / ACKNOWLEDGEMENTS --- p.III / ENGLISH ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VII / Chapter MODULE 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Vascular Endothelium --- p.1 / Chapter 1.1.1. --- Vascular Endothelial Functions --- p.1 / Chapter 1.1.2. --- Calcium Signaling in Vascular Endothelial Cells --- p.2 / Chapter 1.2. --- The Founding Member of TRP Family: Drosophila TRP --- p.3 / Chapter 1.2.1. --- Discovery of Drosophila TRP and TRP-related Proteins --- p.3 / Chapter 1.2.2. --- Drosophila TRPs: Ca2+-permeable Channels? --- p.3 / Chapter 1.3. --- Mammalian TRP Superfamily --- p.5 / Chapter 1.3.1. --- The TRP Subfamily: TRPV --- p.9 / Chapter 1.3.2. --- The TRP Subfamily: TRPM --- p.9 / Chapter 1.3.3. --- The TRP Subfamily: TRPC --- p.11 / Chapter 1.4. --- Functional and Physiological Roles of Mammalian TRPCs --- p.12 / Chapter 1.4.1. --- TRPC1 --- p.15 / Chapter 1.4.2. --- TRPC2 --- p.16 / Chapter 1.4.3. --- "TRPC3, TRPC6 and TRPC7" --- p.17 / Chapter 1.4.4. --- TRPC4 and TRPC5 --- p.19 / Chapter 1.4.5. --- Over-expression of TRPCs: Physiologically Relevant Channels? --- p.20 / Chapter 1.4.6. --- Alternatives to Heterologous Expression Study --- p.21 / Chapter 1.5. --- Aims of the Study --- p.23 / Chapter MODULE 2. --- MATERIALS AND METHODS --- p.24 / Chapter 2.1. --- Functional Characterization of TRPCs by Antisense Technique --- p.24 / Chapter 2.1.1. --- Restriction Enzyme Digestion --- p.26 / Chapter 2.1.2. --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.27 / Chapter 2.1.3. --- "Ligation of TRPC Genes into Mammalian Vector, pcDNA3" --- p.27 / Chapter 2.1.4. --- Transformation for the Desired Clones --- p.28 / Chapter 2.1.5. --- Plasmid DNA Preparation for Transfection --- p.28 / Chapter 2.1.6. --- Confirmation of the Clones］ --- p.29 / Chapter 2.1.6.1. --- Restriction Enzymes Strategy --- p.29 / Chapter 2.1.6.2. --- Polymerase Chain Reaction (PRC) Check --- p.30 / Chapter 2.1.6.3. --- Automated Sequencing --- p.31 / Chapter 2.2. --- Establishing Stable Cell Lines --- p.33 / Chapter 2.2.1. --- Cell Culture --- p.33 / Chapter 2.2.2. --- Transfection Conditions Optimization --- p.33 / Chapter 2.2.3. --- Geneticin Selection --- p.35 / Chapter 2.3. --- Expression Pattern Studies of TRPC Genes in Vascular Tissues --- p.38 / Chapter 2.3.1. --- Immunofluorescence Staining in Cultured CPAE Cells --- p.38 / Chapter 2.3.2. --- Immunolocalization in Human Cerebral and Coronary Arteries --- p.40 / Chapter 2.3.2.1. --- Paraffin Section Preparation --- p.40 / Chapter 2.3.2.2. --- "Immunohistochemistry for TRPC1, 3, 4 and 6 Channels" --- p.40 / Chapter 2.3.2.3. --- Subcellular Localization of hTRPC1 and hTRPC3 Channels in Endothelial Cells --- p.42 / Chapter 2.4. --- Study of Bradykinin-induced Ca2+ Entry by Calcium Imaging --- p.47 / Chapter 2.4.1. --- Primary Aortic Endothelial Cell Culture --- p.47 / Chapter 2.4.2. --- Fura-2 Measurement of [Ca2+］］］ --- p.47 / Chapter 2.5. --- Study of Functional Role of TRPC6 in Stably Transfected H5V Cells … --- p.49 / Chapter 2.5.1. --- Protein Sample Preparation --- p.49 / Chapter 2.5.2. --- Western Blot Analysis --- p.50 / Chapter 2.5.3. --- Confocal Microscopy for Bradykinin-induced Calcium Entry --- p.51 / Chapter 2.6. --- Data Analysis --- p.52 / Chapter MODULE 3. --- RESULTS --- p.53 / Chapter 3.1. --- Bradykinin-induced Calcium Entry in Vascular Endothelial Cells --- p.53 / Chapter 3.1.1. --- Bradykinin-induced Calcium Entry --- p.53 / Chapter 3.1.2. --- Effects of cGMP and PKG on Bradykinin-induced Ca2+ Entry --- p.54 / Chapter 3.1.3. --- Effects of HOEUO on Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.1.4. --- Involvement of Phospholipase C Pathway in Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.2. --- Expression Pattern of TRPC Channels in Vascular Systems --- p.63 / Chapter 3.2.1. --- Immunolocalization of TRPC Homologues in CPAE Cells --- p.63 / Chapter 3.2.2. --- Immunolocalization of TRPC Homologues in Human Cerebral and Coronary Arteries --- p.66 / Chapter 3.2.3. --- Subcellular Localization of TRPC1 and TRPC3 Fused to Enhanced Green Fluorescence Protein (EGFP) --- p.77 / Chapter 3.3. --- Functional Role of TRPC6 Channels in Bradykinin-induced Calcium Entry --- p.81 / Chapter 3.3.1. --- Effect of Antisense TRPC6 Construct on Protein Expression --- p.81 / Chapter 3.3.2. --- Effect of Antisense TRPC6 on Bradykinin-induced Ca2+ Entry --- p.81 / Chapter 3.3.3. --- Effect of Antisense TRPC6 on Thapsigargin-depleted Ca2+ Stores --- p.82 / Chapter MODULE 4. --- DISCUSSION --- p.89 / Chapter 4.1. --- Characterization of Bradykinin-induced Ca2+ Entry in Endothelial Cells --- p.89 / Chapter 4.2. --- The Expression Pattern of TRPC Isoforms in Vascular Tissues --- p.93 / Chapter 4.3. --- Functional Characterization of TRPC6 Homologues in Bradykinin-induced Ca2+ Entry --- p.98 / Chapter 4.4. --- Perspectives --- p.103 / Chapter 4.5. --- Conclusion --- p.104 / Chapter MODULE 5. --- REFERENCES --- p.105