8 1 ABSTRACT (EN) This thesis is focused mainly on understanding mechanisms of regulatory roles of C-terminal Src kinase (CSK) and phosphoprotein associated with glycosphingolipid- enriched microdomains (PAG) in the high-affinity IgE receptor (FcɛRI)-mediated signaling of murine mast cells. FcɛRI activation is initiated by aggregation of the receptor by complexes of multivalent antigen with IgE, followed by activation and enhanced activities of protein tyrosine kinases, phosphatases, adaptor proteins and number of other signal transduction molecules. The signaling events result in mast cell degranulation and release of variety of proinflammatory mediators, responsible for initiation of allergy and other inflammatory diseases. Understanding the function of key regulatory molecules controlling FcεRI-mediated mast cell activation, degranulation, and cytokines production could have therapeutic impact. CSK is a major negative regulator of Src family tyrosine kinases (SFKs) that play a critical role in various immunoreceptor signaling events. However, its function in mast cell activation has not been completely understood. Because of its cytoplasmic localization, CSK was assumed to be brought to the vicinity of the plasma membrane- bound SFKs via binding to membrane-bound adaptors and PAG was a major candidate....

The paradigm shift in cardiovascular biology has been the understanding that atherosclerosis involves not just a mechanical deposition of lipids in the vessel wall, but a dynamic process involving the inflammatory response with cellular infiltration and inflammatory mediator expression. Typical cellular elements that have been studied include endothelial cells, vascular smooth muscle, T lymphocyte and the macrophage. Recent data suggests a role for the human mast cell. The human mast cell is a tissuedwelling cell, typically perivascular in distribution. This multifunctional cell responds rapidly to challenge with the release of inflammatory mediators that can orchestrate an immune response and may have relevance to atherogenesis. Mast cells have been shown to modulate various aspects of cardiovascular disease such as atherogenesis (endothelial activation, cytokine generation and foam cell formation) as well as rupture of an unstable atheromatous plaque. Mast cell activation in the context of cardiovascular disease may occurby cognate cell-cell interactions (interactions with macrophages, T cells, endothelial cells or smooth muscle) or by non-cognate means (such as lipoproteins and other proatherogenic components). More studies are required in order to better understand the molecular role of mast cells in vascular inflammatory disease.

La mastocytose se définit par une accumulation excessive de mastocytes dans un ou plusieurs organes ou tissus. Si dans la plupart des cas la maladie ne diminue pas l’espérance de vie, elle reste néanmoins associée à un handicap souvent mal connu et mal compris. Malgré l’importance des manifestations neuropsychiatriques accompagnant la mastocytose, les efforts concentrés sur leur compréhenson et celle de leurs liens avec la maladie ont été longtemps mis au second plan. L’objectif de cette thèse était de mieux connaître ces troubles neuropsychologiques, notamment la dépression et les troubles de l’attention/ mémoire, d’investiguer les liens entre ces troubles et des aspects biologiques et émotionnels comme l’effet d’un inhibiteur de tyrosine kinase bloquant les mastocytes sur la dépression ; les liens entre le stress perçu, la dépression et la longueur des télomères ; et les liens entre dépression et compétences de régulation émotionnelle. Nos résultats montrent que la dépression est un symptôme fréquent dans la mastocytose, ainsi que les troubles de la mémoire. Ces derniers ne semblent pas liés à la dépression. Par ailleurs, la dépression est améliorée par un traitement visant les mastocytes ; le stress perçu chez ces patients est lié à la longueur des télomères et le métaolisme du tryptophane, en particulier l’activité de l’IDO est associée aux troubles neuropsychologiques chez ces patiens. Ces résultats ouvrent des nouvelles perspectives dans la compréhension des symptômes psychologiques associés à la mastocytose. / Mastocytosis is defined as an excessive accumulation of mast cells in one or more organs or tissues. While in most cases the disease does not reduce life expectancy, it is associated with a disability often poorly known and poorly understood relate to the symptoms due to the release of mast cell mediators. Despite the importance of these neuropsychiatric manifestations, efforts focused on their understanding and their relationship to the disease has been put into the background. The aim of this thesis was to better know these neuropsychological disorders present in mastocytosis, including depression and attention deficit disorder and to investigate the relationship between these disorders and biological and emotional aspects as effect of an inhibitor of tyrosine kinase locking mast cells, the relationship between perceived stress, depression, and telomere length, and the links between depression and emotional regulation skills. Our results show that depression and memory disorders are common symptoms in mastocytosis. Attention impairment do not seem related to depression. Furthermore, depression is improved by masitinib treatment. Perceived stress in these patients is related to telomere length and tryptophan metabolism; in particular the activity of IDO is associated with neuropsychological disorders. These results open new perspectives in understanding the psychological symptoms associated with mastocytosis.

No description available.

Spiral-artery (SA) remodeling is a fundamental process during pregnancy that involves the action of cells of the initial vessel, such as vascular smooth-muscle cells (VSMCs) and endothelial cells, but also maternal immune cells and fetal extravillous trophoblast cells (EVTs). Mast cells (MCs), and specifically chymase-expressing cells, have been identified as key to a sufficient SA remodeling process in vivo. However, the mechanisms are still unclear. The purpose of this study is to evaluate the effects of the MC line HMC-1 and recombinant human chymase (rhuCMA1) on human primary uterine vascular smooth-muscle cells (HUtSMCs), a human trophoblast cell line (HTR8/SV-neo), and human umbilical-vein endothelial cells (HUVEC) in vitro. Both HMC-1 and rhuCMA1 stimulated migration, proliferation, and changed protein expression in HUtSMCs. HMC-1 increased proliferation, migration, and changed gene expression of HTR8/SVneo cells, while rhuCMA treatment led to increased migration and decreased expression of tissue inhibitors of matrix metalloproteinases. Additionally, rhuCMA1 enhanced endothelial-cell-tube formation. Collectively, we identified possible mechanisms by which MCs/rhuCMA1 promote SA remodeling. Our findings are relevant to the understanding of this crucial step in pregnancy and thus of the dysregulated pathways that can lead to pregnancy complications such as fetal growth restriction and preeclampsia.

The selective laser melting (SLM) process allows for the control of unique part form and function characteristics not achievable with conventional manufacturing methods and has thus gained interest in several industries such as the aerospace and biomedical fields. The fabrication processing parameters selected to manufacture a given part influence the created material microstructure and the final mechanical performance of the part. Understanding the process-structure and structure-performance relationships is very important for the design and quality assurance of SLM parts. Image based analysis methods are commonly used to characterize material microstructures, but are very time consuming, traditionally requiring manual segmentation of imaged features. Two Python-based image analysis tools are developed here to automate the instance segmentation of manufacturing defects and subgranular cell features commonly found in SLM 316L stainless steel (SS) for quantitative analysis. A custom trained mask region-based convolution neural network (Mask R-CNN) model is used to segment cell features from scanning electron microscopy (SEM) images with an instance segmentation accuracy nearly identical to that of a human researcher, but about four orders of magnitude faster. The defect segmentation tool uses techniques from the OpenCV Python library to identify and segment defect instances from optical images. A melt pool structure generation tool is also developed to create custom melt-pool geometries based on a few user inputs with the ability to create functionally graded structures for use in a virtual testing framework. This tool allows for the study of complex melt-pool geometries and graded structures commonly seen in SLM parts and is applied to three finite element analyses to investigate the effects of different melt-pool geometries on part stress concentrations. / Master of Science / Recent advancements in additive manufacturing (AM) processes like the selective laser melting (SLM) process are revolutionizing the way many products are manufactured. The geometric form and material microstructure of SLM parts can be controlled by manufacturing settings, referred to as fabrication processing parameters, in ways not previously possible via conventional manufacturing techniques such as machining and casting. The improved geometric control of SLM parts has enabled more complex part geometries as well as significant manufacturing cost savings for some parts. With improved control over the material microstructure, the mechanical performance of SLM parts can be finely tailored and optimized for a particular application. Complex functionally graded materials (FGM) can also easily be created with the SLM process by varying the fabrication processing parameters spatially within the manufactured part to improve mechanical performance for a desired application. The added control offered by the SLM process has created a need for understanding how changes in the fabrication processing parameters affect the material structure, and in turn, how the produced structure affects the mechanical properties of the part. This study presents three different tools developed for the automated characterization of SLM 316L stainless steel (SS) material structures and the generation of realistic material structures for numerical simulation of mechanical performance. A defect content tool is presented to automatically identify and create binary segmentations of defects in SLM parts, consisting of small air pockets within the volume of the parts, from digital optical images. A machine learning based instance segmentation tool is also trained on a custom data set and used to measure the size of nanoscale cell features unique to 316L (SS) and some other metal alloys processed with SLM from scanning electron microscopy (SEM) images. Both these tools automate the laborious process of segmenting individual objects of interest from hundreds or thousands of images and are shown to have an accuracy very close to that of manually produced results from a human. The results are also used to analyze three different samples produced with different fabrication processing parameters which showed similar process-structure relationships with other studies. The SLM structure generation tool is developed to create melt pool structures similar to those seen in SLM parts from the successive melting and solidification of material from the laser scanning path. This structural feature is unique to AM processes such as SLM, and the example test cases investigated in this study shows that changes in the melt pool structure geometry have a measurable effect, slightly above 10% difference, on the stress and strain response of the material when a tensile load is applied. The melt pool structure generation tool can create complex geometries capable of varying spatially to create FGMs from a few user inputs, and when applied to existing simulation methods for SLM parts, offers improved estimates for the mechanical response of SLM parts.

No description available.

No description available.

Deer (<i> Odocoileus </i> spp.) are an important cultural and economic resource. They are the most popular game species in the United States and the number one driver of conservation funding. On the other hand, they also cause damage to resources including to the agricultural industry, private property and to humans via deer vehicle collisions. Many areas in eastern North America have experienced a decline in white-tailed deer (<i> Odocoileus virginianus </i>) populations over the past twenty years concurrent with changes in landscape, deer harvest strategies, and increasing and expanding black bear (<i> Ursus americanus </i>) and coyote (<i> Canis latrans </i>) populations. Most studies have addressed this problem at small spatial and temporal scales and in areas where predation was assumed to be limiting population growth. We evaluated white-tailed deer fawn recruitment both directly and indirectly at relatively broader spatial and temporal scales. We studied fawn survival on Marine Corps Base Quantico in Virginia, USA from 2008–2019. We used the Kaplan-Meier estimator, and Cox-proportional hazards models to assess annual survival and factors that influence hazard risk such as sex, weather, landscape composition and configuration and food availability. On Marine Corps Base Quantico, we found an increase in red oak mast abundance increases survival and probability of fawn survival was higher during the first interval of the study (2008–2010; 0.71 [0.52–0.96]; survival probability [CI95%]) than the last three intervals (2011–2013; 0.46 [0.30–0.70]; 2014–2016; 0.48 [0.35–0.66] and 2017–2019; 0.50 [0.39–0.63]). We also found that predation was the leading source of mortality. We assessed recruitment using lactation status from hunter harvest data collected by the Virginia Department of Game and Inland Fisheries during a 22-year period in 30 counties in the Appalachian Mountains of western Virginia. We predicted lactation status as a function of landscape composition and configuration, oak mast abundance, weather, age, and predator detection rates using generalized linear mixed models. We found land cover diversity index positively and black bear detection rate negatively influenced recruitment. Age also predicted lactation status with middle-aged females (3.5–4.5 years old) having a higher lactation probability than mature (≥5.5 years old) age and young (2.5 years old) age class deer. Based on these findings, recruitment is likely to be greater in areas that are heterogeneous. Therefore, if an increase in recruitment is desired silvicultural practices such as fire and timber harvest could be used to permanently change land cover types. These silvicultural practices could also be used to increase the number of oaks on the landscape, and augment production of mature oaks as red oak mast abundance influenced fawn survival. Also reducing predation by manipulating predator densities could improve recruitment. Another potential option which needs further research, would be to reduce predator efficiency by increasing hiding cover for fawns. / Master of Science / Deer (<i> Odocoileus </i> spp.) are an important cultural and economic resource. They are the most popular game species in the United States and the number one driver of conservation funding. Conversely, they also cause damage to resources including to the agricultural industry, private property and to humans via deer vehicle collisions. Some areas in eastern North America have experienced a decline in white-tailed deer (<i> Odocoileus virginianus </i>) populations over the past twenty years concurrent with the colonization of coyotes (<i> Canis latrans </i>) as well as changes in habitat. Therefore, we aimed to determine white-tailed deer fawn survival rates and factors affecting recruitment across a large spatial and temporal scale. Recruitment is when an individual becomes part of the reproductive population. However, for harvestable populations, recruitment is when individuals can be legally harvested for the first time. On Marine Corps Base Quantico from 2008–2019, in Virginia, USA we found that probability of fawn survival was higher during the first interval of the study (2008–2010; 0.71 [0.52–0.96]; survival probability [CI95%]) than the last three intervals (2011–2013; 0.46 [0.30–0.70]; 2014–2016; 0.48 [0.35–0.66] and 2017–2019; 0.50 [0.39–0.63]). We also discovered predation was the leading cause of death and probability of survival increased with increasing red oak mast abundance. Over a large spatial (30 counties) and temporal (22 years) scale we examined fawn recruitment by predicting lactation status of female harvested white-tailed deer in the Appalachians Mountains of western Virginia. We found land cover diversity index positively and black bear (<i> Ursus americanus </i>) detection rate negatively influenced recruitment. Age also predicted lactation status with middle-aged females (3.5–4.5 years old) having a higher lactation probability than mature (≥5.5 years old) age and young (2.5 years old) age class deer. Based on these findings, recruitment is likely to be greater in areas that have a mixture of land cover types. Therefore, if an increase in recruitment is desired silvicultural practices such as fire and timber harvest could be used to permanently change land cover types. These silvicultural practices could also be used to increase the number of oaks on the landscape, and augment production of mature oaks as red oak mast abundance influenced fawn survival. Also reducing predation by manipulating predator densities could improve recruitment. Overall, we found diversity of land cover types, food availability, predators, and age influences population dynamics of white-tailed deer.

Bei der Wundheilung und fibrosierenden Prozessen sowie bei der Urtikaria ist eine Mastzellvermehrung bekannt. Mastzellen (MZ) üben bei der Urtikaria eine Schlüsselfunktion aus und scheinen auch zum Bindegewebsumbau beizutragen. In humanem Narbengewebe (5-369 Tage alt) wurden MZ-Zahlen und MZ-Subpopulationen mittels Enzym- und Immunhistochemie im Vergleich zu normaler Haut untersucht. Außerdem wurden in Gewebsextrakten Aktivität und mRNA-Expression der MZ-Proteasen und in vitro ihre mitogene Wirkung auf Fibroblasten und Keratinozyten bestimmt. Zur Klärung von Mechanismen, die zur MZ-Vermehrung beitragen könnten, analysierten wir die Expression von MZ-Chemoattraktoren und MZ-Wachstumsfaktoren sowie ihrer Rezeptoren in humanem Narbengewebe (a), läsionaler und nicht-läsionaler Urtikariahaut (b) und in normaler Haut (c): SCF, c-Kit, NGF-R TrkA, NGF-R p75, GM-CSF, GM-CSF-R (a, b, c); NGF, TGF-(, TGF-(-R I, TGF-(-R II (a,c) mittels Immunhistochemie (a, b, c) und RT-PCR (a, c). Zusätzlich wurde die Expression der proentzündlichen Zytokine IL-3, -8, TNF-( untersucht (b, c). Tryptase und Chymase enthaltende MZ waren in Narben gegenüber normaler Haut signifikant vermindert ebenso wie Chymaseaktivität und -mRNA-Expression in Narbengewebsextrakten. Die Anzahl Tryptase-haltiger MZ war unverändert, obwohl Tryptaseaktivität und -mRNA in Narben vermehrt waren. Beide Proteasen erhöhten in vitro die mitogene Antwort von Fibroblasten, jedoch nicht von Keratinozyten. c-Kit+-MZ fanden sich in der mittleren und tiefen Dermis von Narben signifikant vermehrt. SCF, TGF-(, TGF-(-R I und II, NGF-R p75 und TrkA zeigten sich sowohl immunhistochemisch als auch in der RT-PCR in Narbengewebe hochreguliert im Vergleich zu normaler Haut, wohingegen NGF, GM-CSF und GM-CSF-R nur schwach exprimiert waren ohne Unterschied zwischen beiden Geweben. Mittels FACS-Analyse wurde erstmalig die Expression von TGF-(-R I und II auf isolierten Haut-MZ nachgewiesen. Im Gegensatz zu diesen Befunden waren in Urtikariagewebe SCF- und NGF-R p75-exprimierende Zellen vermindert im Vergleich zu normaler Haut. Die Zahl von c-Kit+-, NGF-R TrkA+-, GM-CSF+- und GM-CSF-R+ -Zellen zeigte sich unverändert. Hingegen war die Expression von IL-3 und TNF-( auf Endothelzellen in läsionaler und nicht-läsionaler Urtikariahaut signifikant hochreguliert. Die dargestellten Ergebnisse mit signifikanter Verminderung von Chymase- und Tryptase-haltigen MZ in humanem kutanen Narbengewebe sprechen für MZ-Degranulation nach Trauma. Nachfolgend findet sich in Narbengewebe eine Chymase--, Avidin--, Tryptase+-, c-Kit+-MZ-Subpopulation, am ehesten Folge einer Einwanderung und Proliferation von unreifen MZ oder MZ-Vorläufern, die von den vermehrt exprimierten Wachstumsfaktoren SCF und TGF-(, eventuell auch von NGF über seine vermehrt exprimierten Rezeptoren, induziert werden könnten. Neben NGF und TGF-( scheint auch SCF eine Rolle bei der Wundheilung zu spielen. Bei entzündlichen Hautkrankheiten unterschiedlicher Prägung wie Wundheilung und Urtikaria liegen offenbar verschiedenartige Regulationsmuster der MZ-Proliferation und -Differenzierung vor. Unsere Ergebnisse legen nahe, dass bei Trauma Feedbackmechanismen über Wachstumsfaktoren wie SCF, TGF-( und NGF und ihre Rezeptoren auf MZ ablaufen, bei der Urtikaria unter Mitberücksichtigung bereits bekannter Daten aus der Literatur vorzugsweise über eine Interaktion von Mast- und Endothelzellen. / In wound healing and fibrosing processes as well as in urticaria an increase of mast cells (MC) has been observed. MC are key-players in urticaria, and might also contribute to tissue repair. In human cutaneous scar tissue (5-369 days old) and normal skin MC dynamics and MC subtypes were analysed by enzyme- and immunohistochemistry. Moreover, the activity of the MC proteases in extracts of both tissues and their in vitro effect on the mitogenesis of fibroblasts and keratinocytes were assessed. To elucidate mechanisms involved in mast cell accumulation, expression of MC chemotaxins, MC growth factors and their receptors was evaluated comparing cutaneous scar tissue (a), lesional and non-lesional skin of urticaria (b) and normal skin (c): SCF, c-Kit, NGF-R TrkA, NGF-R p75, GM-CSF, GM-CSF-R (a, b, c); NGF, TGF-(, TGF-(-R I, TGF-(-R II (a,c) by immunohistochemistry (a, b, c) and by RT-PCR (a, c). Additionally, expression of proinflammatory cytokines (IL-3, -8, TNF-() was studied (b, c). Tryptase and chymase containing MC were markedly decreased in scars as well as chymase activity and mRNA expression, whereas overall numbers of tryptase containing MC did not differ from those in normal skin, although tryptase activity and mRNA expression were increased in scar extracts. Both proteases induced a dose-dependent mitogenic response in 3T3-fibroblasts, but not in HaCaT-keratinocytes. Numbers of c-Kit+ MC were significantly increased in the mid and lower dermis of scars. Furthermore, SCF, TGF-(, its receptors I and II, the NGF-R p75 and TrkA were shown to be upregulated in scars both by immunohistochemistry and by RT-PCR, while NGF, GM-CSF and the GM-CSF-R were only weakly expressed without differences between scar and normal tissue. In addition, expression of TGF-(-R I and II could be shown on isolated human skin MC by FACS-analysis. In contrast to these findings, SCF- and NGF-R p75-expressing cells in urticaria tissue were downregulated compared to normal skin. Numbers of c-Kit+, NGF-R TrkA+, GM-CSF+ and GM-CSF-R+ cells remained unchanged. However, IL-3 and TNF-( expression was upregulated on endothelial cells in lesional and non-lesional skin of urticaria. These data show that numbers of resident MCTC are very low in human cutaneous scars suggesting massive mediator release from these cells after wounding. Instead, scar tissue is populated by a chymase-, avidin-, tryptase+, c-Kit+ MC subpopulation that is reflecting most probably an immigration and / or proliferation of immature MC and their precursors which might be promoted by SCF and TGF-beta, possibly also NGF via its receptors. Next to TGF-( and NGF, also SCF seems to play a role in wound healing. Our findings suggest different regulation patterns of MC increase in inflammatory conditions of the skin. After wounding, feedback mechanisms via growth factors (SCF, TGF-(, possibly NGF) and their receptors on MC could be operative, while in urticaria in accordance with data from the literature interactions between MC and endothelial cells appear to be essential.