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No description available.

Vitronectin is a multi-functional glycoprotein that is present in the plasma and extra-cellular matrix of eukaryotes. It is capable of binding a wide variety of structurally different ligands, including plasminogen activators, plasminogen activator-inhibitors, proteases, cell surface receptors and components of the extra-cellular matrix. Vitronectin exists in two conformations – as a monomer in circulation, and as a multimer in the extracellular matrix. The pathway by which vitronectin undergoes the transition from monomer to multimer is not well characterized, but this laboratory has put forward evidence to suggest that the binding of vitronectin with plasminogen activator inhibitor type-1 (PAI-1), facilitates higher order complex formation. Because multimeric vitronectin remains long after PAI-1 has dissociated from the complex, the question of which region(s) within vitronectin are interacting to maintain the multimeric conformation has been asked. To address this question, candidate domain regions in vitronectin were analyzed for potential aggregative properties. Based on this analysis, the Central domain of the protein was hypothesized to be the region responsible for self-association. In support of this hypothesis, the predicted structure of the Central domain is a 4-bladed β–propeller fold, a motif that is capable of mediating protein-protein interactions. However, not all β–propeller proteins have self-associative behavior. Examples include hemopexin, gelatinase-A and collagenase-3, all of which share distant sequence homology with vitronectin. In this study, a negative-design approach was used in which the Central domain of vitronectin was replaced with the homologous β–propeller regions from hemopexin and gelatinase-A. The substitution of this domain with β–propeller regions that do not demonstrate self-association was expected to alter the aggregative properties of the chimeras. An additional recombinant form of vitronectin was also designed which did not possess the C-terminal domain to conclusively rule out this domain as the candidate region. In addition to this approach, limited proteolysis was used to try and isolate the Central domain region of vitronectin so that it could be better characterized both structurally and functionally. All three recombinant proteins were synthesized and expressed in insect Sf9 cells. However, purification of these proteins was met with difficulty due to the low expression levels observed. Optimization attempts to improve expression were unsuccessful, and so structural and functional analysis studies were limited. Nevertheless, all three proteins were able to bind to wild-type PAI-1 in solution, as well as demonstrate Western blot reactivity with two monoclonal antibodies. Initial results from the limited proteolysis study also showed potential peptide fragments for the Central domain of vitronectin. Based on the results from this study, the region responsible for the selfassociation of vitronectin should be further examined and resolved to critical residues within the Central domain. These results may offer insight into the process of oligomerization in other proteins, many of which are implicated in fatal diseases, e.g. Alzheimer’s and Creutzfeldt-Jakob disease.

In situations involving the co-occurrence ecologically similar species, one of two different evolutionary responses is often expected. If sympatry results in competition over a shared resource, character displacement would be a favored outcome, while morphological convergence is an alternative outcome if the species have similar responses to the shared environment. In this study, I examine cranial morphology and dorsal coloration of two ecologically similar salamander species (Plethodon serratus, the Southern Red-backed Salamander, and P. ventralis, the Southern Zig-zag Salamander) to evaluate the alternative hypotheses of character displacement or convergence. Results from linear morphometrics analyses showed no significant shifts in morphology of either species suggestive of either character displacement or convergence in any of the measured traits. However, geometric morphometric analyses showed significant morphological convergence of the two species in sympatry. In contrast, the presence or absence of a dorsal stripe showed evidence of character displacement, corroborating an earlier claim made by Highton. These results are unexpected in that features associated with feeding (cranial morphology) are expected to often exhibit character displacement if dietary resource partitioning is an important mechanism of coexistence, whereas coloration might be expected to show local convergence if its primary function is camouflage or aposematism. Convergence might reflect the overwhelming influence of developmental responses to shared environments or convergent adaptation to local prey communities. Displacement with respect to color polymorphism might be consistent with frequency-dependent selection maintaining alternative ways of being cryptic.

The chief purpose of this study is to contribute a share of knowledge to the sound management of the Logan River and similar bodies of water. If the river is to continue to support the present army of anglers and retain a harvestable crop of fish for them, the management of the crop and the fishermen will have to be established on a sound basis. The second objective of this study is to learn whether or not the state's fish stocking program is meeting fishermen needs, and how stocking should be handled to return the largest number of fish to the creel per dollar expended.

<p>Linker for activation of T cells (LAT) is a transmembrane adaptor protein that lacks any intrinsic enzymatic or transcriptional activity. Upon TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediate the binding of Grb2/Sos, PLC&#947;1, and GADS/SLP-76 complexes. SLP-76 (SH2 domain-containing leukocyte protein of 76kD) is a cytosolic adaptor protein that can interact with a variety of other adaptor proteins and signaling effectors. Through its constitutive binding of GADS, SLP-76 is recruited to the plasma membrane via LAT following TCR stimulation. Together, LAT and SLP-76 nucleate a large multi-molecular signaling complex, which couples TCR proximal signaling to downstream biochemical events, including calcium mobilization and Ras-MAPK pathway activation.</p><p>LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the DN3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by Cre recombinase. Deletion of LAT after the DN3 stage allowed largely normal development of DP thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to efficiently differentiate into SP thymocytes. Moreover, deletion of LAT in peripheral mature T cells rendered these T cells completely unresponsive to CD3 crosslinking due to abolished calcium mobilization and Ras-ERK activation. Long-term survival and lymphopenia-driven homeostatic proliferation of the LAT-deficient naïve T cells were also severely impaired. Together, these data indicate that, in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during the transition from the DP to SP stage, as well as in mature T cell activation and homeostasis.</p><p>Similar to LAT, SLP-76 is also critical for T cell function and thymocyte development. While the functions of various SLP-76 domains have been extensively studied, the role of the sterile alpha motif (SAM) domain in SLP 76 function remains unknown. By generating SLP 76 knock in mice with the SAM domain deleted, we showed that the absence of the SAM domain resulted in impaired positive and negative thymic selections, leading to a partial block of thymocyte development at the DP to SP transition. TCR-mediated IP3 production, calcium flux, and ERK activation were all decreased in these &#916;SAM-SLP-76 knockin T cells, leading to defective IL 2 production and proliferation. Moreover, despite normal association between GADS and SLP-76, TCR-mediated SLP 76 clustering was inhibited by the deletion of the SAM domain, likely causing the aforementioned TCR signaling defects. These data demonstrated for the first time that the SAM domain is indispensable for optimal SLP-76 signaling.</p> / Dissertation

In situations involving the co-occurrence ecologically similar species, one of two different evolutionary responses is often expected. If sympatry results in competition over a shared resource, character displacement would be a favored outcome, while morphological convergence is an alternative outcome if the species have similar responses to the shared environment. In this study, I examine cranial morphology and dorsal coloration of two ecologically similar salamander species (Plethodon serratus, the Southern Red-backed Salamander, and P. ventralis, the Southern Zig-zag Salamander) to evaluate the alternative hypotheses of character displacement or convergence. Results from linear morphometrics analyses showed no significant shifts in morphology of either species suggestive of either character displacement or convergence in any of the measured traits. However, geometric morphometric analyses showed significant morphological convergence of the two species in sympatry. In contrast, the presence or absence of a dorsal stripe showed evidence of character displacement, corroborating an earlier claim made by Highton. These results are unexpected in that features associated with feeding (cranial morphology) are expected to often exhibit character displacement if dietary resource partitioning is an important mechanism of coexistence, whereas coloration might be expected to show local convergence if its primary function is camouflage or aposematism. Convergence might reflect the overwhelming influence of developmental responses to shared environments or convergent adaptation to local prey communities. Displacement with respect to color polymorphism might be consistent with frequency-dependent selection maintaining alternative ways of being cryptic.

Introduction: Uveal melanoma (UM) is an intraocular tumor that affects 4-6 people per million in the United States, 40% of which will develop liver metastasis within five years. Vascular endothelial growth factor A (VEGF-A) has been extensively studied in cancer due to the requirement for additional vasculature in tumors. Bevacizumab is a humanized monoclonal antibody that binds VEGF-A and is approved for the treatment of various malignancies. Herein, we investigated the expression of VEGF-A in three UM cell lines and the effects of inhibiting VEGF-A on their functional abilities. We also determined if positive VEGF-A expression can predict metastasis in a UM model and in patients. Materials and Methods: VEGF-A secretion was evaluated in three UM cell lines using sandwich ELISA. Proliferation, migration, and invasion assays were performed before and after the administration of bevacizumab and cytokine expression was assessed by ELISA. One cytokine that was upregulated following bevacizumab treatment (CCL3) was knocked down using siRNA, and the functional assays were repeated using CCL3 siRNA and combination treatments. Uveal melanoma tumors (n = 27) from an animal model and tumors from human patients (n = 29) were immunostained for VEGF-A. Two different custom immunostaining quantification algorithms implemented with ImageJ software were used to automatically count positive cells and positive staining area. Binary logistic regression was performed to determine if positive staining could predict metastases. P < 0.05 was considered significant. Results: All three UM cell lines produced and secreted VEGF-A and expressed VEGF-R2. Bevacizumab usurped VEGF-A and reduced phosphorylated-VEGF-R2. Proliferation and migration were reduced following bevacizumab treatment (p < 0.05 for one cell line, and p < 0.05 for two cell lines, respectively). Both CCL3 and MMP-9 were significantly upregulated after bevacizumab treatment in all cell lines (p < 0.05). Proliferation was significantly reduced in all three cell lines following CCL3 siRNA and combination treatments (p < 0.05). Migration was significantly reduced in all three cell lines after combination treatment (p < 0.05) and invasion was significantly reduced in two cell lines following siRNA (p < 0.05). In general, combination therapy was more effective than bevacizumab monotherapy for inhibiting UM functional abilities. In both rabbit and human tissue, VEGF-A positivity could be used to significantly predict metastasis (p > 0.05). Conclusions: Auto and paracrine VEGF-A signalling is abundant in UM cell lines, but inhibiting VEGF-A has only moderate effects on their functional abilities. This is attributable to compensatory effects, including the upregulation of CCL3. In addition, VEGF-A staining correlated with metastatic development in our patient cohort. VEGF-A is a powerful cytokine that may play a role in UM development and progression. However, compensatory effects induced by bevacizumab suggest that dual or multiple therapies may be required to effectively treat this tumor. / Introduction: Le mélanome uvéal (MU) est une tumeur intra-oculaire qui affecte 4 à 6 personnes par million aux États-Unis, dont 40% développeront des métastases du foie dans les cinq ans. Le facteur de croissance vasculaire endothélial A (VEGF-A) a été largement étudié sur le cancer en raison de l'exigence de la vascularisation des tumeurs supplémentaires. Le bevacizumab est un anticorps monoclonal humanisé qui se lie au VEGF-A et il est approuvé pour le traitement de diverses tumeurs malignes. Ici, nous avons étudié l'expression du VEGF-A dans trois lignées cellulaires du MU et les effets de l'inhibition du VEGF-A sur leurs capacités fonctionnelles. Nous avons également déterminé si VEGF-A positif d'expression ne peut prédire les métastases dans un modèle UM et chez les patients. Matériaux et méthodes: VEGF-A sécrétion a été évaluée dans trois lignées cellulaires du MU en utilisant ELISA en sandwich. Des tests de prolifération, la migration et l'invasion ont été réalisés avant et après l'administration de l'expression des cytokines et bevacizumab a été évaluée par ELISA. Une cytokine qui a été régulée à la hausse après le traitement bevacizumab (CCL3) a été renversée à l'aide de siRNA, et les tests fonctionnels ont été répétés en utilisant CCL3 siRNA et des traitements combinés. Des tumeurs de MU (n = 27) provenant d'un modèle animal et des tumeurs provenant de patients humains énucléés (n = 29) ont été immunocolorées pour le VEGF-A. Deux différents algorithmes de quantification immunomarquage mis en œuvre avec le logiciel ImageJ ont été utilisés pour compter automatiquement des cellules positives pour la région et une coloration positive. La régression logistique binaire a été effectuée pour déterminer si une coloration positive pouvait prédire les métastases. P <0,05 était considérée comme significative. Résultats: Les trois lignées cellulaires de MU ont produites et sécrétées du VEGF-A et ont exprimées du VEGF-R2. Le bevacizumab usurpé VEGF-A et réduit phosphorylée-VEGF-R2. La prolifération et la migration ont été réduites à la suite du traitement par le bevacizumab (p <0,05 pour une lignée cellulaire, et p <0,05 pour les deux lignées cellulaires, respectivement). Les deux CCL3 et MMP-9 étaient significativement augmentés à la hausse après le traitement par le bevacizumab dans toutes les lignées cellulaires (p <0,05). La prolifération a été réduite de façon significative dans les trois lignées cellulaires à la suite CCL3 siRNA et des traitements combinés (p <0,05). La migration a été significativement réduite dans les trois lignées cellulaires après le traitement combiné (p <0,05) et l'invasion a été considérablement réduite dans les deux lignées cellulaires suivantes siRNA (p <0,05). En général, la thérapie combinée était plus efficace que la monothérapie bevacizumab pour inhiber les capacités fonctionnelles du MU.Dans les deux lapins et les tissus humains, la présence du VEGF-A pourrait être utilisée pour prédire de façon significative des métastases (p> 0,05). Conclusions: La signalisation autocrine et paracrine du VEGF-A est abondante dans les lignées cellulaires du MU, mais l'inhibition du VEGF-A n'a que des effets modérés sur ses capacités fonctionnelles. Cette situation est attribuable à des effets compensatoires, y compris la régulation positive du CCL3. En outre, le VEGF-A coloration peuvent être utilisées pour identifier les patients prédisposés au développement métastatique. Le VEGF-A est une cytokine puissante qui peut jouer un rôle dans le développement et la progression du MU. Cependant, les effets compensatoires induits par le bevacizumab suggèrent que les thérapies doubles ou multiples peuvent être nécessaires pour traiter efficacement ce type de tumeur.

The aim of this work was to examine the effects of chlorine (Cl2) gas inhalation on airway dysfunction in a mouse model of irritant-induced asthma. Cl2 inhalation induces airway hyperresponsiveness (AHR), bronchial epithelial cell damage, increased oxidative stress and airway inflammation, characterized by neutrophilia. While the effects of Cl2 inhalation had been previously characterized, mechanisms driving airway injury induced by Cl2 remained elusive. We hypothesized that oxidative stress following Cl2 was pivotally involved in mechanisms leading to airway injury. First, using antioxidants we aimed to further characterize the effects of Cl2 inhalation by evaluating airway mechanics, pulmonary antioxidant activity, lung inflammatory profiles and bronchial epithelial cell proliferation. We established that administration of antioxidants post-Cl2 inhalation was effective at ameliorating AHR, oxidative stress, bronchial epithelial cell proliferation, and airway inflammation, suggesting indirect mechanisms were responsible for Cl2-induced airway damage. Next, we depleted several inflammatory cell types in an attempt to determine which cell type, if any, was contributing to the observed effects of Cl2. We depleted macrophages, eosinophils and neutrophils and found that only depletion of neutrophils was effective at reducing airway hyperresponsivness and prevented endogenous antioxidant activity as measured by nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation. Concomitant experiments established Cl2 increased the pro-inflammatory lipid mediators cysteinyl-leukotrienes and mRNA levels of cysLT-related biosynthetic genes. Given the role of cysLTs and their receptor cysLTr1 as established pro-asthma mediators, we chose to explore the role of cysLTr1 in our model by utilizing cysLTr1 deleted mice (cysLTr1-/-). We found that deletion of cysLTr1 worsened measured outcomes following Cl2. Compared to wild-type mice, cysLTr1-/- demonstrated increased AHR, airway neutrophilia and epithelial cell apoptosis. In contrast to wild-type mice, we observed that cysLTr1-/- did not show nuclear translocation of NRF2 or increased antioxidant mRNA levels following Cl2 exposure. At baseline levels, we observed that cysLTr1-/- mice had increased e-cadherin protein expression on bronchial epithelial cells compared to wild-type mice. We propose that deletion of cysLTr1 in mice results in an increased susceptibility to Cl2 induced airway damage and dysfunction and this damage is mediated via an aberrant endogenous antioxidant response. This vulnerability to oxidant damage, likely due to increased neutrophil numbers and NRF2 idleness, may be due to an innate increase in e-cadherin, which is partially mediated via cysLTr1. Finally, we use in vitro studies to examine how exogenous cysteinyl-leukotriene D4 stimulates bronchial epithelial cells to release the neutrophil chemoattractant IL-8. We found this pathway to be dependent on the activation of the epidermal growth factor receptor. These in vitro studies establish the basis for further examination of specific pathways that may be involved in how epithelial cells may be involved in neutrophil recruitment. / L'objectif du travail présenté dans cette thèse était d'examiner les effets de l'inhalation de chlore gazeux (Cl2) sur la dysfonction des voies respiratoires chez la souris, un modèle d'asthme induit par un irritant. L'inhalation de Cl2 induit une hyperréactivité bronchique (HRB), des lésions des cellules épithéliales bronchiques, une augmentation du stress oxydatif et l'inflammation des voies aériennes qui se caractérise principalement par une neutrophilie. Même si les effets de l'inhalation de Cl2 sont bien caractérisés, en revanche, les mécanismes entraînant les lésions des voies aériennes induites par le Cl2 sont toujours méconnus. Nous émettons l'hypothèse que le stress oxydatif qui survient suite à une exposition au Cl2 joue un rôle clef dans l'apparition des lésions des voies aériennes. Tout d'abord, les effets de traitements antioxydants sur l'inhalation de Cl2 ont été caractérisés en évaluant les fonctions respiratoires, l'activité antioxydante et le profil inflammatoire pulmonaire ainsi que la prolifération des cellules épithéliales bronchiques. Nous avons établi que l'administration d'antioxydants à la suite d'une inhalation de Cl2 améliore l'HBR, le stress oxydatif, la prolifération des cellules épithéliales bronchiques et l'inflammation des voies respiratoires. Ces résultats suggèrent que des mécanismes indirects sont responsables des lésions des voies respiratoires induites par Cl2. Ensuite, afin de déterminer quel(s) type(s) de cellules contribuent, le cas échéant, aux effets du Cl2, nous avons effectué une déplétion de plusieurs types de cellules inflammatoires : macrophages, éosinophiles et neutrophiles. Nous avons constaté que seule la déplétion des neutrophiles permet de réduire l'hyperréactivité bronchique et de prévenir l'activation des facteurs antioxydants endogènes, telle que mesurée par la translocation nucléaire du facteur nucléaire (erythroid-derived 2)-like 2 (NRF2). L'exposition au Cl2 augmente le taux de médiateurs pro-inflammatoires lipidiques cystéinyl-leucotriènes (cysLTs) ainsi que la quantité d'ARNm liés à la biosynthèse de ces médiateurs, dans le poumon proximal. Étant donné que les cysLTs et leur récepteur cysLTr1 sont établis comme étant des médiateurs pro-asthmatiques, nous avons choisi d'explorer le rôle de cysLTr1 dans notre modèle en utilisant des souris mutées pour le gène cysLTr1 (cysLTr1-/-). Nous avons constaté que la suppression de cysLTr1 aggrave les résultats obtenus suite à l'exposition au Cl2. Par rapport aux souris de type sauvage, les souris cysLTr1-/- montrent une augmentation de l'HBR, une neutrophilie des voies respiratoires et une apoptose des cellules épithéliales. Contrairement aux souris de type sauvage, nous avons observé que les souris cysLTr1-/- ne montrent pas de translocation nucléaire de NRF2 ni d'augmentation des niveaux d'ARNm anti-oxydantes après une exposition au Cl2. Nous avons observé que l'expression des protéines E-cadhérine dans les cellules épithéliales bronchiques des souris cysLTr1-/- est augmentée par rapport aux souris de type sauvage. Nous proposons que la suppression de cysLTr1 chez la souris cause une sensibilité accrue aux lésions et aux dysfonctionnements des voies respiratoires induites par le Cl2. Nous pensons qu'une réponse aberrante des antioxydants endogènes est à l'origine de ces dommages. Cette vulnérabilité aux dommages oxydatifs, probablement causée par l'augmentation du nombre de neutrophiles et l'absence de translocation NRF2, peut s'expliquer par une augmentation innée de l'e-cadhérine, partiellement contrôlée par cysLTr1. Contrairement à une carence chronique en cysLTr1, une exposition aiguë à LTD4 des cellules épithéliales respiratoires humaines provoque une cascade d'événements conduisant à la libération d'HB-EGF, l'activation du récepteur d'EGF et la libération IL-8. À la fois protecteurs et pro-inflammatoires/régénérateurs, les leucotriènes s'avèrent jouer un rôle complexe dans la réponse de l'hôte à une exposition au Cl2.