It is well established that atherosclerosis, an inflammatory response to chronic injury in the blood vessel wall, plays a leading role in the development and progression of cardiovascular disease. Mineralocorticoid receptor (MR) over-activation has been implicated in atherosclerosis. In mineralocorticoid-target tissues, 11β- Hydroxysteroid dehydrogenase type 2 (11β-HSD2) inactivates glucocorticoids, conferring aldosterone specificity upon the normally unselective MR. Recent evidence suggests that 11β-HSD2 may also afford protection of MR in the cells of the vasculature, providing possible mechanisms by which MR activation may directly promote atherosclerosis. Consistent with this, Apoe-/-/11β-HSD2-/- double knockout (DKO) mice show accelerated atheroma development. The present thesis tested the hypothesis that inactivation of 11β-HSD2, allowing inappropriate activation of MR in cells of the vasculature, accelerates atherogenesis through promotion of a pro-inflammatory environment with increased endothelial cell expression of adhesion molecules and subsequent macrophage infiltration into plaques. DKO mice received either the MR antagonist eplerenone (200mg/kg/day) or vehicle in normal chow diet from 2 months of age for 12 weeks. Eplerenone significantly decreased atherosclerotic burden in brachiocephalic arteries of DKO mice, an effect that was accompanied by alterations in the cellular composition of plaques such that a more stable collagen- and smooth muscle cell- rich plaque was formed. Eplerenone treatment was also associated with a reduction in vascular inflammation as demonstrated by a significant reduction in macrophage infiltration into DKO plaques. The accelerated atherogenesis in DKO mice was clearly evident by 3 months of age, a time point at which Apoe-/- mice were completely lesion free. By 6 months, some Apoe-/- mice had developed lesions whilst all DKO mice at this age showed much larger plaques. Compared to Apoe-/- mice, the cellular composition of DKO plaques was altered favouring vulnerability and inflammation, with increased macrophage and lipid content and decreased collagen content. To investigate the possible underlying mechanisms responsible for increased inflammatory cell content, the expression of vascular cell adhesion molecule 1 (VCAM-1) was compared in DKO and Apoe-/- brachiocephalic arteries. VCAM-1 immunostaining was significantly greater on the endothelial cells of DKO arteries at 3 months compared to age-matched Apoe-/- mice. At 6 months, DKO and Apoe-/- mice had similar expression of VCAM-1. Finally, mouse aortic endothelial cells (MAECs) were used to investigate the mechanism of adhesion molecule up-regulation in the absence of 11β-HSD2. Both aldosterone and TNF-α, included as a positive control, dramatically increased VCAM-1 expression in MAECs. Spironolactone pre-treatment blocked the effect of aldosterone, suggesting an MR-mediated mechanism. Corticosterone alone had no effect on VCAM-1 expression. However, inhibition of 11β-HSD2 by pre-treatment with glycyrrhetinic acid allowed corticosterone to induce a significant increase in the number of VCAM-1-stained MAECs, demonstrating functional expression of 11β- HSD2 in MAECs. Consistent with 11β-HSD2 involvement, VCAM-1 up-regulation by corticosterone in the presence of glycyrrhetinic acid was reversed by blockade of MR with spironolactone. In conclusion, loss of 11β-HSD2 activity leading to inappropriate activation of MR in atherosclerotic mice promotes plaque vulnerability and increases vascular infiltration of macrophages which accelerates plaque growth, possibly through enhanced MR- mediated endothelial cell expression of VCAM-1.

This study explored the possibility that upregulation of Hypoxia Inducible Factor-1 (Hif-1)-responsive genes in Human Umbilical Vein Endothelial Cells (HUVEC) would promote and stabilize HUVEC formation into inchoate vascular beds within artificial collagen gels. This experiment was designed to explore the above possibility by sub-cloning Hif-1[alpha], the related chimeric construct Hif-1[alpha]/VP16, and the marker gene dsRed into retroviral expression vectors, producing retroviral vectors containing these genes, and stably transducing HUVEC using these retroviruses. Transduced HUVEC were to be observed in cell culture as well as after implantation into artificial collagen gels that have previously supported vascular bed formation by HUVEC. Our results show, preliminarily, that HUVEC transduced with Hif-1[alpha]/VP16 go into cell-cycle arrest. Attempts to transduce HUVEC with Hif-1[alpha] failed to achieve high enough transduction efficiency to determine the cells angiogenic potential. This study concluded that more experiments need to be conducted to better characterize the effects of hypoxia-responsive gene upregulation in controlling HUVEC angiogenesis and cell-cycle signaling and that straightforward transduction of HUVEC by Hif-1[alpha]/VP16 is probably not sufficient, in itself, to induce in vitro vascular bed formation.

Freshwater lake ecosystems often exhibit strong oxygen, and temperature gradients across which many zooplankton species live. Daphnia sp. vary in their ability to up-regulate hemoglobin in response to low oxygen environments. However; the role that hemoglobin up-regulation plays in diel vertical migration, and how it might mediate coexistence of Daphnia within lakes is still unclear. Using an oligotrophic lake in Ontario, I studied three distinct phenotypes of Daphnia pulicaria, which differed in the ability to up-regulate hemoglobin (classified as red, pink, and pale). Twenty-four hour surveys were conducted during the fall of 2012 and samples were drawn at 1m intervals to monitor changes in diel vertical migration. At each 1m interval Daphnia were color indexed, photographed, and preserved for genetic analysis using cellulose acetate electrophoresis. Red and pink Daphnia showed little change in distribution over the water column through time, suggesting individuals experienced little vertical migration. Pale individuals showed strong changes in vertical distribution through time suggesting vertical migration. The phenotypes are strongly correlated with multi-locus genotypes, suggesting genetic differences in migration behavior. Mesocosm experiments were used to manipulate migration over heterogeneous environments to test the hypothesis that vertical migration impacts genetic and phenotypic diversity in Daphnia pulicaria. The first mesocosm experiment contained two treatment groups; a migrating and non-migrating treatment containing the three phenotypes. The migrating treatment permitted unrestricted movement throughout the water column, and the non-migrating treatment restricted Daphnia to discrete 1m intervals. The second mesocosm experiment comprised two non-migrating treatments; red non-migrating and pale non-migrating. Results from the first set of mesocosm experiments indicate decreased genetic and phenotypic diversity in the migrating treatment. Shifts in hemoglobin up-regulation between pales and reds in the second mesocosm experiments suggest hemoglobin up-regulation is plastic, whereby pale, pink, and red individuals have the ability to up and down regulate hemoglobin. The differences in Daphnia migration patterns and the plastic response in hemoglobin up-regulation permits migrating genotypes to withstand low oxygen conditions. Overall implications of this study suggest that migration over a strong environmental gradient plays a key role in fostering phenotypic plasticity and genetic diversity in organisms living in heterogeneous environments. / Thesis (Master, Biology) -- Queen's University, 2014-04-08 12:07:42.757

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references.

[Truncated abstract] Mutations in the skeletal muscle -actin gene (ACTA1) have been shown to be one cause of a broad group of muscle disorders all termed the congenital myopathies. Over 170 different mutations have now been identified across all 6 coding exons of ACTA1 in patients presenting with muscle weakness and any one or more of the following histopathological features: nemaline rods, intranuclear rods, fibre-type disproportion, excess of thin filaments and central cores. While the identification of the causative gene has been of great comfort for affected patients and their families, with pre-natal genetic testing becoming available, the ultimate aim is to develop a therapy for these disorders. Of the therapies currently being explored for the muscular dystrophies, up-regulation of an alternative gene seemed to be one of the most promising avenues for treatment of the ACTA1 diseases. Up-regulation of utrophin, the foetal homologue of dystrophin, has been shown to be a promising therapy for the treatment of Duchenne muscular dystrophy. The main aim of my research was to determine whether up-regulation of cardiac -actin, the predominant -actin expressed in foetal skeletal muscle and in the adult heart, could be used as a therapy for the ACTA1 diseases. A proof-of-concept experiment was performed whereby skeletal muscle -actin knock-out (KO) mice (all of which die by postnatal day 9) were crossed with transgenic mice over-expressing cardiac -actin (known as Coco mice) in postnatal skeletal muscle. ... While patients that are ACTA1 nulls have been identified in a number of mainly consanguineous populations, the majority of ACTA1 mutations result in dominant disease in which the mutant protein interferes with the function of the wild-type skeletal muscle -actin. Research described in this thesis also focuses on characterizing two transgenic mouse models of dominant ACTA1 disease at the ultra-structural, cellular and functional level; this is the first step towards a proof-of-concept experiment to determine whether cardiac -actin up-regulation can dilute out the pathogenesis of dominant ACTA1 disease. It has long been noted that patients with ACTA1 disease do not have ophthalmoplegia, even in the most-severely affected individuals. Protein analysis performed on extraocular muscle (EOM) biopsies obtained from humans, sheep and pigs showed that the EOMs co-express cardiac and skeletal muscle -actin, with cardiac -actin comprising 70 % of the striated -actin pool. Thus we propose that sparing of the EOMs in ACTA1 disease is at least in part due to cardiac -actin diluting out the pathogenesis associated with expression of the mutant skeletal muscle -actin. This finding provides further support for the hypothesis that dilution of mutant skeletal muscle -actin in dominant ACTA1 disease by up-regulation of cardiac -actin may be a viable therapy for this group of devastating muscle diseases. The research contained herein has advanced the understanding of the pathobiology of skeletal muscle -actin diseases and provides strong evidence in support of cardiac -actin up-regulation as a promising therapy for these diseases.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Missouri--Columbia, 2005. / "July 2005." Typescript. Vita. Includes bibliographical references (leaves 152-167). Also issued on the Internet.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2006. / Typescript (photocopy).

Tetranychus urticae es una especie plaga importante en cítricos que puede también alimentarse de otras especies asociadas a la cubierta vegetal de este cultivo. Para determinar el efecto de la gestión de la cubierta vegetal en el control biológico de este ácaro, hemos estudiado la dinámica tanto de ácaros Tetranychidae como Phytoseiidae en cuatro parcelas comerciales de mandarino clementino en las que se aplicó tres estrategias diferentes de gestión de la cubierta vegetal: (1) suelo desnudo, (2) cubierta espontánea y (3) cubierta sembrada de Festuca arundinacea. Los resultados apuntan a que tanto los enemigos naturales (mecanismos "top-down") como la planta huésped (mecanismos "bottom-up") juegan un papel importante en la regulación de los ácaros Tetranychidae. Por un lado, la selección de dos razas de T. urticae especializadas en F. arundinacea y en Citrus clementina, en la cubierta y en el árbol respectivamente, cuando esta gramínea se utiliza como cubierta podría explicar en parte los resultados obtenidos (regulación "bottom-up") ya que esto impediría a los especímenes de una planta huésped colonizar con éxito la otra. Los ensayos de trasplante recíproco realizados muestran que las dos demos de T. urticae recogidas de clementina y F. arundinacea difieren considerablemente en su éxito en el desarrollo en el huésped alternativo y esto indica la existencia de fenómenos de adaptación local. Esta adaptación se traduciría en mecanismos "bottom-up" que evitarían que los ácaros que habitan en la cubierta colonicen con éxito la copa de los árboles. Por otro lado, la composición cualitativa de las comunidades de Phytoseiidae asociados a las diferentes cubiertas podría ser clave en la regulación de las poblaciones de T. urticae y Panonychus citri (regulación "top-down"). Los ácaros Phytoseiidae tipo I y II, depredadores especializados en Tetranychidae, se encuentran de manera consistente en la cubierta de F. arundinacea y esto puede explicar la mejor regulación de las poblaciones de ácaros Tetranychidae en los árboles asociados a esta cubierta. Por el contrario, la disposición más regular de fuentes de alimentación alternativas (polen) en la cubierta natural en relación con la cubierta de F. arundinacea, podría explicar la mayor abundancia de Phytoseiidae tipo IV en la primera. Como consecuencia, los Phytoseiidae tipo I y II, más eficaces en el control de Tetranychidae, podrían sufrir las consecuencias de ser competitivamente inferiores que el Phytoseiidae generalista tipo IV que explota el polen en la cubierta espontánea. Este hecho, en combinación con los períodos de escasez de presa, podría dar lugar a su desaparición del agroecosistema y resultar en un deficiente control de los ácaros Tetranychidae en los árboles asociados a una cubierta natural. Haciendo balance de gastos e ingresos, la cubierta más favorable fue la de F. arundinacea (entre 44,4 y 74,5% de reducción de costes en relación con la más cara). Festuca arundinacea como cubierta vegetal es una estrategia de control biológico por conservación muy recomendable para los productores de clementina. Aunque su uso no redujo las poblaciones de ácaros en los árboles por debajo del umbral económico, la disminución en la necesidad de tratamientos, hace que la adopción de esta táctica sea una alternativa beneficiosa tanto ecológica como económicamente. Nuestros resultados apuntan a la cubierta de F. arundinacea, que no permitió el establecimiento de Tetranychus evansi y ofrece una mejor regulación de P. citri y T. urticae que en suelo desnudo o cubierta natural, como la más adecuada para un control más sostenible de los ácaros Tetranychidae en cítricos.