Digital Commons Network^™

Autophagy Over The Lifespan: Using Fetal, Stem Cell, And Adult Retinal Pigment Epithelium (Rpe) Cultures To Model Amd Pathogenesis, Katherine Jean Davis

Yale Medicine Thesis Digital Library

Purpose: Dysfunctional autophagy in the retinal pigment epithelium (RPE) has been implicated as a therapeutic target in age-related macular degeneration (AMD). To explore how RPE autophagy changes over the lifespan and in response to phagocytosis of photoreceptor outer segments (POS), we compared stem cell-derived RPE (hESC-RPE, iPS-RPE), human fetal RPE (hfRPE), the ARPE-19 cell line, and adult RPE (ad-RPE).

Methods: RPE was cultured from 16-week human fetuses and cadaveric eyes. Stem cell- derived RPE was prepared from human embryonic stem cells (hESC-RPE) and induced pluripotent stem cells (iPS-RPE). LC3 conversion (immunoblotting) and changes in autophagy-related gene expression (qRT2-PCR) were used …

Novel Mechanisms Of Trophoblast Responses To Antiphospholipid Antibodies And Therapeutics In Obstetric Antiphospholipid Syndrome, Stefan Mathias Gysler

Yale Medicine Thesis Digital Library

Obstetric antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by circulating antiphospholipid antibodies (aPL) and an increased risk of recurrent pregnancy loss and preeclampsia. Current treatments, such as low molecular weight heparin (LMWH) may maintain pregnancy but do not reduce the risk of these late gestational complications. aPL trigger placental inflammation by activating trophoblast Toll-like receptor 4 (TLR4), leading to cytokine production. Since some microRNAs (miRs) regulate TLR responses, this study sought to determine the functional role of aPL-induced miR expression in regulating trophoblast inflammation. Since vitamin D deficiency is common in APS patients, the effect of vitamin D …

Syndecan2 Controls Vascular Smooth Muscle Development And Cytoskeletal Dynamics, Deepak Atri

Yale Medicine Thesis Digital Library

Syndecan2 is a member of the Syndecan family of transmembrane proteoglycans, expressed broadly in mammals. Syndecan2, along with Syndecan4, is expressed in the vasculature of mouse and man. Global deletion of Syndecan2 in mice does not affect viability and fertility of mice. However, it does result in defects in vascular smooth muscle cell coverage of the dermal vasculature of the mouse embryo.

We here report that lineage specific deletion of Syndecan2 in vascular smooth muscle results in delayed development of vascular smooth muscle coverage in the developing retinal vasculature. Postnatally, mice that lack Sdc2 in vascular smooth muscle mice exhibit …

Tissue Engineering The Neural Retina With Human Embryonic Stem Cells: Exploring The Role Of The Retinal Pigment Epithelium, Yu Cheng Peter Zhao

Yale Medicine Thesis Digital Library

In order to restore vision in the late stages of retinal degeneration, it is necessary to address loss of cells from both the retinal pigment epithelium (RPE) and the neural retina. As a step towards generating neural retinal tissue for translational studies, we evaluated a biodegradable polymer scaffold as a three-dimensional vehicle for the directed differentiation of H9 human embryonic stem cells (H9-hESC) into neural retina. Polymer scaffolds were electrospun from 14% w/v polycaprolactone (PCL), approximated the thickness of native neural retina, and consisted of loose, randomly oriented fibers of subcellular diameter. H9-hESC were seeded to PCL scaffolds, cultured in …

Langerhans Cell Contributions To Ultraviolet Light-Induced Cutaneous Carcinogenesis, Marianna Freudzon

Yale Medicine Thesis Digital Library

Epidermal dendritic cells, called Langerhans cells (LC), were recently shown to facilitate DMBA/TPA-induced chemical carcinogenesis. LC contribution to UV-induced carcinogenesis, however, remains to be elucidated. We used the Langerin-DTA transgenic mouse model that lacks LC to characterize early and late effects of DNA damage in epidermis following exposure to UVB light. We found that LC density in LC-intact mice remains unchanged at 1hr and 24hr post 100 J/m2 UVB, suggesting that LC are poised to affect the surrounding epidermal cells following acute low-dose UVB exposure. During this time interval, LC-deficient mice had significantly fewer γH2Ax-positive cells within the epidermis, indicative …

Characterization Of The Effect Of Braf Inhibitors On Melanoma Metabolism In Vitro And In Vivo, Alexander Marzuka

Yale Medicine Thesis Digital Library

Vemurafenib is the first FDA-approved personalized treatment for metastatic melanoma to show an improvement in survival. This serine threonine kinase inhibitor targets the mutated BRAF V600E protein, which occurs in approximately 50% of melanomas. The downstream effect of BRAF V600E blockade is inhibition of cell proliferation. Little is known about the effect of vemurafenib on glucose metabolism in melanoma cells. The Warburg effect, or the use aerobic glycolysis to generate energy and building blocks for cell proliferation, is a hallmark of cancer. Normal cells, in contrast, metabolize glucose through oxidative phosphorylation in the presence of oxygen and through glycolysis in …

Sirna Therapy In Glioblastoma Stem Cells: Identification Of Target Genes And Potential Therapeutic Implications., Benjamin Himes

Yale Medicine Thesis Digital Library

Glioblastoma multiforme (GBM), the most common primary brain malignancy, carries a grim prognosis; survival statistics have scarcely improved in decades. Even with the development of temozolomide, the current front-line chemotherapeutic agent for GBM, improvement in long-term survival has been minimal, with recurrence virtually assured. One explanation for the persistence of this disease is the presence of a stem-like cell population within GBM (glioblastoma stem cells, or GSCs). These cells are capable of self-renewal, tumor initiation, and are resistant to chemotherapy. We hypothesized that derangement in the expression of genes critical for the maintenance of GSCs could eliminate these cells outright, …

Statin Inhibition Of Macrophage Integrin-Induced Rac2-Myosin Iia Interaction: An Anti-Inflammatory Effect, Kenneth E. Ike

Yale Medicine Thesis Digital Library

STATIN INHIBITION OF MACROPHAGE INTEGRIN-INDUCED RAC2-MYOSIN IIA INTERACTION: AN ANTI-INFLAMMATORY EFFECT.

Kenneth E. Ike, Alan Morrison, and Jeffrey R. Bender. Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University, School of Medicine, New Haven, CT.

HMG-CoA reductase inhibitors (statins) are pharmaceuticals that are utilized for the treatment of lipid disorders along with the primary and secondary prevention of coronary heart disease. HMG-CoA reductase is the rate-limiting enzyme in cholesterol synthesis, converting HMG-CoA to mevalonate. The isoprenoid products, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), are derived from the mevalonate pathway and serve as substrates in the prenylation of 2% …

The Effect Of E-Cadherin Loss On Melanoma Formation And Metastasis, Lara Rosenbaum

Yale Medicine Thesis Digital Library

Melanoma is the most lethal form of skin cancer and accounts for the majority of skin cancer related mortality. Early metastases are characteristic of melanoma and are an ominous sign, as current therapeutic interventions have little effect on overall survival. The lack of accurate prognostic indicators and effective therapies emphasize the need for a better understanding of the genetic and phenotypic changes in melanoma formation and progression. One such change is the loss of E-cadherin, which normally plays a role in cell-cell adhesion and is thought to be a central feature of epithelial-mesenchymal transition (EMT). Maintenance of E-cadherin in melanoma …

Investigating The Influence Of Langerhans Cells On Keratinocyte Proliferative Response To Chemical And Ultraviolet B (Uvb) Exposure, Kseniya Golubets

Yale Medicine Thesis Digital Library

Initially, the Girardi lab demonstrated that LC facilitate chemical carcinogenesis by showing lower levels of proliferation in LC-deficient (Langerin-diptheria toxin A transgenic) mice relative to LC-intact littermate controls. Thus, we sought to explore the breadth of potential influences of LC on keratinocyte responses (hypertrophy, apoptosis, and clonal expansion) in two classic models of carcinogenesis (chemical and UVB), by using immunohistochemical (IHC) and immunofluorescent (IF) methods of cell identification. At baseline, LC-deficient compared to LC-intact mouse skin showed comparable proliferation levels as measured by Ki-67 IHC (13.6±2.6 vs 12.7±2.8 Ki-67+ cells, NS) and epidermal thickness (7.9±1.8 vs 6.9±2.8 micrometers, NS). After …

Nonsense Mutations In The Human Beta Globin Gene Affect Mrna Metabolism, Susan J. Baserga

Yale Medicine Thesis Digital Library

A common mutation causing thalassemia in Mediterranean populations is an amber (UAG) nonsense mutation at the 39th codon of the human β globin gene, the β-39 mutation. Studies of mRNA metabolism in reticulocytes from patients with β-39 thalassemia and studies using heterologous transfection systems have suggested the possibility that this mutation affects not only protein synthesis but also alters mRNA metabolism. This phenomenon has been investigated further by two approaches. A careful series of RNA expression studies were performed comparing expression of β-39 to β-normal (β-nl). These experiments led to the conclusion that the defect in expression of the β-39 …