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Full-Text Articles in Cell Biology

Increased Expression Of Frontotemporal Dementia Risk Factor Tmem106b Alters Lysosomal And Autophagosomal Pathways, Johanna Irene Busch Jan 2016

Increased Expression Of Frontotemporal Dementia Risk Factor Tmem106b Alters Lysosomal And Autophagosomal Pathways, Johanna Irene Busch

Publicly Accessible Penn Dissertations

Frontotemporal lobar degeneration (FTLD) is an important cause of dementia in individuals under age 65. Common variants in the TMEM106B gene were previously discovered by genome-wide association (GWAS) to confer genetic risk for FTLD-TDP, the largest neuropathological subset of FTLD (p=1x10-11, OR=1.6). Prior to its discovery in the GWAS, TMEM106B, or Transmembrane Protein 106B, was uncharacterized. To further understand the role of TMEM106B in disease pathogenesis, we used immortalized as well as primary neurons to assess the cell biological effects of disease-relevant levels of TMEM106B overexpression and the interaction of TMEM106B with additional disease-associated proteins. We also ...


Endocytic Regulation Of Notch Signaling In Drosophila Melanogaster Neural Progenitor Cells, Seth Andrew Johnson Jan 2016

Endocytic Regulation Of Notch Signaling In Drosophila Melanogaster Neural Progenitor Cells, Seth Andrew Johnson

Publicly Accessible Penn Dissertations

Notch signaling is a ubiquitously used signaling pathway that is highly conserved and used throughout metazoan development. Understanding the regulation of Notch signaling is becoming increasingly important in determining the mechanism and treatment for the myriad of human Notch-related diseases. In Drosophila. melanogaster, the development of external sensory organs provides a context in which Notch can be manipulated and phenotypes can be easily interpreted. Here, we expand upon the growing field of Notch regulation through endocytic trafficking by examining the role of Numb and Sara endosomes. Numb is a potent Notch inhibitor whose function is conserved in higher organisms, but ...


Epigenetic Regulation Of The Dlk1-Meg3 Imprinted Locus In Human Islets, Vasumathi Kameswaran Jan 2016

Epigenetic Regulation Of The Dlk1-Meg3 Imprinted Locus In Human Islets, Vasumathi Kameswaran

Publicly Accessible Penn Dissertations

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease characterized by inadequate insulin secretion by the pancreatic β-cell in response to increased blood glucose levels. Despite compelling evidence that T2DM has a high rate of familial aggregation, known genetic risk variants account for less than 10% of the observed heritability. Consequently, post-transcriptional regulators of gene expression, including microRNAs and other noncoding RNAs, have been implicated in the etiology of T2DM, in part due to their ability to simultaneously regulate the expression of hundreds of targets.

To determine if microRNAs are involved in the pathogenesis of human T2DM, I sequenced ...


Car Drivers And Fuel Sources: How Distinct Signaling Domains In Chimeric Antigen Receptors Reprogram T Cells, Omkar Uday Kawalekar Jan 2016

Car Drivers And Fuel Sources: How Distinct Signaling Domains In Chimeric Antigen Receptors Reprogram T Cells, Omkar Uday Kawalekar

Publicly Accessible Penn Dissertations

With breakthroughs in synthetic biology, improved cell culture techniques and advanced genetic engineering, it has now become possible to generate bi-specific primary human T cells with desired specificities. One mode of redirecting specificity is the modification of T cells to express chimeric antigen receptors (CARs). Recent studies indicate that natural T cells have distinct biochemical and metabolic features that endow them with short lived effector or long lived memory fates. The central objective of this thesis was to investigate whether the signaling endodomain of CARs could reprogram T cells with pre-specified effector and memory fates. This thesis describes a novel ...


An Olfactory Cilia Pattern In The Mammalian Nose Ensures High Sensitivity To Odors, Rosemary Challis Jan 2016

An Olfactory Cilia Pattern In The Mammalian Nose Ensures High Sensitivity To Odors, Rosemary Challis

Publicly Accessible Penn Dissertations

In many sensory organs, specialized receptors are strategically arranged to enhance detection sensitivity and acuity. It is unclear whether the olfactory system utilizes a similar organizational scheme to facilitate odor detection. Curiously, olfactory sensory neurons (OSNs) in the mouse nose are differentially stimulated depending on the cell location. We therefore asked whether OSNs in different locations evolve unique structural and/or functional features to optimize odor detection and discrimination. Using immunohistochemistry, computational fluid dynamics modeling, and patch clamp recording, we discovered that OSNs situated in highly stimulated regions have much longer cilia and are more sensitive to odorants than those ...


Haloferax Volcanii Strategies To Regulate Type Iv Pilus Dependent Adhesion And Microcolony Formation, Rianne Nicole Esquivel Jan 2016

Haloferax Volcanii Strategies To Regulate Type Iv Pilus Dependent Adhesion And Microcolony Formation, Rianne Nicole Esquivel

Publicly Accessible Penn Dissertations

Microorganisms can utilize type IV pili to initiate and maintain biofilms - microbial communities that provide protection against stressful conditions. Because environmental conditions change suddenly, microorganisms have evolved multiple mechanisms to rapidly transition from a planktonic to sessile cell state. Despite the presence of archaea alongside bacteria throughout the environment, including the human microbiome, little is known about how these organisms form and maintain biofilms. Here we use genetic, microscopic and biochemical techniques to investigate multiple strategies the model archaeon Haloferax volcanii employs to permit effective adhesion and microcolony formation, early steps in biofilm formation and maturation, as well as eventual ...


Adventures With Rna Fish For Diagnosing Viral Infections And Exploring Single-Cell Heterogeneity In Cancer, Sydney Shaffe Shaffer Jan 2016

Adventures With Rna Fish For Diagnosing Viral Infections And Exploring Single-Cell Heterogeneity In Cancer, Sydney Shaffe Shaffer

Publicly Accessible Penn Dissertations

Single-molecule RNA FISH is a robust method for visualizing individual molecules of RNA within intact cells that has been used extensively for describing single-cell hetero- geneity in gene expression. In this thesis, we leverage RNA FISH-based technologies for two major problems in biology and medicine: rapid detection of viral infections and understanding mechanisms of resistance to targeted therapy in cancer. Until recently, RNA FISH was not a viable technology for rapid diagnostics, as the hybridization process required a minimum of 6 hours. We start by presenting a modification to the RNA FISH protocol developed by Raj et al. 2008, that ...


Uncovering Bunyavirus-Host Interactions, Mary Jane Drake Jan 2016

Uncovering Bunyavirus-Host Interactions, Mary Jane Drake

Publicly Accessible Penn Dissertations

Bunyaviruses are a large family of enveloped RNA viruses that are distributed globally and include many important human and agricultural pathogens. Compared with other pathogenic viruses, bunyaviruses are relatively understudied. Currently, there are no licensed vaccines or antivirals to treat bunyavirus infections in the United States. To better understand bunyavirus interactions with their mammalian hosts in the hopes of uncovering novel therapeutic targets, we utilized a forward genetic screening approach in a human haploid cell line (HAP1). We performed insertional mutagenesis of the HAP1 cells with a retroviral gene-trap vector and subsequently challenged the cells with recombinant vesicular stomatitis viruses ...


The Mitotic Genome: Accessibility And Transcriptional Control, Chris Hsiung Jan 2016

The Mitotic Genome: Accessibility And Transcriptional Control, Chris Hsiung

Publicly Accessible Penn Dissertations

Mitosis entails dramatic global alterations to genome structure and regulation, including

chromosome condensation, dissociation of the transcriptional machinery from chromosomes, and transcriptional silencing. Here I report studies that address the macromolecular accessibility of the mitotic genome and the control of transcriptional reactivation upon mitotic exit in a mammalian cell line. The results obtained from measuring the sensitivity of chromatin to DNase I cleavage by sequencing (DNase-seq) in pure mitotic cell populations demonstrate that macromolecular accessibility of the mitotic genome is widely preserved. Thus, steric hindrance from chromatin condensation is insufficient for explaining the eviction of transcription factors from mitotic chromatin ...


Molecular Mechanisms Of Oncogenesis & Precision Medicine Approaches For Pediatric Low-Grade Gliomas, Payal Jain Jan 2016

Molecular Mechanisms Of Oncogenesis & Precision Medicine Approaches For Pediatric Low-Grade Gliomas, Payal Jain

Publicly Accessible Penn Dissertations

Pediatric low-grade gliomas (PLGGs) are a heterogeneous group of tumors that collectively represent the most common childhood brain cancer. Despite favorable outcomes with surgical and adjuvant therapies, majority of patients suffer from long-term treatment-related morbidities and recurrent/inoperable disease. This necessitates a deeper understanding of PLGG biology to aid development of molecular diagnostics and low-toxicity targeted therapeutics.

Hitherto, PLGGs have been defined by activating mutations that dysregulate the MAPK signaling pathway, leading to clinical testing of RAF/MAPK inhibitors for PLGGs. Interestingly, recent large-scale sequencing efforts discovered novel gene fusions in PLGGs and we identified the unique recurrent association of ...


Treating Cancer With Engineered T Cell Therapies: Murine And Canine Models Of Safety And Efficacy, Jenessa Barbara Smith Jan 2016

Treating Cancer With Engineered T Cell Therapies: Murine And Canine Models Of Safety And Efficacy, Jenessa Barbara Smith

Publicly Accessible Penn Dissertations

Redirecting a patient’s T-cells against cancer shows tremendous clinical responses in certain tumor types, but potent therapies for ovarian cancer remain limited. Here we describe the preclinical development of three novel cancer immunotherapy platforms. We first isolated an ErbB2(369-377)-specific T-cell receptor (TCR) from a patient who was previously vaccinated against ErbB2, a protein ubiquitously overexpressed in ovarian cancer. We hypothesized that an ErbB2(369-377)-specific TCR can recognize endogenously processed ErbB2 protein in human cancer. This strategy re-directed human T-cells against ErbB2(369-377), conferring recognition of ErbB2(+) HLA-A2(+) tumor cell lines in vitro and in vivo. Together ...


A Study Of The Role Of Gata6 In Definitive Endoderm Specification And Β-Cell Functionality By Genome Engineering Of Pluripotent Stem Cells, Amita Tiyaboonchai Jan 2016

A Study Of The Role Of Gata6 In Definitive Endoderm Specification And Β-Cell Functionality By Genome Engineering Of Pluripotent Stem Cells, Amita Tiyaboonchai

Publicly Accessible Penn Dissertations

Human pluripotent stem cells (PSCs) provide a powerful model system for the study of early human development, disease modeling and physiology. We chose to focus our studies on monogenic diabetes using this model system. Within the pancreas, β cells are one of the most critical endocrine cells as loss of this cell type disrupts blood glucose homeostasis, leading to diabetes. Due to the limited availability of primary human cells it is difficult to study them in vitro, especially in the context of genetic disease where patient material is even more difficult to obtain. Here, we characterize endodermal progenitor (EP) derived ...


Investigation Of The Spatiotemporal Dynamics Of Camp And Pka Signaling And The Role Of Hcn4 Subunits In Anxiety-Related Behavior And Memory, Vincent Gerard Luczak Jan 2016

Investigation Of The Spatiotemporal Dynamics Of Camp And Pka Signaling And The Role Of Hcn4 Subunits In Anxiety-Related Behavior And Memory, Vincent Gerard Luczak

Publicly Accessible Penn Dissertations

In the hippocampus, long-term memory and synaptic plasticity occur through a series of coordinated intracellular signaling cascades that strengthen and stabilize subsets of synaptic connections while leaving thousands of others unaltered. Therefore, understanding how molecular signals are accurately transmitted is critical to understanding how hippocampal neurons store information. Molecules like cAMP and protein kinase A are critical components of memory and plasticity, but it is unclear how these diffusible signals are dynamically regulated to achieve the spatial and temporal specificity that underlies pathway-specific plasticity. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are ion channels that are modulated by cAMP and are ...


Akt Controls Adipocyte Function And Systemic Metabolism, Abigail Shearin Jan 2016

Akt Controls Adipocyte Function And Systemic Metabolism, Abigail Shearin

Publicly Accessible Penn Dissertations

ABSTRACT

AKT CONTROLS ADIPOCYTE FUNCTION AND SYSTEMIC METABOLISM

Abigail L. Shearin

Morris J. Birnbaum

Adipose tissue is a key regulator of energy homeostasis. Diseases with an increase or decrease in adiposity result in perturbations of systemic metabolism. The insulin signaling and insulin-like growth factor 1 (IGF-1) cascades are vital to the function of many tissues during development and in the mature organism. AKT, a Ser/Thr kinase, is a central node in the insulin and IGF-1 pathways. In the liver, much is known about the consequences when insulin-AKT signaling is lost, but adipose tissue has presented a unique challenge to ...


Molecular And Cellular Approaches Toward Understanding Dynein-Driven Motility, Swathi Ayloo Jan 2016

Molecular And Cellular Approaches Toward Understanding Dynein-Driven Motility, Swathi Ayloo

Publicly Accessible Penn Dissertations

Active transport is integral to organelle localization and their distribution within the cell. Kinesins, myosins and dynein are the molecular motors that drive this long range transport on the actin and microtubule cytoskeleton. Although several families of kinesins and myosins have evolved, there is only one form of cytoplasmic dynein driving active retrograde transport in cells. While dynactin is an essential co-factor for most cellular functions of dynein, the mechanistic basis for this evolutionarily well conserved interaction remains unclear. Here, I use single molecule approaches with purified dynein to reconstitute processes in vitro, and implement an optogenetic tool in neurons ...


Modulation Of Antitumor Immunity By The Mek Inhibitor Trametinib: Implications For Targeted Therapy Of Cancer, Michael J. Allegrezza Jan 2016

Modulation Of Antitumor Immunity By The Mek Inhibitor Trametinib: Implications For Targeted Therapy Of Cancer, Michael J. Allegrezza

Publicly Accessible Penn Dissertations

Through rational drug design, much progress has been made to develop small molecules that specifically inhibit the oncogenic signaling pathways driving malignant growth. However, the normal function of immune cells depends upon many of the same pathways inhibited by such targeted cancer therapies. Because the immune system can influence the growth of many cancers, I hypothesized that most small molecule inhibitors would have activity on leukocytes relevant in cancer, and this activity would contribute to their antitumor mechanisms. In order to test this hypothesis, I first screened a panel of over 40 small molecule inhibitors for their activity on proliferating ...


Genetic Requirement For The Rna Helicase Mov10l1 In Pirna Biogenesis, Qi Fu Jan 2016

Genetic Requirement For The Rna Helicase Mov10l1 In Pirna Biogenesis, Qi Fu

Publicly Accessible Penn Dissertations

GENETIC REQUIREMENT FOR THE RNA HELICASE MOV10L1 IN PIRNA BIOGENESIS

Qi Fu

P. Jeremy Wang

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs required for transposon silencing, germ cell development, and fertility in many eukaryotic species. However, many of the mechanisms underlying piRNA biogenesis have not been elucidated. Studies of MOV10L1 support its function as an RNA helicase in the processing of piRNA precursors. In this study, we elucidate the requirement for MOV10L1 RNA helicase activity in piRNA biogenesis.

To determine the requirement for MOV10L1 RNA helicase activity in piRNA biogenesis in vivo, we generated two knock-in mouse ...


Coordination Of Vibrio Cholerae Early Colonization Phenotypes In Response To Host Intestinal Factors, Amanda J. Hay Jan 2016

Coordination Of Vibrio Cholerae Early Colonization Phenotypes In Response To Host Intestinal Factors, Amanda J. Hay

Publicly Accessible Penn Dissertations

Vibrio cholerae causes human infection through ingestion of contaminated food and water, leading to the diarrheal disease, cholera. In aquatic environments this bacterium displays an expression profile that is distinct from that observed during infection. It can also form matrix-encased aggregates known as biofilms, typically on chitinous surfaces, which can be important for transmission and infectivity. Upon entry into the host, a tightly regulated circuit coordinates induction of two major virulence factors: cholera toxin (CT) and a toxin co-regulated pilus (TCP). This study finds that bile components present in the host intestine can affect both of these processes. Certain bile ...


Estrogens Impair Antitumor Immunity By Promoting The Accumulation Of Myeloid-Derived Suppressor Cells, Nikolaos Svoronos Jan 2016

Estrogens Impair Antitumor Immunity By Promoting The Accumulation Of Myeloid-Derived Suppressor Cells, Nikolaos Svoronos

Publicly Accessible Penn Dissertations

Estrogens are pleiotropic steroid hormones with pro- and anti-inflammatory effects that influence autoimmune disease and pregnancy. Both autoimmunity and pregnancy are similar to cancer with regard to the immune system. In established tumors, as is the case in autoimmune disease and pregnancy, the host is exposed to self or allogeneic antigens, which are capable of eliciting immune responses. However, for pregnancies to remain viable, autoimmune disease patients survive, and tumors to persist, the immune system must be at least partially tolerized to these challenges. Therefore, I hypothesize that, just as they appear to influence pregnancy and autoimmunity, estrogens’ ability to ...


Insights Into Terminal Erythropoiesis Influenced By Human Genetic Variation, Elizabeth Traxler Jan 2016

Insights Into Terminal Erythropoiesis Influenced By Human Genetic Variation, Elizabeth Traxler

Publicly Accessible Penn Dissertations

Red blood cells (RBCs) carry hemoglobin, enabling delivery of oxygen to all tissues of the body. They are the products of a highly specialized differentiation process that begins with a hematopoietic stem cell and results in an enucleated, biconcave RBC. This thesis is focused on the use of human genetic studies to gain a better understanding of the molecular processes occurring during terminal erythroid differentiation. We studied the regulation and roles of two erythroid-restricted genes, Trim58 and Hemoglobin Gamma Chain (HBG1 and HBG2, γ-globin), by using a combination of loss-of-function techniques, including RNA-interference-mediated gene suppression, a mutant mouse model, and ...


Interplay Between P53 And Epigenetic Pathways In Cancer, Jiajun Zhu Jan 2016

Interplay Between P53 And Epigenetic Pathways In Cancer, Jiajun Zhu

Publicly Accessible Penn Dissertations

The human TP53 gene encodes the most potent tumor suppressor protein p53. More than half of all human cancers contain mutations in the TP53 gene, while the majority of the remaining cases involve other mechanisms to inactivate wild-type p53 function. In the first part of my dissertation research, I have explored the mechanism of suppressed wild-type p53 activity in teratocarcinoma. In the teratocarcinoma cell line NTera2, we show that wild-type p53 is mono-methylated at Lysine 370 and Lysine 382. These post-translational modifications contribute to the compromised tumor suppressive activity of p53 despite a high level of wild-type protein in NTera2 ...


Coming Full Circle: Epithelial Plasticity And The Natural History Of Metastasis, Nicole Aiello Jan 2016

Coming Full Circle: Epithelial Plasticity And The Natural History Of Metastasis, Nicole Aiello

Publicly Accessible Penn Dissertations

The primary cause of cancer-related deaths is metastasis— the spread of cancer cells to distant organs— and yet the mechanisms underlying this process remain elusive due to the difficulty in detecting early metastatic events, which are rare, stochastic and microscopic. To investigate the cellular and molecular mechanisms of metastasis, I utilized an autochthonous mouse model of pancreatic cancer (KPCY) in which all tumor cells are genetically labeled with yellow fluorescent protein (YFP). The YFP lineage label allows for the detection and isolation of disseminated tumor cells as they delaminate from epithelial structures within the primary tumor, invade into the stroma ...


Bidirectional Interactions Between Mitochondrial Function And Cell Mechanics, Judith Kandel Jan 2016

Bidirectional Interactions Between Mitochondrial Function And Cell Mechanics, Judith Kandel

Publicly Accessible Penn Dissertations

Energetic and structural maintenance are both critical to cellular homeostasis, and clinical disease is often characterized by alterations in both of these realms. While the manifestation of pathology in each of these fields has been extensively studied, little research has been done to characterize basic, direct interactions between mitochondrial function and cell mechanics. The experiments described in this dissertation endeavored to address that gap, first by investigating the cytoskeletal and mechanical effects of mitochondrial dysfunction and then by considering the mitochondrial consequences of cytoarchitectural breakdown.

Mechanical integrity of the cell following mitochondrial dysfunction was investigated through multiple experimental platforms, including ...