Cell Biology Commons^™

Rnase-Mediated Protein Footprint Sequencing Reveals Protein-Binding Sites Throughout The Human Transcriptome, Ian M. Silverman, Fan Li, Anissa Alexander, Loyal Goff, Cole Trapnell, John L. Rinn, Brian D. Gregory

Departmental Papers (Biology)

Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP–RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.

Role Of Pikfyve In Platelet Lysosomal Homeostasis, Sang Hee Min

Publicly Accessible Penn Dissertations

PIKfyve is a lipid kinase that is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], and for the regulation of membrane dynamics within the endolysosomal system in mammals. Depletion of intracellular pools of PtdIns(3,5)P2 in humans and in mice is associated with neurodegeneration and early lethality. However, the biological role of PtdIns(3,5)P2 in non-neural tissues is not well understood. Platelets are hematopoietic cells that function in a variety of physiological responses. Essential to many of these functions is the activation-dependent release of effectors from distinct storage granules - alpha granules, dense granules ...

Role Of Ceramide Kinase In Breast Cancer Progression, Ania Warczyk Payne

Publicly Accessible Penn Dissertations

Recurrent breast cancer is typically an incurable disease and, as such, is disproportionately responsible for deaths from this disease. Recurrent breast cancers arise from the pool of disseminated tumor cells (DTCs) that survive adjuvant or neoadjuvant therapy, and patients with detectable DTCs following therapy are at substantially increased risk for recurrence. Consequently, the identification of pathways that contribute to the survival of breast cancer cells following therapy could aid in the development of more effective therapies that decrease the burden of residual disease and thereby reduce the risk of breast cancer recurrence. We now report that Ceramide Kinase (Cerk) is ...

Roles Of Dendritic Cells In Immunity To Toxoplasma Gondii, Christopher David Dupont

Publicly Accessible Penn Dissertations

Toxoplasma gondii is an intracellular protozoan parasite that actively invades host cells. During toxoplasmosis, dendritic cells (DCs) promote CD4⁺ and CD8⁺ T cell responses, which are critical for long-term immunity. Despite this critical role of DCs, questions remain regarding how this population is regulated during infection, and the specific types of interactions (phagocytosis or active invasion) between parasites and DCs that are necessary to induce T cell responses.

Previous studies have observed an infection-induced expansion of DC populations during toxoplasmosis, but the factors that regulate this expansion are currently unknown. Mice deficient in the cytokine Flt3L, which promotes the proliferation ...

Chromatin Compaction And Genome Reorganization During Spermatogenesis In M. Musculus And Sporulation In S. Cerevisiae, Jessica Michelle Bryant

Publicly Accessible Penn Dissertations

Gametogenesis is a complex process that results in a highly differentiated gamete capable of transmitting genetic and epigenetic information to the next generation. In the cases of mammalian spermatogenesis and yeast sporulation, an extreme post-meiotic compaction of the genome is key to gamete function. While genome compaction in sperm is reliant upon a histone-to-protamine transition, yeast spores accomplish compaction with a full complement of histones. Although the mechanisms behind such striking chromatin dynamics are largely unknown, several histone variants and post-translational modifications, especially acetylation of histone H4, have been implicated in these processes. The following studies elucidate the roles of ...

Dynamics And Fate Of The Inner Membrane Complex In Apicomplexan Parasites, Dinkorma Toure Ouologuem

Publicly Accessible Penn Dissertations

The eukaryotic phylum apicomplexa encompasses many thousands of parasite species of medical and veterinary importance, including Plasmodium sp. and Toxoplasma gondii. These obligate intracellular parasites survive and replicate within suitable eukaryotic hosts, ultimately rupturing infected cells to release parasites that can invade neighboring cells. Repeated cycles of infection and lysis are responsible for the pathogenesis associated with these parasites. Apicomplexan parasites replicate using an unusual process known as endodyogeny or schizogony, in which daughters are constructed de novo within the mother. This distinctive mode of replication relies on dynamic assembly of an organelle known as the Inner Membrane Complex (IMC ...

Effect Of Cyclosporin A On The Tumor Microenvironment, Yao Zhou

Publicly Accessible Penn Dissertations

Tumor angiogenesis is a hallmark of cancer, and plays a critical role in tumor growth, expansion, and metastasis. Both physiological and pathological angiogenesis is assumed to be regulated by the balance between pro and anti-angiogenic factors. One of the best characterized and most potent pro-angiogenic regulators is vascular endothelial growth factor, or VEGF. Calcineurin signaling is an important mediator of VEGF signaling in endothelial cells. Negative regulation of calcineurin by increased expression of its endogenous inhibitor, Down Syndrome Candidate Region-1 (DSCR1), suppresses tumor growth and angiogenesis. However, a potent pharmacological calcineurin inhibitor, the commonly used immunosuppressant cyclosporin A (CsA), significantly ...

Cellular Interactions During Motor Nerve Regeneration, Allison F. Rosenberg

Publicly Accessible Penn Dissertations

Vertebrate peripheral nerves can regenerate, enabling severed axons to reconnect with their original synaptic targets. The interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves, macrophages, and Schwann cells in live intact zebrafish using transgenic lines that label each cell type in vivo. We find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wlds ...

Engineered Materials To Measure And Regulate Cell Mechanotransduction, Brandon Lou Blakely

Publicly Accessible Penn Dissertations

The extracellular environment plays a key role in a wide array of cellular functions including migration, tissue formation, and differentiation. This thesis overviews the design of a molecular sensor to measure cellular forces and a hydrogel system to engineer angiogenic sprouting. We developed molecular force probes (FPs) that report traction forces of adherent cells with high spatial resolution, can be linked to virtually any surface, and do not require monitoring deformations of elastic substrates. FPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific amounts of force. In chapter two we overview the ...

Regulation Of Cell Signaling By Mig6 And Sprouty2 In Cancers With Egfr Mutations, Alice Macdonald Walsh

Publicly Accessible Penn Dissertations

Epidermal growth factor receptor (EGFR) mutation and overexpression promote tumorigenesis in multiple cancers. Understanding the complex EGFR regulatory network is critical for developing effective therapeutic interventions. To this end, this work investigated the functions of two incompletely characterized regulators of EGFR trafficking and signaling, mitogen-inducible gene 6 (MIG6) and Sprouty2 (SPRY2), in two cancer settings where EGFR mutation is common, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). In NSCLC cells, results indicate that MIG6, an endogenous inhibitor of EGFR activity and endocytic adaptor, is surprisingly responsible for at least half of EGFR endocytosis, suggesting that a substantial fraction ...

A Chemical-Genetic Screen For Identifying Substrates Of The Er Kinase Perk, Nancy L. Maas

Publicly Accessible Penn Dissertations

Cells constantly encounter changing environments that challenge the ability to adapt

and survive. Signal transduction networks enable cells to appropriately sense and respond

to these changes, and are often mediated through the activity of protein kinases. Protein

kinases are a class of enzyme responsible for regulating a broad spectrum of cellular

functions by transferring phosphate groups from ATP to substrate proteins, thereby

altering substrate activity and function. PERK is a resident kinase of the endoplasmic

reticulum, and is responsible for sensing perturbations in the protein folding capacity of

the ER. When the influx of unfolded, nascent proteins exceeds the folding ...

Ebna1-Specific T Cell Responses During Persistent Rhesus Lcv Infection And The Development Of A Novel Therapeutic Prototype Vaccine For Ebv-Associated Diseases, Rachel Mandy Leskowitz

Publicly Accessible Penn Dissertations

The impact of EBV on human health is substantial, but vaccines that prevent primary EBV infections or treat EBV-associated diseases are not yet available. The Epstein-Barr nuclear antigen 1 (EBNA1) is an important target for vaccination because it is the only protein expressed in all forms of latency and in all EBV-associated malignancies. The overarching goal throughout this dissertation was to determine if EBNA1 is a suitable target for vaccine development. This was addressed in two ways. First, because an improved understanding of EBNA1-specific T cell responses benefits EBV vaccine development, we characterized responses against EBNA1 of the EBV-homologous rhesus ...

Opposing Actin Networks Modulate The Mechano-Activation Of The Integrin Lfa-1 During Immunological Synapse Formation, William Andrew Comrie

Publicly Accessible Penn Dissertations

Formation of a functional immune response requires the regulated transfer of information between T cells, and APCs, which leads to a variety of functional outcomes. In many cases this information transfer occurs at a regulated area of cell - cell contact termed the immunological synapse (IS). In T cells responding to APCs there is a robust accumulation of F-actin on the T cell side of the synapse. This F-actin response is characterized by robust polymerization at the periphery of the contact site followed by centripetal flow of the network towards the center of the IS. While it is well known that ...

Proliferation And Survival Mechanisms In Soft Tissue Sarcoma And Glioblastoma Tumors, Vera Mucaj

Publicly Accessible Penn Dissertations

Soft tissue sarcomas and glioblastomas are two deadly tumors that are characterized by aggressive overproliferation, and regions of severe intratumoral nutrient and oxygen deprivation. The mechanisms by which tumors evade proliferation control signals and survive in a hostile microenvironment are active areas of investigation. This work describes two projects investigating loss of proliferation control in soft tissue sarcoma, as a result of Hippo pathway deregulation, and mechanisms of survival under stress in glioblastoma, as a result of decreased microRNA-124 (miR-124) levels. First, we demonstrate that the Hippo pathway is deregulated in soft tissue sarcoma patient samples, leading to overexpression of ...

Roles Of Tnfaip8 Protein In Cell Death, Listeriosis, And Colitis, Thomas Porturas

Publicly Accessible Penn Dissertations

TNF-alpha-induced protein 8 (TNFAIP8 or TIPE) is a newly described regulator of cancer and infection. However, its precise roles and mechanisms of actions are not well understood. Here I report the generation of TNFAIP8 knockout mice and describe their increased responsiveness to colonic inflammation and resistance to lethal Listeria monocytogenes infection. TNFAIP8 knockout mice were generated by germ line gene targeting and were born without noticeable developmental abnormalities. Their major organs including those of the immune and digestive systems were macroscopically and microscopically normal. However, compared to wild type mice, TNFAIP8 knockout mice exhibited significant differences in the development of ...

The Role Of Trim58 In Erythropoiesis, Christopher Stephen Thom

Publicly Accessible Penn Dissertations

Red blood cells (erythrocytes) deliver oxygen to all tissues of the body. Defects in red blood cell production (erythropoiesis) can cause disease. Mammalian erythropoiesis culminates in enucleation, an incompletely understood process that entails the physical separation of the nucleus and cytoplasm. The work in this thesis investigated the role of a previously uncharacterized protein named Trim58 in erythropoiesis.

Human genetic studies suggested that TRIM58 played an important role in erythroid development. In humans and mice, Trim58 expression was found to be restricted to red blood cell precursors during late stage maturation. In fact, murine Trim58 was upregulated just prior to ...

Hypoxia Inducible Factors In Cancer And Inflammation, Jessica Elizabeth Stewart Shay

Publicly Accessible Penn Dissertations

Hypoxia-inducible factors (HIFs) mediate adaptation to low O2, or hypoxia, are important at every stage of tumor initiation, and impact the progression of a variety of diseases, including colorectal cancer. This body of work investigates the role of hypoxia and HIF-mediated signaling in both tumor cells and macrophages across the natural history of inflammation-induced cancers. First, the effect of HIF inhibition in tumor parenchyma and stroma in extant colitis-associated colon carcinomas (CAC) is investigated using acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity. Pharmacologic HIF inhibition represents a novel therapeutic strategy for cancer treatment and data ...

Identification Of Molecular Mechanisms Underlying The Development Of Barrett's Esophagus, Maria Eugenia Vega

Publicly Accessible Penn Dissertations

Esophageal cancer is one of the deadliest cancers in the U.S and worldwide. Esophageal cancer is characterized by two subtypes: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). One of the major risk factors for the development of EAC is Barrett's esophagus (BE). BE is defined as incomplete intestinal metaplasia characterized by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. Currently, the cell of origin for human BE has yet to identified. Using an innovative 3D organotypic culture system, we explored the role of inhibition of Notch signaling promotion ...

Microtubule Motors Drive Nuclear Dynamics And Positioning In Developing Skeletal Muscle Cells, Meredith Hayley Wilson

Publicly Accessible Penn Dissertations

Dynamic interactions with the cytoskeleton are essential to move and anchor nuclei during tissue development, and defects resulting in nuclear mispositioning are often associated with human disease, such as muscular dystrophy and myopathy. Skeletal muscle cells are large syncytia formed by fusion of myoblasts, and contain hundreds of nuclei positioned regularly along the length the cell. During muscle cell development, nuclear movement in myotubes requires microtubules, but the mechanisms involved have not been elucidated. Here, we find that nuclei actively translocate through myotubes. As they translocate, they also rotate in three-dimensions. These movements require an intact microtubule cytoskeleton, which forms ...

Mutual Reinforcement Between Telomere Capping And Canonical Wnt Pathway Activity In The Intestinal Stem Cell Niche, Ting-Lin Yang

Publicly Accessible Penn Dissertations

Mice lacking telomerase (e.g. mTR-/-) for several generations develop dysfunctional telomeres and severe gastrointestinal pathology. Intestinal stem cell (ISC) abnormalities in late-generation mTR mice have been described, and we set out to characterize it in more detail. Expression profiling of mTR-/- crypts and an unbiased gene set enrichment analysis revealed broadly decreased expression of Wnt pathway genes in crypt epithelia and underlying stroma. We describe abnormalities in the Wnt-dependent intestinal stem cell (ISC) niche in these mice, including decreased expression of ISC marker genes Ascl2, Lgr5, and Sox9. The importance of these changes was revealed by rescue of crypt ...

T Cells Bearing A Chimeric Antigen Receptor Against The Tumor Vasculature Destroy The Tumor Endothelium And Result In Tumor Regression, Stephen Santoro

Publicly Accessible Penn Dissertations

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of pro-tumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here I describe the development of two chimeric antigen receptors (CAR)s against the tumor vasculature, targeting either tumor endothelial marker 1 (TEM1) or prostate-specific membrane antigen (PSMA). CAR T cells incorporating scFv78, an scFv isolated against TEM1, were able to recognize immobilized plate-bound TEM1 protein, but were unable to recognize TEM1 on the surface of endothelial cell targets. In contrast, anti-PSMA CAR ...

Degradation Of The Oncoprotein Mdmx In Neurodegenerative States: Evidence For A Pro-Survival Role Of Mdmx In Neurons, Daniel James Colacurcio

Publicly Accessible Penn Dissertations

Neurodegenerative diseases, such as Alzheimer Disease (AD) and HIV–associated neurocognitive disorder (HAND) represent a tremendous burden to healthcare and a devastating impact on society. A common feature of neurodegenerative diseases is progressive dysfunction and death of neurons in key regions of the brain. Observations of dysregulated cell cycle proteins in the brains of patients, along with research in animal models and cultured cells, suggest that aberrant functions of cell cycle proteins contribute to neuronal death and progression of neurodegenerative diseases.

The p53 tumor suppressor is a key component in cell cycle signaling and cell death. p53 maintains multiple functions ...

Distinct Patterns Of Ccr5 Versus Alternative Coreceptor Dependence In Non-Natural Host Versus Natural Host Simmian Immunodeficiency Virus Infection, Sarah Tc Elliott

Publicly Accessible Penn Dissertations

Natural host sooty mangabeys infected with simian immunodeficiency viruses (SIV) exhibit high plasma viral loads without widespread CD4+ T cell loss. By contrast, non-natural host rhesus macaques experimentally infected with related SIV exhibit high viral loads but display subsequent CD4+ T cell loss and progression to AIDS, analogous to the effects of HIV-1 infection in humans. Several mechanisms have been proposed to explain these discrepant outcomes, including infection of distinct target cells in vivo. Cell targeting is substantially determined at the level of viral entry. Prior work demonstrated that sooty mangabey infection occurs in the absence of functional coreceptor CCR5 ...

Mechanochemical Control Of Stem Cell Biology In Development And Disease: Experimental And Theoretical Models, Polimyr Caesar Dave Pelisco Dingal

Publicly Accessible Penn Dissertations

Whether a stem cell remains or egresses away from its physiological niche is a function of mechanical and soluble factors in a time-dependent manner, which implicates a `memory' of prior mechanochemical conditioning. Virtually every organ in the body contains resident stem or progenitor cells that contribute to organ homeostasis or repair. The wound healing process in higher vertebrate animals is spatiotemporally complex and usually leads to scarring. Limitations for the use of stem cells as regenerative therapy include the lack of expansion capabilities in vitro as well as materials issues that complicate traditional biochemical protocols. A minimal `scar in a ...

Epigenetic Regulation Of Progenitor Cell Commitment By Hdac3, Mudit Gupta

Publicly Accessible Penn Dissertations

Tissue-specific progenitor cells emerge during development to expand and differentiate into the multiple cell lineages that populate the embryo. Appropriate differentiation of these precursor cells requires coordinated expression of numerous lineage-specific genes and repression of alternative fate programs. Epigenetic regulators are enzymes capable of activating or silencing large genomic domains by altering histone modifications, DNA methylation status and chromatin organization. Although differentiating progenitor cells undergo epigenetic changes and epigenetic factors are required for appropriate cell behavior, the precise mechanism of how these proteins influence cell fate remains unclear. In this dissertation, I examine the role of histone deacetylase 3 in ...

Recirculation Of Innate Lymphocyte Subsets In The Skin, Skye Geherin

Publicly Accessible Penn Dissertations

The trafficking of innate-like lymphocytes, such as γδ T cells and B-1 B cells, has garnered comparatively little attention from the immunological community relative to conventional T and B cells. However, recent studies have shown that innate-like cell subsets are critical for immune regulation and host defense. In this study, we use a classic ovine lymph cannulation model to describe the phenotype and function of γδ T cells migrating through the skin. We find that γδ T cells traveling in the skin-draining afferent lymph are IFN-γ- and/or IL-17-producing effector cells that express high levels of the skin- and inflammation-seeking ...

Role Of Cytoskeletal Remodeling In T Cell Receptor Signaling And Integrin Activation At The Immunological Synapse, Alexander Babich

Publicly Accessible Penn Dissertations

The efficiency of an immune response critically depends on the ability of T cells to respond to antigens. Upon encountering cognate antigenic peptides on the surface of antigen-presenting cells, T cells form a specialized interface, termed the immunological synapse (IS), which serves as the site of information transfer between the cells. This contact zone is characterized by the enrichment of signaling receptors, kinases and adaptor proteins, and is the site of extensive cytoskeletal remodeling. The versatile nature and spatio-temporal regulation of signaling cascades at the IS has long been recognized but the exact mechanisms that coordinate these processes remain poorly ...

Epithelial Cell Shape Changes During Lung Branching Morphogenesis: The Role Of Wnt/Fzd2 Signaling In Directing New Branch Formation, Rachel S. Kadzik

Publicly Accessible Penn Dissertations

Formation of the intricately branched mammalian lung requires precise coordination between the epithelium and mesenchyme over the course of development. This coordination is mediated by molecular signaling between the two tissue compartments. How these signaling pathways coordinate changes in cellular and tissue morphology to give rise to the highly ramified branched network of the lung is not well understood. In this work, I show that signaling through Frizzled 2 (Fzd2) is required for promoting changes in epithelial cell shape that lead to tissue-level changes needed for branching morphogenesis in the lung. Through analysis of both fixed lungs and live imaging ...

Membrane Forces And Key Protein Determinants Of Hematopoietic Cell Function: Lamins And Myosin-Ii In Hematopoiesis And Cd47 In Immunotherapy Of Cancer, Kyle Spinler

Publicly Accessible Penn Dissertations

Hematopoiesis in human bone marrow generates every second about 10⁵ – 10⁶ anucleated platelets and red blood cells as well as nucleated white blood cells that are capable of infiltrating distant tissues. The thesis begins in the marrow with a description of (1) nuclear membrane ‘lamina’ physicochemical properties that influence marrow-to-circulation trafficking, and proceeds to detail (2) the physicochemical roles of membrane cortex ‘myosin’ in key marrow processes of motility and division as well as platelet biogenesis and disease. The thesis finishes with (3) studies of macrophages in peripheral tissues far from the marrow and aspects of how such ...

Regulation Of Zygotic Transcription And Cell Cycle Checkpoints In Early Embryogenesis, Xiang Maomao Zhang

Publicly Accessible Penn Dissertations

For many organisms, the first goal of embryogenesis is to accumulate a large cell population to accommodate gastrulation. To achieve this quickly, embryos employ specialized cell cycles called cleavages that consist of continuous rounds of DNA replication and division. Cell proliferation occurs rapidly because cleavage cycles lack the gap phases and cell cycle checkpoints found in canonical cell cycles. Further, the genetic materials required to sustain cleavage cycles are preloaded during oogenesis, aiding efficient cell cycle progression. After a constant, organism-specific number of cleavages, many metazoan embryos undergo the mid-blastula transition (MBT), which initiates extensive cell cycle remodeling. Cell cycles ...