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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Surface Patterns On Single Cells: A Consequence Of A Phase Transition To Modulated Phases, Asja Radja Jan 2019

Surface Patterns On Single Cells: A Consequence Of A Phase Transition To Modulated Phases, Asja Radja

Publicly Accessible Penn Dissertations

Patterns are ubiquitous in the world around us, and we have only begun to scratch the surface of understanding their complexity and formation. In this thesis, we draw inspiration from rigid, extracellular surface patterns found on single living cells in many taxa and try to understand if there is a common thread in their pattern formation mechanisms that can be described by a single physical formalism. Pollen grains, butterfly wing scales, and deep-sea protists called phaeodarians all have beautifully ornate and varied hard surface structures that are likely patterned by the deposition of some soft organic matrix originating inside of ...


Post-Transcriptional Regulation Of The Eulkaryotic Transcriptome By The Covalent Rna Modicication N6-Methyladenosine, Stephen J. Anderson Jan 2019

Post-Transcriptional Regulation Of The Eulkaryotic Transcriptome By The Covalent Rna Modicication N6-Methyladenosine, Stephen J. Anderson

Publicly Accessible Penn Dissertations

Post-Transcriptional regulation of the eukaryotic transcriptome by the covalent RNA modification N6-methyladenosine

Stephen James Anderson

Brian Gregory

Once a messenger RNA molecule is transcribed, a myriad of RNA fate decisions must be made. How these fate decisions are made is often unclear, and elucidating factors determining these fate outcomes is an essential task in order to fully understand gene regulation. One poorly- understood but undoubtedly important factor in post-transcriptional gene regulation is the covalent modification of ribonucleotides. Much like DNA can have chemical groups added to a nucleotide within its primary sequence, RNA can be modified in a similar manner ...


Unraveling The Beta Cell Translatome: Elucidation Of An Erk/Hnrnpk/Jund Axis, Austin Lewis Good Jan 2019

Unraveling The Beta Cell Translatome: Elucidation Of An Erk/Hnrnpk/Jund Axis, Austin Lewis Good

Publicly Accessible Penn Dissertations

In type 2 diabetes, oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation, however, the mechanisms underlying this process in β cells are not fully understood. Here we use TRAP-seq to examine changes in the ribosome occupancy of mRNAs during conditions associated with β cell dysfunction, leading us to identify a cohort of translationally regulated genes with 3’UTR enrichment of a cytosine-rich motif. Of particular interest was the gene encoding JUND, a transcription factor with anti-oxidant functions in other cell ...


Nucleobase, Nucleoside, And Neighboring Nucleotides: Intrinsic Preferences For Tet Enzyme-Mediated Oxidation Of 5-Methylcytosine, Jamie Elizabeth Denizio Jan 2019

Nucleobase, Nucleoside, And Neighboring Nucleotides: Intrinsic Preferences For Tet Enzyme-Mediated Oxidation Of 5-Methylcytosine, Jamie Elizabeth Denizio

Publicly Accessible Penn Dissertations

The Ten-eleven-translocation (TET) family of enzymes can oxidize the fifth base of DNA, 5-methylcytosine (mC) sequentially, to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). The biochemical preference of TET enzymes for these substrates, in the canonical cytosine guanine dinucleotides (CpG), mimics the order in which they are generated and is reflected in levels of these oxidized modifications (oxmCs) detected in various genomes. Other than this exception, there is conflicting or limited data concerning intrinsic substrate preferences of TET, particularly with regards to different nucleic acid structures, sequence contexts, and extent to which TET mediates oxmCs in clustered proximity to one ...


The Role Of P120catenin In The Initiation And Dissemination Of Pancreatic Cancer, Basil Bakir Jan 2019

The Role Of P120catenin In The Initiation And Dissemination Of Pancreatic Cancer, Basil Bakir

Publicly Accessible Penn Dissertations

Pancreatic cancer is the third leading cause of cancer mortality, the overwhelming majority of which is due to metastasis. The mechanisms of dissemination have been intensively studied, but questions remain as to how epithelial properties are regulated between the primary tumor site and destination as well as to how organotropic patterns of metastasis are determined. We demonstrate, using multiple complementary mouse models, that liver and lung metastatic organotropism in pancreatic cancer is dependent on p120catenin (p120ctn)-mediated epithelial identity. Monoallelic p120ctn loss accelerates KrasG12D-driven PanIN/PDAC formation and metastasis to the liver as well as being sufficient for E-CADHERIN-mediated cell ...


Miniaturizing High Throughput Droplet Assays For Ultrasensitive Molecular Detection On A Portable Platform, Venkata Ravitheja Yelleswarapu Jan 2019

Miniaturizing High Throughput Droplet Assays For Ultrasensitive Molecular Detection On A Portable Platform, Venkata Ravitheja Yelleswarapu

Publicly Accessible Penn Dissertations

Digital droplet assays – in which biological samples are compartmentalized into millions of femtoliter-volume droplets and interrogated individually – have generated enormous enthusiasm for their ability to detect biomarkers with single-molecule sensitivity. These assays have untapped potential for point-of-care diagnostics but are mainly confined to laboratory settings due to the instrumentation necessary to serially generate, control, and measure millions of compartments. To address this challenge, we developed an optofluidic platform that miniaturizes digital assays into a mobile format by parallelizing their operation. This technology has three key innovations: 1. the integration and parallel operation of hundred droplet generators onto a single chip ...


Investigating The Dysregulation And Therapeutic Potential Of Neuroprotective Stress Response Proteins In Huntington’S Disease, Julianne M. Rieders Jan 2019

Investigating The Dysregulation And Therapeutic Potential Of Neuroprotective Stress Response Proteins In Huntington’S Disease, Julianne M. Rieders

Publicly Accessible Penn Dissertations

Huntington’s disease (HD) is a fatal, genetic neurodegenerative disease that shares many features with other common neurological disorders, including early synapse loss. In both human HD brain and murine models, apoptotic pathways are dysregulated and mammalian target of rapamycin complex 1 (mTORC1) activity is reduced. These pathways are of particular interest because they regulate cell survival and metabolism, and enhancing mTORC1 is protective in HD models. RNA binding motif protein 3 (RBM3) is a stress response protein that promotes synaptic plasticity and cell survival, and is dysregulated in Alzheimer, prion and HD models. Hippocampal overexpression of RBM3 in Alzheimer ...


The Development Of Unnatural Amino Acid-Based Probes And Methods For Biological Studies, Ismail Abd Al Azim Ahmed Jan 2019

The Development Of Unnatural Amino Acid-Based Probes And Methods For Biological Studies, Ismail Abd Al Azim Ahmed

Publicly Accessible Penn Dissertations

Proteins form a diverse ensemble of dynamic structures to carry out all life-sustaining functions. Therefore, many efforts have gone into studying the structure-dynamics-function relationship of proteins using a wide range of techniques, including fluorescence and infrared (IR) spectroscopies. While very useful, intrinsic fluorescence and IR signals arising from the natural amino acid side chains within the protein are often insufficient or unable to provide the information needed to understand the biological question of interest. To this end, various extrinsic spectroscopic probes, such as fluorescent dyes, have been used to increase the information content in specific measurements and applications. However, incorporation ...


Role Of The Nuclear Receptor Pparγ In Clear Cell Renal And Bladder Urotheial Carcinoma, Danielle Sanchez Jan 2019

Role Of The Nuclear Receptor Pparγ In Clear Cell Renal And Bladder Urotheial Carcinoma, Danielle Sanchez

Publicly Accessible Penn Dissertations

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) has a well-characterized role in the developmental process of adipogenesis and transcriptional regulation of lipid metabolism. However, its expression patterns and functions in various cancer subtypes are less understood. My studies investigate the role of PPARγ in two distinct cancers of the urinary tract: clear cell renal cell carcinoma (ccRCC) and bladder urothelial carcinoma (UC). In ccRCC, I hypothesized that PPARγ activity contributes to the aberrant lipid accumulation phenotype characteristic of this disease, thereby promoting tumor progression. Through ChIP-seq, I demonstrated that PPARγ and its heterodimeric DNA binding partner retinoid X receptor ...


The Regulation Of Egfr Signaling And Kras Tumorigenesis By Receptor Palmitoylation, Akriti Kharbanda Jan 2019

The Regulation Of Egfr Signaling And Kras Tumorigenesis By Receptor Palmitoylation, Akriti Kharbanda

Publicly Accessible Penn Dissertations

Non-Small Cell Lung Cancer (NSCLC) is often characterized by mutually exclusive mutations in epidermal growth factor receptor (EGFR) or KRAS. The mutual exclusivity of these mutations is due to synthetic lethality, revealing a potential therapeutic vulnerability if possible to selectively activate EGFR in KRAS mutant cells. This thesis work demonstrates a previously unidentified mechanism of EGFR signal regulation through palmitoylation, the addition of the 16-carbon palmitate. The palmitoyltransferase, DHHC20, catalyzes this palmitoylation to Cys1025, Cys1122 and Cys1034 on the C-terminal tail of EGFR. Loss of EGFR palmitoylation leads to hyperactivation of the receptor, but decreased cell growth of KRAS mutant ...


Mass Spectrometry: An Ideal Method For Rna Modification Analysis, Samuel Peter Wein Jan 2019

Mass Spectrometry: An Ideal Method For Rna Modification Analysis, Samuel Peter Wein

Publicly Accessible Penn Dissertations

Currently there is no good way to measure and find the exact location of multiple RNA modifications. Existing technology can effectively find single varieties of modifications, but cannot identify co-occurrence. As the field of proteomics has shown, mass spectrometry is a powerful and versatile technique assessing broad ranges of chemical modifications in the context of the cellular environment. In this project I used our expertise in proteomics to build a mass spectrometry based platform for identifying RNA modifications. I have since set up both software and analytical platforms querying RNA modifications, and used this platform to survey human tRNA samples ...


Induction Of Antibodies To Hiv-1 Envelope Using Simian Adenovirus Vaccines, Kristel Lucie Emmer Jan 2018

Induction Of Antibodies To Hiv-1 Envelope Using Simian Adenovirus Vaccines, Kristel Lucie Emmer

Publicly Accessible Penn Dissertations

Human immunodeficiency virus type 1 (HIV-1) has infected 76 million people since the beginning of the epidemic. The first evidence that an HIV-1 vaccine could prevent infection in humans was provided in the RV144 vaccine efficacy trial. RV144 demonstrated 31.2% efficacy and immune correlate analyses indicated that antibodies targeting the variable 2 (V2) region of HIV-1 envelope (Env) correlated with decreased risk of infection. However, significant improvements are needed to develop a globally effective vaccine against HIV-1.

Several approaches can be employed to improve upon vaccination strategies: heterologous prime-boost regimens, immunogen design, and alternative adjuvants. To enhance Env-specific antibodies ...


Use Of Induced Pluripotent Stem Cell Models To Elucidate Retinal Disease Pathogenesis And To Develop Gene-Based Therapies, Thu Thi Duong Jan 2018

Use Of Induced Pluripotent Stem Cell Models To Elucidate Retinal Disease Pathogenesis And To Develop Gene-Based Therapies, Thu Thi Duong

Publicly Accessible Penn Dissertations

Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degenerative disease caused by mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. CHM is characterized by childhood-onset night blindness (nyctalopia), progressive peripheral vision loss due to the degeneration of neural retina, RPE and choroid in a peripheral-to-central fashion. Most of CHM mutations are loss-of-function mutations leading to the complete lacking of REP1 protein. However, the primary retinal cell type leading to CHM and molecular mechanism remains unknown in addition to the fact of lacking proper disease models. In this study, we explored the utility of induced pluripotent stem ...


Mechanosensing By The Nuclear Lamina: From Embryonic Development To Aging, Sangkyun Cho Jan 2018

Mechanosensing By The Nuclear Lamina: From Embryonic Development To Aging, Sangkyun Cho

Publicly Accessible Penn Dissertations

‘Nuclear mechanosensing’ encompasses a wide range of biophysical pathways that are emerging as key processes in the regulation of cell function and fate. Many of these mechanisms involve the main structural protein of the nucleus, lamin-A, which is abundant in stiff and mechanically stressed tissues such as striated muscle, but is comparatively low in soft tissues such as the brain. Lamin-A’s increase with tissue stiffness correlates strongly with elevated levels of collagen-I fibers in the extracellular matrix (ECM), but mechanisms and functional consequences of any matrix-nucleus interplay remain unclear. Here, in the first set of studies, we show that ...


A Tale Of Two Sirtuins: The Impact Of Sirt1 And Sirt3 On The Pathophysiology Of Shock, Carrie Adelia Sims Jan 2018

A Tale Of Two Sirtuins: The Impact Of Sirt1 And Sirt3 On The Pathophysiology Of Shock, Carrie Adelia Sims

Publicly Accessible Penn Dissertations

Both acute blood loss and severe infection activate common cellular pathways leading to shock – a pathologic condition characterized by systemic inflammation, oxidative stress, and mitochondrial dysfunction. Sirtuins, a highly conserved group of NAD-dependent enzymes, play a critical role in cellular survival and many of the benefits associated with sirtuin activation are thought to be secondary to decreased inflammation, reduced oxidative stress, and improved mitochondrial physiology. As such, we hypothesized that sirtuin pathways play a crucial role in shock and could be could be targeted to improve outcomes following acute blood loss and severe infection. In a series of in vivo ...


Reprogramming The Retina: Next Generation Strategies Of Retinal Neuroprotection And Gene Therapy Vector Potency Assessment, Devin Scott Mcdougald Jan 2018

Reprogramming The Retina: Next Generation Strategies Of Retinal Neuroprotection And Gene Therapy Vector Potency Assessment, Devin Scott Mcdougald

Publicly Accessible Penn Dissertations

Mutations within over 250 known genes are associated with inherited retinal degeneration. Clinical success following gene replacement therapy for Leber’s congenital amaurosis type 2 establishes a platform for the development of downstream treatments targeting other forms of inherited and acquired ocular disease. Unfortunately, several challenges relevant to complex disease pathology and limitations of current gene transfer technologies impede the development of gene replacement for each specific form of retinal degeneration. Here we describe gene augmentation strategies mediated by recombinant AAV vectors that impede retinal degeneration in pre-clinical models of acquired and inherited vision loss. We demonstrate distinct neuroprotective effects ...


Reverse Micelle Encapsulation And Its Use In Examining The Interplay Between Hydration And Protein Dynamics, Bryan Stephen Marques Jan 2018

Reverse Micelle Encapsulation And Its Use In Examining The Interplay Between Hydration And Protein Dynamics, Bryan Stephen Marques

Publicly Accessible Penn Dissertations

As the universal solvent, water is unquestionably essential to most aspects of protein biophysics from protein folding to enzymatic activity. Much has been learned about the relationship between proteins and surrounding solvent waters; however, it is often difficult to experimentally examine these interactions in a site-specific manner without perturbing molecular structure. Furthermore, the effect of nearby hydration dynamics on protein dynamics (and, in effect, protein conformational entropy) is poorly understood at atomic resolution. With the use of a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and protein reverse micelle (RM) encapsulation, it is possible to examine both the dynamic behavior ...


All The Right Noises: Causes And Consequences Of Stochastic Trimethylamine Oxide Reductase Expression In Escherichia Coli, Jeffrey Carey Jan 2018

All The Right Noises: Causes And Consequences Of Stochastic Trimethylamine Oxide Reductase Expression In Escherichia Coli, Jeffrey Carey

Publicly Accessible Penn Dissertations

Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes ...


Wnt5a Signaling Induced Phosphorylation Increases Acyl Protein Thioesterase Activity And Promotes Melanoma Metastatic Behavior, Rochelle Shirin Sadeghi Jan 2018

Wnt5a Signaling Induced Phosphorylation Increases Acyl Protein Thioesterase Activity And Promotes Melanoma Metastatic Behavior, Rochelle Shirin Sadeghi

Publicly Accessible Penn Dissertations

Wnt5a has been implicated in melanoma progression and metastasis, although the exact downstream signaling events that contribute to melanoma metastasis are poorly understood. Wnt5a signaling results in acyl protein thioesterase 1 (APT1) mediated depalmitoylation of pro-metastatic cell adhesion molecules CD44 and MCAM, resulting in increased melanoma invasion. The mechanistic details that underlie Wnt5a-mediated regulation of APT1 activity and cellular function remains unknown. Here, we show Wnt5a signaling regulates APT1 activity through induction of APT1 phosphorylation and we further investigate the functional role of APT1 phosphorylation on its depalmitoylating activity. We found phosphorylation increased APT1 depalmitoylating activity and reduced APT1 dimerization ...


Exploring The Role Of Tet1 In Genomic Imprinting, Jennifer Myers Sanmiguel Jan 2018

Exploring The Role Of Tet1 In Genomic Imprinting, Jennifer Myers Sanmiguel

Publicly Accessible Penn Dissertations

DNA methylation is an essential epigenetic mark crucial for normal mammalian development. This modification controls the expression of a unique class of genes, designated as imprinted, which are expressed monoallelically and in a parent-of-origin-specific manner. Proper parental allele-specific DNA methylation at imprinting control regions (ICRs) is necessary for appropriate imprinting. Processes that deregulate DNA methylation of imprinted loci cause disease in humans. DNA methylation patterns dramatically change during mammalian development: first, the majority of the genome, with the exception of ICRs, is demethylated after fertilization, and subsequently undergoes genome-wide de novo DNA methylation. Secondly, after primordial germ cells are specified ...


Gene Therapy Approaches To Immune Tolerance Induction In Canine Hemophilia, Robert French Jan 2018

Gene Therapy Approaches To Immune Tolerance Induction In Canine Hemophilia, Robert French

Publicly Accessible Penn Dissertations

A key issue in gene therapy is the immune response to the therapeutic transgene. This is especially important in applications where current treatments often elicit an antibody response, like hemophilia, where protein replacement therapy results in neutralizing

antibodies (“inhibitors”) in ~25% of severe hemophilia A and 1-3% of severe hemophilia B patients. To test the ability of skeletal muscle-directed gene therapy to prevent an immune response, we used an inhibitor-prone dog model of severe hemophilia B to express a hyperactive factor IX (FIX) variant from skeletal muscle via adeno-associated viral (AAV) vector and observed curative levels of expression that lasted ...


Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins, Mara Olenick Jan 2018

Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins, Mara Olenick

Publicly Accessible Penn Dissertations

Axonal transport is vital for the development and survival of neurons. The transport of cargo and organelles from the axon to the cell body is driven almost completely by the molecular motor, cytoplasmic dynein. Yet, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Previous work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. During my thesis work, I have investigated the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. Using optogenetic and single molecule assays ...


Structure, Function & Dynamics At The Membrane, Evan O'Brien Jan 2018

Structure, Function & Dynamics At The Membrane, Evan O'Brien

Publicly Accessible Penn Dissertations

The biological membrane is necessary for maintaining cellular identity, yet must also allow for interaction with the extracellular environment in order to respond to stimuli. Proteins that are directly embedded in the membrane or that interact more peripherally are responsible for these extracellular signaling events, which lie at the heart of cell communication. The first major goal of this work was to interrogate the peripheral interaction of cytochrome c and the mitochondrial lipid cardiolipin at atomic resolution using solution nuclear magnetic resonance (NMR) techniques; this interaction is key to promoting apoptosis. After demonstrating that the protein was correctly folded in ...


Β Cell Replacement Therapy: A Novel Application For Targeted Epigenetic Editing, Kristy Ou Jan 2018

Β Cell Replacement Therapy: A Novel Application For Targeted Epigenetic Editing, Kristy Ou

Publicly Accessible Penn Dissertations

Pancreatic β cells are the exclusive source of insulin, which normalizes blood glucose levels under hyperglycemic conditions. In 2015, over 252,000 deaths in the United States were contributed by diabetes, a family of disorders directly linked to defects in the pancreatic β cells. β cell deficiency or dysfunction leads to insufficient insulin secretion, resulting in chronic hyperglycemia and increased risk for severe health complications. Although severely diabetic patients can clinically manage their glucose levels with mealtime delivery of insulin analogues, many still experience potentially life-threatening hypoglycemic episodes due to erroneous insulin administration. Only β cell replacement therapy, through the ...


The Role Of Molecular Motors In Peripheral Nerve Regeneration, Melissa D. Priest Jan 2018

The Role Of Molecular Motors In Peripheral Nerve Regeneration, Melissa D. Priest

Publicly Accessible Penn Dissertations

Following injury, axons of the peripheral nervous system have retained the capacity for regeneration. While it is well established that injury signals require molecular motors for their transport from the injury site to the nucleus, whether kinesin and dynein motors play additional roles in peripheral nerve regeneration is not well understood. Here we use genetic mutants of motor proteins in a zebrafish peripheral nerve regeneration model to visualize and define in vivo roles for kinesin and dynein. We find that both kinesin-1 and dynein are required for zebrafish peripheral nerve regeneration. While loss of kinesin-1 reduced the overall robustness of ...


Dissecting The Molecular Basis Of The Scaffold Protein Jade In Hbo1 Histone Acetyltransferase (Hat) Activity, Joseph Han Jan 2018

Dissecting The Molecular Basis Of The Scaffold Protein Jade In Hbo1 Histone Acetyltransferase (Hat) Activity, Joseph Han

Publicly Accessible Penn Dissertations

HBO1 is a member of the human MYST family of acetyltransferases that is evolutionarily conserved from yeast to human. HBO1 functions in the context of a multi-protein histone acetyltransferase (HAT) complex containing JADE1/2/3, HBO1, ING4/5 and Eaf6 to regulate DNA replication, transcription, and other important cellular processes. HBO1 is shown to be responsible for the majority of H4 acetylation throughout the human genome. HBO1 functions in many important biological processes such as interaction with the origin recognition complex (ORC) and loading of the minichromosome maintenance protein complex (MCM), highlighting its importance in DNA replication. In addition, HBO1 ...


A Genetically Encoded Fluorescent Amino Acid Reveals Protein Dynamics Regulating The Bacterial Dna Damage Response, Zachary Michael Hostetler Jan 2018

A Genetically Encoded Fluorescent Amino Acid Reveals Protein Dynamics Regulating The Bacterial Dna Damage Response, Zachary Michael Hostetler

Publicly Accessible Penn Dissertations

Diversification of the genetic code in response to selective pressures can render organisms more fit to particular stresses. In many bacteria, the inducible prokaryotic DNA damage (SOS) response facilitates survival and adaptation to genotoxic stresses by upregulating genes involved in both high-fidelity and pro-mutagenic DNA damage repair. Within pathogenic bacteria, treatment with genotoxic antibiotics can induce the SOS response and lead to the acquisition of antibiotic resistance. Interest in disarming the SOS-dependent ability of bacteria to evade antibiotics has prompted investigation into the mechanisms underlying SOS pathway activation. Two proteins, the repressor LexA and the DNA recombinase RecA, operate together ...


Optimization Of Nuclear Magnetic Resonance Spectroscopy Methods For Measuring Protein Hydration In Reverse Micelles, Christine Jorge Jan 2018

Optimization Of Nuclear Magnetic Resonance Spectroscopy Methods For Measuring Protein Hydration In Reverse Micelles, Christine Jorge

Publicly Accessible Penn Dissertations

Water is fundamental to all aspects of protein function including folding, stability, catalysis, and dynamics. The unique characteristics of water make it the ideal solvent for supporting life but also make it incredibly difficult to study. While much has been learned about the role of water in protein function, a site resolved understanding of these interactions has remained elusive thereby leaving a large hole in the biophysical puzzle. Experimental techniques that provide a site-resolved view of protein hydration without mutation of the protein are necessary to understand the thermodynamic role of water on protein function. It has been shown that ...


Defining And Exploiting The Cytosine Deaminase Activity Of Apobec3a On The Extended Epigenome, Emily Katharine Schutsky Jan 2018

Defining And Exploiting The Cytosine Deaminase Activity Of Apobec3a On The Extended Epigenome, Emily Katharine Schutsky

Publicly Accessible Penn Dissertations

AID/APOBEC family cytosine deaminases canonically play crucial roles in immunity by converting cytosine to uracil in single-stranded DNA (ssDNA). Outside of this established physiological role, AID/APOBEC enzymes have also been implicated in the poorly-understood process of DNA demethylation through their proposed deamination of epigenetically-modified cytosine bases like 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC). However, there has been no thorough biochemical characterization of AID/APOBEC activity on these substrates, or on the recently-discovered 5-formylcytosine (fC) and 5-carboxylcytosine (caC) to inform this proposed role. Here, we provide the first steady-state kinetic measurements of the most active family member–APOBEC3A(A3A)–against ...


Synthesis And Characterization Of Photolabile Ruthenium Polypyridyl Crosslinkers With Applications In Soft Materials And Biology, Teresa Rapp Jan 2018

Synthesis And Characterization Of Photolabile Ruthenium Polypyridyl Crosslinkers With Applications In Soft Materials And Biology, Teresa Rapp

Publicly Accessible Penn Dissertations

Since its discovery in 1844, ruthenium has solidified its position as the most widely used transition metal in catalysis and excited state chemistry. Its lower toxicity and relatively low price (compared to other platinum group metals) have enabled many applications of ruthenium coordination compounds. In this dissertation I discuss ruthenium polypyridyl complexes that undergo photoinduced ligand exchange, and how this unique property can be harnessed to develop next-generation smart materials and responsive chemical biology tools.

Ru(LL)2X22+ complexes, where LL is a bidentate aromatic heterocycle such as 2,2’-bipyridine, 1,10-phenanthroline, or 2,2’-biquinoline, and X is ...