Biochemistry, Biophysics, and Structural Biology Commons^™

Dna Damage Response Activates The Electron Transport Chain And Oxidative Metabolism By Two Parallel Mechanisms, Shreya Nagar

Theses and Dissertations

The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. Major part of DDR is coordinated by DNA damage checkpoint (DDC). In addition to DDC, eukaryotic cells also have DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. DDR involves stalling or arrest of the cell cycle, initiation of DNA repair, and altered regulation of transcription, translation, and the ubiquitin-proteasome system. DDR also triggers transcription shut-off of histone genes. One of the key outcomes of DDC/DRC activation is the increased synthesis of the deoxyribonucleoside triphosphates (dNTPs), …

Protacs – A Novel And Rapidly Developing Field Of Targeted Protein Degradation, Hannah R. Gatley

Theses and Dissertations

There is a continued need for new technology and strategies for tackling cancer and other diseases, and within the current century a novel therapeutic strategy has emerged in the realm of targeted protein degradation called Proteolysis-Targeting Chimeras (PROTACs). This technology specifically targets and degrades disease-causing proteins via the ubiquitin-proteasome system, and has seen an explosion of research and intrigue in both academia and industry over the past two decades. The diversity of PROTAC classes based on the E3 ligase recruiting ligand and the target protein allows for a universal molecular structure that can be customized for a specific target and …

Characterization Of The Responses To Chronic Stress In Caenorhabditis Elegans, Amy Laura Knight

Theses and Dissertations

Exposure to chronic temperature stress influences organismal phenotypes that are important for human health, agriculture, and ecology. In this thesis, the model organism Caenorhabditis elegans was used to study the effects of temperature stress on reproduction and lifespan. It was found that worms demonstrated a rapid shut down in egg-laying between 18-24 hours of exposure to 28°C. Despite this reproductive defect, the overall lifespan of worms was unaffected. At the molecular level, heat shock factor 1 (HSF-1), a regulator of the protective molecular pathway known as the heat shock response (HSR), was identified as important for progeny production during heat …

Development Of New Treatments For Asthma And Neuropathic Pain Based On Ɣ-Aminobutyric Acid A Receptor (Gabaar) Ligands, Nicolas Mark Zahn

Theses and Dissertations

The γ-aminobutyric acid A receptor (GABAAR) is a ligand-gated pentameric chloride channel consisting of several identified subunits: α1-6, β1-3, γ1-3, δ, ε, π, θ, ρ1-3.1-2 Typical arrangement of subunits consists of two α subunits, two β subunits, and one γ subunit.3 GABAARs have two binding sites for the endogenous ligand γ-aminobutyric acid (GABA), between the α and β subunits. GABAARs also have a binding site for positive allosteric modulators, such as benzodiazepines, between the α and γ subunits.4-5 Due to their ability to treat anxiety, epilepsy, insomnia, and muscle relaxation, benzodiazepines are widely prescribed pharmaceuticals.6-7 Still, adverse effects result from …

Repurposing Metformin And Antifolates For The Treatment Of Hepatocellular Carcinoma, Sherouk Mohamed Tawfik

Theses and Dissertations

Hepatocellular carcinoma (HCC), one of the most prevalent types of cancers worldwide, continues to maintain high levels of resistance to standard therapy. As clinical data revealed poor response rates, the need for developing new methods has increased to improve the overall wellbeing of patients with HCC. Due to its safety, wide availability and previously reported anti-cancer effects, metformin (MET) serves to be a possible therapeutic agent when combined with other well-known anti-cancer agents. The aim of this study was to investigate the potential anti-cancer effects of MET, an anti-diabetic agent, when combined with two antifolate drugs: trimethoprim (TMP) or methotrexate …

The Role Of Irf-1 In Spontaneous Mouse Glioma, Aakash B. Vaidya

Theses and Dissertations

Glioblastoma Multiforme has been shown to be one of the deadliest primary brain cancers. One of the reasons why GBM is so deadly, is a unique immunosuppressive tumor microenvironment that promotes GBM growth and progression. Both astrocyte and microglia have been implicated in immunosuppression. In this study, we explored the role of Interferon Regulatory Factor 1 (IRF-1) in astrocytes and glioma cells on the growth of spontaneous glioma tumors. IRF-1 is regulated by the JAK/STAT pathway and induces expression of Programmed death ligand 1 (PD-L1). PD-L1 downregulates immune responses to glioma. We found that IRF-1 had no effect on spontaneous …

Negative Regulation Of The Kinase Lin-45 By The E3/E4 Ubiquitin Ligase Ufd-2, Augustin Deniaud

Theses and Dissertations

The serine/threonine kinase BRAF is a key part of the Ras-Raf-MEK-ERK pathway, an inducer of cell growth, differentiation, and survival. In humans, activating mutations, most commonly BRAF(V600E), have been detected in several cancers, including melanoma and thyroid cancer. In the Caenorhabditis elegans ortholog LIN-45, the equivalent mutation LIN-45(V627E) results in elevated Raf-MEK-ERK signaling. We performed an unbiased genetic screen to identify negative regulators of LIN-45(V627E). Here, we report the identification of the E3/E4 ubiquitin ligase UFD-2, and show it is a negative regulator of LIN-45 protein activity and levels. Loss of UFD-2 leads to accumulation of wild-type LIN-45 protein as …

Divergent Regulatory Roles Of Nurd Chromatin Remodeling Complex Subunits Gatad2 And Chd4 In Caenorhabditis Elegans, Nicole Lynn Golden

Theses and Dissertations

During stress, a protective cellular network known as the heat shock response (HSR) is induced to maintain protein-folding homeostasis, or proteostasis. While the HSR is essential for stress resistance, its misregulation is associated with neurodegenerative disease and cancer. Using the nematode model organism Caenorhabditis elegans, we have identified the chromatin remodeling complex NuRD (nucleosome remodeling and deacetylase) as a novel regulator of the HSR. Here, we begin with a brief introduction of the HSR and chromatin remodeling complexes in C. elegans, prior to presenting our findings in a series of two chapters. In chapter one, we outline a set of …

Distinct Spatiotemporal Regulation Of The Cytoprotective Heat Shock Response In Caenorhabditis Elegans, Rosemary Nadine Plagens

Theses and Dissertations

Every organism studied to-date utilizes the heat shock response (HSR) to maintain protein-folding homeostasis (proteostasis) during temperature or other protein-folding stress. The HSR has been well characterized using acute heat stress (HS) in single-celled models, but less is known about how distinct cell types and tissues respond to HS. Furthermore, how metazoans respond to prolonged HS at the molecular level remains relatively unexplored. The model organism C. elegans, with its genetic tractability and distinct tissues and behaviors, has been used extensively in the field to characterize the acute HSR, but with considerable variability across labs regarding HS temperature and duration. …

Reducing Seed Coat Fiber Content And Pod Shatter, And Engineering Medium Chain Fatty Acids-Containing Oil, In The Oilseed Crop Pennycress ( Thlaspi Arvense L. ), Maliheh Esfahanian

Theses and Dissertations

The overall goal of this thesis was to genetically improve agronomic traits of pennycress (Thlaspi arvense L.; Field Pennycress) and demonstrate the production of value-added designer seed oils to domesticate pennycress and enable its establishment as a new winter annual oilseed/meal/cover crop to be grown in temperate regions of the world. In the U.S. Midwest, pennycress can be double cropped on existing farmland during the time between corn harvest and subsequent planting of soybeans the following spring. Pennycress has the potential to produce 2,000 lbs/acre seeds, which at 33% by weight oil content and 20% protein, would yield 85 gallons/acre …

A Mechanism Behind The Mechanotransduction Of Surface Characteristics In Osteoblasts, Otto J. Juhl Iv

Theses and Dissertations

Biomaterials for use in bone regeneration and healing range from metal and metal alloy implants to hydrogel-based solutions. These materials can be optimized to increase bone healing and integration by improving the mechanical and biological properties. Regardless of the material itself, the cell-substrate interaction is key to the success of the biomaterial once implanted. Substrate surface characteristics such as roughness, wettability, and particle density are well-known contributors to a substrate’s overall osteogenic potential, and therefore the substrate's overall success. Unfortunately, it is still unknown how these substrate surface characteristics are transduced into intracellular signals by cells, preventing specific tailoring of …

A Molecular Toolset For The In Vivo Detection Of A Sulfolobus Islandicus Leucyl Trna Synthetase Paralog, Nicholas Michael Bretz

Theses and Dissertations

An ancient and ubiquitous set of enzymes known as the aminoacyl-tRNA synthetases are required for the viability of all organisms. The aminoacyl-tRNA synthetases catalyze the attachment of amino acids onto tRNA molecules. The aminoacylated, or charged, tRNA is then transported and utilized at the ribosome for the synthesis of proteins. The genome of the hyperthermophilic microorganism Sulfolobus islandicus (S. islandicus) harbors a unique leucyl-tRNA synthetase paralog, LeuRS-I, of unknown function. While its duplicate, LeuRS-F, carries out all the classical functions of LeuRS-family enzymes, LeuRS-I fails to charge leucine onto tRNALeu, despite its ability to activate this amino acid and bind …

Gene Regulatory Mechanisms Driving Temporal Dynamics Of Lineage Specific Differentiation In The Cranial Neural Crest, Maria R. Replogle

Theses and Dissertations

Genetic and environmental perturbations impacting neural crest (NC) development can result in pleiotropic structural and functional birth defects, many of which are associated with pediatric syndromes. As developmental precursors, the NC has the unique capacity to give rise to a diverse array of ectodermal and mesoectodermal cell types, from neurons and glia of the peripheral nervous system to the cartilage and bone of the face. In order to transition from a multipotent progenitor to a specific cell type, NC cells must undergo a series of dynamic morphological and behavioral transformations that gradually unfold over time. However, the NC is rare …

The Impact Of Aging And Mechanical Injury On Alveolar Epithelial And Macrophage Responses In Acute Lung Injury And Inflammation, Michael S. Valentine

Theses and Dissertations

Patients with severe lung pathologies, such as Acute Respiratory Distress Syndrome (ARDS), often require mechanical ventilation as a clinical intervention; however, this procedure frequently exacerbates the original pulmonary issue and produces an exaggerated inflammatory response that potentially leads to sepsis, multisystem organ failure, and mortality. This acute lung injury (ALI) condition has been termed Ventilator-Induced Lung Injury (VILI). Alveolar overdistension, cyclic atelectasis, and biotrauma are the primary injury mechanisms in VILI that lead to the loss of alveolar barrier integrity and pulmonary inflammation. Stress and strains during mechanical ventilation are believed to initiate alveolar epithelial mechanotransduction signaling mechanisms that contribute …

Regulation Of The Microtubule Cytoskeleton And Cell Wall Development In Arabidopsis Thaliana, Christy J. Fornero

Theses and Dissertations

Regulation of the cortical microtubule cytoskeleton is critical for organized plant cell division. Arabidopsis ton1 and ton2 mutants display random cell division plane placement and lack the plant-specific cortical microtubule array that encircles the nucleus prior to mitosis. In wild type plants, this preprophase band (PPB) of cortical microtubules precisely marks the future division plane. The specific roles of TON1 and TON2 in PPB formation are not yet known. It is suspected that TON1 Recruiting Motif (TRM) proteins may be involved in TON1 and TON2 recruitment to the PPB. Here we describe results for the targeted disruption of a group …

Understanding The Molecular And Cellular Functions Of Odd-Skipped Related 1 In Outflow Tract Development, Menglan Xiang

Theses and Dissertations

The cardiac outflow tract (OFT) is a transient conduit that connects the embryonic heart chambers to the vascular network. Transcription factor Osr1 promotes the proliferation and cell cycle progression of second heart field (SHF), an essential cell population that contribute to the developing OFT. In this study, we investigated the role of Osr1 in OFT development on cellular and molecular levels using a systems biology approach. We observed OFT rotation and elongation defects, as well as double-outlet right ventricle and overriding aorta as a result of SHF-specific deletion of Osr1. Using genetic inducible fate mapping, we showed that Osr1-expressing SHF …

Lorelei Localization And Ovule Ultrastructure In Arabidopsis Thaliana, Juleen May Dickson

Theses and Dissertations

Communication between the male and female gametophyte is vital to successful fertilization during sexual reproduction in plants. One of the proteins known to be important for communication between the male and female gametophyte is LORELEI (LRE). Several studies have shown that there are defects in pollen tube guidance and synergid degeneration, however this is the first study that shows that cell wall thickness in the female gametophyte may also be affected. Previous confocal studies have documented that LRE is present both in the filiform apparatus and found in puncta throughout the cytoplasm. This study confirmed this, but our studies suggest …

An Assessment Of Bsa Protein Hydrogel Biocompatibility In The Vertebrate Intestinal Tract, Ryan Joseph Garde

Theses and Dissertations

The fields of biomedicine and pharmacology have a mission to design methods to treat disease while minimizing adverse side effects using novel drug delivery systems. In developing new therapeutic systems, it is crucial to test that drug delivery systems target pathological cells and tissue and is non-toxic in healthy tissue. One promising method for targeted drug delivery is the use of hydrogels as carriers. Here, we studied the effects of bovine serum albumin (BSA) hydrogel consumption to assess the potential for hydrogel use in treating intestinal disease via oral administration. We investigated intestinal architecture and cell populations following hydrogel treatments …

Investigation Of The Interactions Between The Dream Complex And Hpv16, Kevin Ko

Theses and Dissertations

According to the American Cancer Society, it has been estimated that in 2019 alone, there will be approximately 53,000 new cases of oropharyngeal cancers. Oropharyngeal cancers are the largest subset of head and neck squamous cell carcinomas (HNSCCs), which are the sixth most common cancer across worldwide populations. They, along with other HNSCCs, fall under a category of cancers known as Human papillomavirus (HPV)-associated cancers, and it has been found that upwards of 70% of these cancers can be attributed to high-risk HPV infections.

Specifically, the high-risk HPV gene, E7, plays a key role in relieving cell cycle repression by …

Regorafenib Enhances Lethality Of Sildenafil And Curcumin In Colorectal Cancer Cells, Kervin Benjamin Owusu

Theses and Dissertations

In the United States, more than 130,000 people will be diagnosed with colorectal cancer (CRC) each year and an estimated 50,000 people will die from the disease. Standard of care (SOC) therapies for CRC combine multiple cytotoxic chemotherapeutic drugs. These combinations have varying degrees of effectiveness and can often result in significant patient morbidity. For second recurrence patients, the multi-kinase inhibitor, regorafenib, is an approved agent, but is often poorly tolerated at current doses. In the current study, we propose to develop therapeutic regime of combining agents with modest toxicity profiles: curcumin and sildenafil with regorafenib. Using clinically achievable enterohepatic …

Eralpha Isoforms Modulate The Tumorigenicity Of 24r,25(Oh)2d3 In Estrogen-Responsive Cancer, Anjali Verma

Theses and Dissertations

Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D₃ levels have better prognoses than patients with low serum vitamin D_3. However, double-blind clinical trials on the efficacy of vitamin D₃ supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D₃ supplements who had ‘estrogen receptor positive’ …

Zebrafish Model Of Mll-Rearranged Acute Myeloid Leukemia, Alex J. Belt

Theses and Dissertations

Acute myeloid leukemia (AML) is the second most common type of leukemia and accounts for 80% of adult acute leukemia cases and is characterized by the accumulation of poorly or undifferentiated myeloid blast cells. Standard treatment includes chemotherapy, which if unsuccessful, is followed by more rigorous chemotherapy as well as stem cell transplantation. Considering most patients are over the age of 45, these more rigorous therapies are not always possible, and as such, new therapies must be developed. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) that results in the expression of an MLL fusion protein …

Molecular And Functional Analysis Of The Pixb Gene In Xenorhabdus Nematophila, John Lucas

Theses and Dissertations

Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae form a mutualistic relationship facilitating the infection, death and consumption of an insect host. The infective juvenile (IJ) form of S. carpocapsae invades the insect host through natural openings and proceeds to the hemocoel where exposure to hemolymph stimulates the release of X. nematophila from the anterior vesicle. Excreted X. nematophila releases immunosuppressive compounds and insect toxins into the insect hemolymph that facilitates death of the host. As X. nematophila reaches high cell density it secretes exoenzymes that degrade insect tissues and produces antibiotics that reduce microbial competition. S. carpocapsae utilizes the …

Resolution Of Inflammation Rescues Axon Initial Segment Disruption, Nicholas M. George

Theses and Dissertations

Axonal domains are required for proper neuron function. These domains are unstable and degenerate concurrent with the inflammation in multiple sclerosis (MS) and the inflammatory disease models experimental autoimmune encephalomyelitis (EAE) and lipopolysaccharide (LPS) induced inflammation. Previous studies from our laboratory have shown that the axon initial segment (AIS) is maintained independently of the presence of myelin, but that AIS disruption is seen in MS as well as EAE and LPS-mediated inflammation. AIS loss can be interrupted in the early stage of EAE using the anti-inflammatory drug Didox. However, the potential for Didox directed repair of the AIS in later …

Biophysical Properties Of Cellular Membranes In Gram-Positive Bacterial Pathogens And Their Impact On Major Physiological Attributes And Virulence Determinants, Suranjana Sen

Theses and Dissertations

The cytoplasmic membrane of bacterial cells, forming an essential barrier from the surroundings, is a critical component of cellular physiology ensuring proper survival and maintenance of major cellular functions. The integrity of the membrane is an important feature that plays an essential role in the transport of solutes and nutrients through active and passive pathways, functions of membrane-associated proteins, electron transport and ATP synthesis, maintaining turgor pressure and combating environmental stresses, and thus is a crucial factor of a majority of cellular adaptations. The various biophysical properties affecting the integrity of this membrane are mainly determined by the composition and …

Expression, Localization, And Kinetic Characterization Of The Phospholipid Biosynthesis Enzyme Ctp: Phosphocholine Cytidylyltransferase From The Protozoan Parasite Leishmania Major, Justin Daniel Theodore Lange

Theses and Dissertations

The eukaryotic parasite Leishmania is the causative agent of the disease leishmaniasis. L. major is the most common of 21 species that causes visceral leishmaniasis in humans, and 30 that cause the same disease in other mammals. Visceral leishmaniasis causes fever, weight loss, and over a short amount of time, multiple organ failure, and has a 100% mortality rate within 2 years. This makes it the second largest parasitic killer in the world behind malaria. Over 90% of the worldâ??s cases of visceral leishmaniasis have been reported in underdeveloped countries of India, Bangladesh, Nepal, Sudan, Ethiopia and Brazil, with 500,000 …

Cell Wall Mutants In Arabidopsis Thaliana, Christy Jane Moore

Theses and Dissertations

Plant cell walls are versatile structures, playing important roles in communication, defense, organization and support. The importance of each of these functions varies by cell type, with specialized cells often utilizing one or two functions more than others. Trichomes, or leaf hairs, and hypocotyl cells for instance, exhibit distinct cell wall characteristics. Trichomes have developed very thick cell walls with several raised structures, known as papillae, on their surfaces. It is believed that these cells function in defense against predators, making it difficult to crawl on the leaf surface, and in protection against ultra violet radiation, through refraction of light …

Role Of Non-Muscle Myosin Ii And Calcium In Zebrafish Midbrain-Hindbrain Boundary Morphogenesis, Srishti Upasana Sahu

Theses and Dissertations

Elucidating the molecular mechanisms that play a role in cellular morphogenesis is critical to our understanding of brain development and function. The midbrain-hindbrain boundary (MHB) is one of the first folds in the vertebrate embryonic brain and is highly conserved across species. We used the zebrafish MHB as a model for determining the molecular mechanisms that regulate these cell shape changes. Cellular morphogenesis is tightly regulated by signaling pathways that rearrange the cytoskeleton and produce mechanical forces that enable changes in cell and tissue morphology. The generation of force within a cell often depends on motor proteins, particularly non-muscle myosins …

Rheb Dynamics On Lysosomal Membranes Determines Mtorc1 Activity After Loss Of P53 Or Activation Of Ampk, Catherine M. Bell

Theses and Dissertations

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss …

Nitric Oxide Synthase Activity And Its Modulation In The Treatment Of Colorectal Cancer, Asim Alam

Theses and Dissertations

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under …