Life Sciences Commons^™

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 …

Rapid Diffusion Observed In Microcrystals By X-Ray Free Electron Laser Mix-And-Inject Serial Crystallography, Tek Narsingh Malla

Theses and Dissertations

With time resolved X-ray crystallography (TRX), it is possible to follow reaction progress in real time. The time resolution is achieved by initiating reaction in crystal prior to X-ray exposure, and then collecting diffraction pattern at different time delays. Time resolved serial femtosecond crystallography (TR-SFX) at X-ray free electron laser (XFELs) allows damage free data collection from microcrystals. Mix-and-inject serial crystallography (MISC) is a type of TR-SFX established at XFELs. In MISC, reaction in enzymatic crystals is triggered by mixing with a substrate, and the resulting structural changes are probed by XFEL pulses.Enzymatic reactions are of great interest due to …

Dissecting Molecular Mechanism Of Heat Stress On Anther Development In Fragaria Vesca, Shikha Malik

Theses and Dissertations

Strawberry is an important fruit crop in the Rosaceae family. Originally from the temperate region, strawberry is vulnerable to heat waves, which reduce fruit yield and quality. Previous studies have shown that heat stress impairs pollen development; however, the molecular mechanisms by which heat stress affects the development of anthers, where pollen develops, are unclear. Due to the genome complexity of cultivated strawberry (Fragaria × ananassa, 2n = 8x= 56), Fragaria vesca (2n = 2x = 14) was used, which emerges as a diploid model plant for Rosaceae, to study the effects of heat stress on anther development at morphological …

A Mechanistic Investigation Of Cytochrome C Nitrite Reductase Catalyzed Reduction Of Nitrite To Ammonia: The Search For Catalytic Intermediates, Shahid Shahid

Theses and Dissertations

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic homodimeric decaheme enzyme that catalyzes the reduction of nitrite to ammonium in a process that involves six electrons and eight protons. Under standard assay conditions, which use a strong reducing agent as an electron source, catalysis takes place rapidly without producing detectable intermediates. However, intermediates do accumulate when weaker reducing agents are employed, allowing the ccNiR mechanism to be studied. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated in isolation by using the weak reducing agents N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) and the 2-electron reduced form of indigo trisulfonate. Experiments were …

Macromolecular Structure Determination At X-Ray Free Electron Lasers From Single-Particle Imaging To Time-Resolved X-Ray Crystallography, Ishwor Poudyal

Theses and Dissertations

X-ray free-electron lasers (XFELs) open the possibility of obtaining diffraction information from a single biological macromolecule. This is because XFELs can generate extremely intense X-ray pulses which are so short that diffraction data can be collected before the sample is destroyed. By collecting a sufficient number of single-particle diffraction patterns from many tilts of a molecule relative to the X-ray beam, the three-dimensional electron density can be reconstructed ab-initio. The resolution and therefore the information content of the data will ultimately depend largely on the number of patterns collected at the experiment. We estimate the number of diffraction patterns required …

Pump-Probe And Mix-And-Inject Experiments At X-Ray Free Electron Lasers, Suraj Pandey

Theses and Dissertations

Time resolved serial femtosecond crystallography (TR-SFX) utilizes X-ray crystallography to visualize the reaction of molecules in real time at the atomic level. Crystals of biological macromolecules are exposed to powerful X-ray pulses. The X-ray radiation emitted by the crystal is then measured by an X-ray sensitive area detector that produces an image called a diffraction pattern. These patterns are analyzed to determine a three-dimensional atomic structure of the biological macromolecule.The ultimate goal of TR-SFX is to make a “molecular movie” that shows the reaction dynamics of a biological process. For this, a reaction is started in a macromolecular crystal and …

Structural And Functional Characterization Of L-Enduracididine Biosynthetic Enzymes, Nemanja Vuksanovic

Theses and Dissertations

The mannopeptimycins produced by Streptomyces hygroscopicus are non-ribosomal peptide antibiotics with activity against a number of Gram-positive pathogens, including drug-resistant strains like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Attempts to synthesize more potent mannopeptimycin analogues have so far not been successful. One reason this has proven difficult is that the mannopeptimycins contain the unusual non-proteinogenic amino acid L-enduracididine (L-End), or its hydroxylated derivative β-hydroxy enduracididine (βhEnd,). This amino acid is not commercially available and, while synthetic routes have been developed, they are multi-step processes starting from an advanced intermediate. Understanding enzymatic synthesis of L-End is an important goal, …

Stem Cell Technology For Age Related Macular Degeneration Intervention, Laura Valdez

Theses and Dissertations

The main focus of this thesis is to study RPE cell derived from IMR90 induced pluripotent stem cells in various physiological conditions mimicking RPE transplantation rejection. Retinal pigmented epithelium (RPE) cells are located between the choroid and photoreceptors in the eye and they are essential to provide nutrients from blood to rods and cones, as well retinoids of the visual cycle. Vision loss and various ocular diseases are attributable to the degeneration or dysfunction of the RPE cells, leading to blindness. One of the major ocular problem from RPE dysfunction is macular degeneration. Age-related macular degeneration (AMD) can be frequently …

Analysis Of Olfactory Mrna Cap Modifications Using Rnaseh And Lc-Ms, Ilinca M. Giosan

Theses and Dissertations

In the olfactory system, olfactory sensory neurons (OSNs) express odorant receptors (ORs) in a singular fashion through unknown mechanisms. Published data supports the notion that singularity is achieved, in part, through a modification the mRNA strand, perhaps in its 5’cap region. This theoretical study aims to test this 5’cap hypothesis.

Snow-Albedo Feedback In Northern Alaska: How Vegetation Influences Snowmelt, Lucas C. Reckhaus

Theses and Dissertations

This paper investigates how the snow-albedo feedback mechanism of the arctic is changing in response to rising climate temperatures. Specifically, the interplay of vegetation and snowmelt, and how these two variables can be correlated. This has the potential to refine climate modelling of the spring transition season. Research was conducted at the ecoregion scale in northern Alaska from 2000 to 2020. Each ecoregion is defined by distinct topographic and ecological conditions, allowing for meaningful contrast between the patterns of spring albedo transition across surface conditions and vegetation types. The five most northerly ecoregions of Alaska are chosen as they encompass …

Quagga Mussel Induced Phosphorus Cycling Changes In Lake Michigan, Rae-Ann Maclellan-Hurd

Theses and Dissertations

Quagga mussels (Dreissena rostriformis bugensis) are an invasive ecosystem engineer that have successfully colonized both profundal and nearshore regions in Southern Lake Michigan. Quagga mussels directly altered the flow of nutrients by filtering particles, excretion of soluble forms of nutrients in the benthos, and through the production of biodeposits. Mussel excretion, egestion, and capture rates of P were compared between a 25 meter and a 55 meter deep site near Milwaukee Harbor to determine the retention of nutrients in these regions. The capture rate of particulate phosphorus at the 25m site (278 ± 388 μmol m-2) was similar to that …

Adsorption Behavior Of Chemically / Charged Modified Antibodies On Gold Nanoparticles, Samuel Okyem

Theses and Dissertations

Gold nanoparticles (AuNPs) have been exploited in the various domains of science such as drug delivery, bio-sensing, immunoassays and environmental sensors, due to their optical properties and intriguing surface chemistry. Different scientific procedures have been used to effectively immobilize antibodies onto AuNPs. Although acceptable outcomes have been achieved in the immobilization of antibodies onto AuNPs, the sensitivity of these immobilized antibodies to target antigen or binding sites is limited due to improper orientation of the antibodies. Also, the possibility of nanoparticle aggregation when exposed to proteins limits its biomedical applicability.

There is some evidence that the surface charge of antibodies …

Understanding The Role Of Protein Kinases Kin1 And Kin2 In The Protein Folding Pathways In The Yeast Saccharomyces Cerevisiae, Chandrima Ghosh

Theses and Dissertations

Eukaryotic protein kinases catalyze the transfer of the -phosphate of an ATP to a serine/threonine/tyrosine residue present in a protein substrate. The phosphorylation of proteins has profound effects on their activity and protein-protein interactions, thus regulating a plethora of cellular processes, including cell growth, differentiation and protein homeostasis (or proteostasis). Our lab is the first to demonstrate that protein kinases Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae, orthologs of human microtubule affinity-regulating kinase (MARK), contribute to protein-folding homeostasis inside the endoplasmic reticulum (ER), in addition to their canonical roles in cellular exocytosis. The main aim of …

Zinc Proteomics: Interactions Of Zn2+, Cd2+, And Metal-Binding Ligands With Zn-Binding Sites In The Proteome, Eric Daniel Lund

Theses and Dissertations

Cadmium ion causes toxicity in humans, most prominently in the kidney. This thesis focuses on mechanisms by which Cd2+ harms kidney proximal tubule cells. Previous experiments have shown the time-dependent distribution of Cd2+ and Zn2+ within supernatant of pig kidney, LLC-PK1 cells after exposure to Cd2+ plus the ionophore pyrithione1. The first part of this research describes the trafficking of cadmium within LLC-PK1 proximal tubule cells, including the time dependent distribution, speciation, and quantification of Cd2+ and Zn2+ in cells exposed to Cd2+ and Cd2+ plus pyrithione. The latter treatment introduces Cd2+ into cells at time zero and permits the …

Photobiomodulation Modulates Bioenergetics And Oxidative Stress In An In Vitro Model Of Diabetic Retinopathy, Alexandria E. Hall

Theses and Dissertations

Diabetic Retinopathy (DR) is the most common complication of diabetes mellitus and a leading cause of blindness. The pathophysiology of DR is complicated, involving mitochondrial dysfunction, oxidative stress, inflammation and vascular degeneration. The most common therapeutic approach for DR includes the use anti-vascular endothelial growth factor (VEGF) drugs to reduce vascular proliferation. These treatments are invasive, frequently ineffective and have numerous adverse effects. A non-invasive more effective therapy is clearly needed. An alternative, non-invasive therapy using far-red light (photobiomodulation, PBM) may be an improvement over current therapy. PBM has documented efficacy in experimental and clinical studies of retinal disease including …

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 …

A Tale Of Two Adaptors: The Role Of Two Adaptor Proteins In Pseudomonas Aeruginosa Chp Chemosensory System Signal Transduction And Implications For Chemosensory Array Formation, Zachary Hying

Theses and Dissertations

Bacteria use chemosensory systems to coordinate environmental signals to direct chemotaxis and make lifestyle decisions such as surface attachment and biofilm formation. Chemosensory systems form extended arrays with pseudo-hexagonal symmetry that are essential for efficient signal transduction. These arrays consist of three essential components: Methyl-Accepting Chemotaxis proteins (MCPs), which receive signals, a histidine kinase to coordinate cell responses through phosphorylation of response regulators, and an adaptor protein to transduce conformational change and facilitate array formation. Pseudomonas aeruginosa uses four chemosensory systems to control flagellar-based motility, type IV pili-mediated twitching motility and acute virulence, and biofilm formation. The Chp chemosensory system …

Examining Various Aspects Of Zika Virus Dissemination In Aedes Aegypti, Ileana C. Lozano

Theses and Dissertations

South Texas is one of the few locations where Zika virus has been locally transmitted in the U.S. It has a climate which is distinct to other areas with autochthonous transmission, with extremely hot, dry summers and moderate winter temperatures. Studying mosquito transmission of Zika in a range of temperatures conditions replicating where virus transmission is occurring is essential in order to have a better understanding of transmission patterns. These factors were examined by infecting mosquitoes and monitoring the dissemination status through real-time PCR analysis.

To further characterize dissemination of Zika virus within south Texas mosquitoes a time series immunohistochemical …

Acetoacetate Enhances Oxidative Metabolism And Response To Toxicants Of Cultured Renal Cells, Trudi Maria Denoon

Theses and Dissertations

In vitro methods can be cost effective and facilitate large scale drug screening in the pharmaceutical industry. Renal proximal tubular cells have been a prime target for drug induced toxicity. LLC-PK1 cells possess many transport functions of the proximal tubule epithelia, however, its energy metabolism differs from the highly oxidative tubule metabolism in vivo. This may impact its reliability in predicting toxicity. Modulating the growth media composition has been shown to shift cultured liver and muscle cells from high glycolytic activity to increased oxidative metabolism, but few studies have been done on renal cells. This study is designed to test …

Characterization Of The Dyrk1a Protein-Protein Interaction Network, Varsha Ananthapadmanabhan

Theses and Dissertations

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is a protein kinase encoded by a dosage-dependent gene. An extra copy of DYRK1A contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe mental retardation and autism. DYRK1A is involved in phosphorylation of several proteins that regulate cell cycle control and tumor suppression. However, the function and regulation of this kinase is not well understood and current knowledge does not fully explain dosage-dependent function of this important kinase. Our previous proteomic studies identified several novel DYRK1A interacting proteins including RNF169, FAM117B, TROAP, LZTS1, LZTS2 and DCAF7. In this …

Characterization Of The Tsc/Dyrk1a Interaction, Supriya Joshi

Theses and Dissertations

The Tuberous sclerosis complex (TSC) includes TSC1, TSC2 and the TBC1D7 subunits that together function as a principal inhibitor of the mTOR protein kinase complex 1 (mTORC1). mTORC1 is a master regulator of cell growth and proliferation that responds to signaling cues such as growth factors and nutrient availability. Proteomic studies in our lab revealed an interaction between the TSC subunits and DYRK1A, a ubiquitous protein kinase encoded by a gene located in the Down syndrome (DS) region on human chr21. In this study, we sought to validate the interaction of the TSC components with DYRK1A and to determine the …

The Role Of Manganese In Streptococcus Sanguinis, Tanya M. Puccio

Theses and Dissertations

Streptococcus sanguinis is primarily associated with oral health as a commensal bacterium. As an opportunistic pathogen, S. sanguinis is capable of colonizing heart valve vegetations, leading to the disease infective endocarditis. Previous studies from our lab have identified the high-affinity manganese transporter SsaACB as important for endocarditis virulence. The impact that manganese depletion has on S. sanguinis had never been evaluated and a secondary manganese transporter has not been identified. Thus, we employed the use of a fermentor to control large-scale growth over time and depleted manganese in an ΔssaACB mutant using a metal chelator, EDTA. The changes in …

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 …