Life Sciences Commons^™

Small Gtpase Regulated Intracellular Protein Trafficking In Endothelium, Caitlin Francis

Electronic Theses and Dissertations

Intracellular protein trafficking is the movement of membrane-bound organelles to and from requisite locations within the cell. Small GTPases are a critical component to the spatiotemporal accuracy of intracellular trafficking pathways as they determine the specificity and direction of organelle transport. There exists over 150 small GTPases categorized into 5 sub-families and are employed across all cell types. Despite their universal expression and relevance to cellular function, small GTPases remain incompletely understood across tissue types. In various instances, the trafficking pathway of a particular Rab in one cell type may belong to a completely disparate pathway in another cell type. …

The Pathophysiological Mechanisms Of Alzheimer's Disease; Investigating Therapeutic Interventions For Disease Onset, Alexandra A. Sandberg

Electronic Theses and Dissertations

Alzheimer’s Disease is a multifarious disease that progressively affects more people as both the proportion of older adults in the population and life expectancy increase in both the United States and worldwide. This devastating disease is a result of rampant neuronal loss in the memory centers of the brain that robs the independence of those who are diagnosed and places a heavy burden on those who care for them. Traditionally speaking, research has focused on the hallmark pathology of amyloid plaques, targeting them to try and prevent disease onset. However, countless failures in clinical trials aimed at this said pathology …

Morphological Variation And Community Science In Orthoptera, Amy Byerly

Electronic Theses and Dissertations

Patterns of morphological divergence across species’ ranges provide insight into local adaptation and speciation. Here, we compare phenotypic divergence among 4,221 crickets from 337 populations of two related species of field cricket, Gryllus firmus and G. pennsylvanicus and their hybrids. We find that these species differ across their geographic range in key morphological traits, such as body size and ovipositor length, and we directly compare phenotype with genotype for a subset of crickets demonstrating nuclear genetic introgression, phenotypic intermediacy of hybrids, and essentially unidirectional mitochondrial introgression. We discuss how these morphological traits relate to life history differences between the species. …

Responses Of Arthropods To Fire And How Pollinators And Pollination Services Are Affected By Fire Severity, Blyssalyn V. Bieber

Electronic Theses and Dissertations

Fires impact ecosystems globally and due to climate change, there are shifts in fire regimes that impact ecological communities which provide essential ecosystem services. Focusing on arthropods, fire can influence this ubiquitous animal group in various way. We performed a comprehensive meta-analysis evaluating how fires impact differing arthropod functional groups. We found that overall, fire negatively effects community level responses for most functional groups with herbivores as the only exception showing some positive effects of fire. We also studied mixed-severity fires that burned >20 years ago and compared floral visitor communities across fire severities. We implemented a pollinator exclusion experiment …

The Role Of Ehd2 In Endothelial Cells, Rachael Judson

Electronic Theses and Dissertations

The role of EHD2 in terms of Cav1 interaction had not been previously characterized and was the main goal of this research. Cav1 has been shown to lead to the ubiquitination of Rac1. Because of this, the role of EHD2 in Rac1 regulation was investigated. An increase in long-term migration in endothelial cells was observed and suggested that EHD2 impacts the RhoA pathway instead of the Rac1 pathway. This lead to the hypothesis that EHD2 controls a part of the RhoA pathway. This hypothesis is supported by the loss of filamentous actin and an increase in serrated junctions in cells …

Organellar Zn2+ Homeostasis And The Role Of Trpml Channels In Neuronal Lysosome Physiology And Axonal Transport, Taylor Franklin Minckley

Electronic Theses and Dissertations

Zinc (Zn²⁺) is crucial for proper cellular function, and as such it is important to measure and track Zn²⁺ dynamics in living cells. Fluorescent sensors have been used to estimate Zn²⁺ content of subcellular compartments, but little is known about endolysosomal Zn2+ homeostasis. Similarly, although numerous sensors have been reported, it is unclear whether and how Zn²⁺ can be released from intracellular compartments into the cytosol due to a lack of probes that can detect physiological dynamics of cytosolic Zn²⁺. My dissertation started with comparing and characterizing different Zn²⁺ sensors including the …

Environmental Gradients Impact Key Leaf Traits In Riparian Trees, Amanda Malone

Electronic Theses and Dissertations

Specific leaf area (SLA) reflects a plant’s carbon investment per area and relates to generalized leaf economics spectrum growth strategies (LES). To fill gaps of knowledge about intraspecific SLA variation and why it is often inconsistent with the LES, we studied the response of two riparian tree species along the urban-rural gradient in Northeastern Colorado: an invasive, nitrogen-fixer with broad tolerances and a nitrogen-limited and shade-intolerant native. Despite these differences, the two species responded similarly, apart from the response to the urban-rural gradient, in which lower nitrogen in rural areas was associated with a more conservative strategy in the native …

Antioxidant Biomarkers And Nutraceutical Therapeutics In Neurodegeneration And Neurotrauma, Lilia A. Koza

Electronic Theses and Dissertations

Mild traumatic brain injury (mTBI), yielding a Glascow Coma Scale of 13-15, is the most commonly occurring severity of TBI. Pathology from mTBI consists of blood brain barrier disruption, neuroinflammation, oxidative stress, excitotoxicity, mitochondrial dysfunction, protein aggregation, axonal degeneration, and resulting neuronal death. These processes deplete the body’s endogenous antioxidant system. We report a retrospective analysis of antioxidant blood biomarkers in patients with a history of mTBI from a local sports medicine clinic, Resilience Code. We found persistent sex-specific antioxidant depletions in mTBI patients associated with worsened symptomology.

Certain populations, such as athletes, are at high risk for repetitive mTBI …

Optimized Microbial Recombinant Production Of Hiv-1 Anti-Envelope Antibody Fragments With Applications To Single Particle Tracking Of Virus Assembly, Merissa Michelle Bruns

Electronic Theses and Dissertations

In my findings, I have established a set series of protocols to recombinantly produce, purify and apply various fluorescent probes in vitro for the fluorescent labeling and study of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein during HIV viral assembly. There remains insufficient knowledge about the molecular dynamics and interactions of HIV-1 Env protein with its counterpart, Gag, on the inner host cell surface during assembly of a mature virus particle. There also remains an insufficient amount of data for the understanding and clarification of the mechanism of action of a known host cell HIV-1 restriction factor, …

Understanding Patterns And Functional Impacts Of An Invasive Tree And Its Biological Control In A Riparian System, Annie L. Henry

Electronic Theses and Dissertations

Invasive species have become an inextricable part of the landscape, particularly in riparian plant communities, and removal is often a key component of restoration programs. Biological control (biocontrol) is a method of removal that is often both efficient and effective. However, the impact of biocontrol on target species and indirect effects from invasive species removal can be hard to predict. While monitoring the impact of invasive species removal usually involves some species-based assessment such as changes in diversity, historically dominant species or native species, these strategies do not typically provide insight into the mechanisms underlying plant community response to removal. …

Rab35 Centered Membrane Trafficking Pathway Directs Apical Constriction During Drosophila Gastrulation, Hui Miao

Electronic Theses and Dissertations

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating high-tension contractile forces at the cell cortex before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be cellular processes to extract unidirectional changes that drive processive transformations in cell shape. During Drosophila melanogastergastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a …

The Ecological Resilience Of A Tropical Montane Pine Forest To Droughts, Fire, And Hurricane Disturbance, Daniel Erland Brugger Swann

Electronic Theses and Dissertations

Owing to the geographic isolation and unique climatic conditions which occur in tropical montane forests (TMFs), these systems are highly sensitive to changes in both climate and disturbance. Therefore, as ongoing climate change alters disturbance regimes – potentially facilitating multiple disturbance interactions much greater than that of isolated disturbances – TMFs may be shifted to alternate stable states. However, study of TMF resilience is essentially unknown given the lack of long-term studies in these systems. We address the resiliency of the tropical montane pine forests of the Cordillera Central, Dominican Republic using long-term climate records, remeasured vegetation data, and reconstructed …

The Role Of Vps54 In Drosophila Melanogaster Neuronal Development And Age Progressive Neurodegeneration, Emily Wilkinson

Electronic Theses and Dissertations

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). The “wobbler” mouse is the phenotypic result of a destabilizing point mutation in Vps54. This mutation causes neurodegeneration and is subsequently used as a model for human motor neuron disease. Presently, it is unclear how disruption of GARP complex function leads to motor neuron degeneration. To better understand the role of Vps54 in motor neuron development, function, and age-related neurodegeneration, we disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on larval neuromuscular junction …

Validation Of A Deployable Proteomic Assay For The Serological Screening Of Sexual Assault Samples, Catherine O'Sullivan Brown

Electronic Theses and Dissertations

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing …

Characterization Of The Whale Shark (Rhincodon Typus) Melanocortin-2 Receptor, Brianne Hoglin

Electronic Theses and Dissertations

Among bony vertebrates, the melanocortin-2 receptor ortholog is unique among the family of five melanocortin receptors on the basis that it is dependent on its accessory protein, MRAP1, for trafficking and activation, and is selective for activation by ACTH alone. Previous studies on the MC2R orthologs of select cartilaginous fish, the elephant shark (Callorhinchus milii) and the red stingray (Dasyatis akajei), revealed divergent traits in a less obligatory relationship on MRAP1 and its ability to be activated by ACTH or the MSH-sized peptides. However, observed traits were not consistent between these two cartilaginous fish species, posing …

Development Of Endoplasmic Reticulum Targeted Probes And Red Fluorescent Probes For Detecting Zinc, Drew Maslar

Electronic Theses and Dissertations

Zinc (Zn²⁺) is the second most abundant transition metal in the body and is important in various biological functions. Fluorescent sensors based on circularly permuted fluorescent proteins (cpFPs) have been previously made to detect labile, or unbound, Zn²⁺ within the cytoplasm of cells. These sensors have proven invaluable for studying Zn²⁺, however, these sensors are limited to their use in the cytoplasm and by the fact that only green cpFP have been utilized to create fluorescent Zn²⁺ sensors. In this thesis, we use a combination of peptide targeting sequences, site-directed mutagenesis, and rational design …

Fxs-Causing Point Mutations In Fmrp Disrupt Neuronal Granule Formation And Function, Emily L. Starke

Electronic Theses and Dissertations

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by the disruption of Fragile X Mental Retardation Protein (FMRP) function in neurons, affecting nearly 1 in 7,500 individuals. Although FXS typically occurs from a complete loss of FMRP expression due to a CGG trinucleotide expansion within the 5’UTR of the FMR1 gene, single nucleotide polymorphisms (SNPs) within the KH domains of FMRP have been shown to severely disrupt FMRP function. FMRP is an RNA-binding translation repressor that interacts with ~4% of the neuronal transcriptome. Many target mRNAs encode for proteins important for regulating synaptic processes and modulate synaptic plasticity. It …

Notch Regulates Vascular Collagen Iv Basement Membrane Through Modulation Of Lysyl Hydroxylase 3 Trafficking, Stephen J.B. Gross

Electronic Theses and Dissertations

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have …

Cellular And Developmental Insights Into The Early Evolution Of Muscle, Jeffrey J. Colgren

Electronic Theses and Dissertations

Whereas a great deal has been learned about the molecular underpinnings of morphological evolution in animals, much less is known about the origin of novel cell and tissue types. During the time in which the earliest animal lineages were diversifying, fundamental cell and tissue types, such as muscles, arose. Sponges are one of two animal lineages that lack muscles, yet they undergo coordinated full body contractions. Whereas the signaling processes have been studied, the physical mechanisms of contraction are completely uncharacterized. The main purpose of this work is to understand the primary contractile tissue of the sponge Ephydatia muelleri, …

Human Genetics, Psychotropic Drugs, And Acts Of Violence, Selma Jolanda Eikelenboom-Schieveld

Electronic Theses and Dissertations

From the start of the use of psychoactive prescription medications in the 1950s, physicians reported paradoxical adverse reactions, ranging from newly developing depressions to an increase in existing mood disorders, and extremely violent and bizarre acts of suicide and homicide. In this research, it is hypothesized that the pharmacological properties of the prescribed drugs or the interaction between the drugs and the enzymes that are primarily responsible for their metabolism (cytochrome P450s) could cause these reactions. Given that acts of violence could be medication-induced, the role of the rate of drug metabolism is discussed. Genetic testing of certain CYP450s could …

Quantification Of Mitochondrial Zinc Homeostasis And Analysis Of Zinc And Polyamine-Mediated Axonal Trafficking, Dylan Hunt Fudge

Electronic Theses and Dissertations

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors …

Subalpine Forest Tree Seedling Response To Drought, Alex Goke

Electronic Theses and Dissertations

Knowledge of tree species’ ability to tolerate drought is necessary to anticipate future forest dynamics with climate change, especially at the seedling stage given their role in shaping forest structure. We used precipitation reduction shelters to mimic drought for subalpine conifer seedlings (A. lasiocarpa and P. engelmannii) in the Rocky Mountains and compared survivorship and morphological and physiological responses to assess relative degrees of drought tolerance. We detected no significant investment in morphological tolerance traits (e.g. root biomass, leaf:stem area ratio) but substantial reductions in net photosynthesis. While shading partially ameliorated drought effects when precipitation reduction was moderate, …

Sorting Of Cargo Proteins Within The Regulated Secretory Pathway: The Peripheral Membrane Protein Hid-1 As A Sorting And Vesicle Biogenesis Factor, Blake H. Hummer

Electronic Theses and Dissertations

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize …

Cellular And Organismal Ramifications Of De Novo Purine Synthesis Dysregulation, Randall Craig Mazzarino

Electronic Theses and Dissertations

Purines are a class of nitrogenous bases and are essential small molecules to life. Purines are used within the cell as genetic information carriers, energy currency, signaling molecules, and cofactors for multiple processes. They are formed through de novo and salvage pathways found in cells across the phylogenetic tree. The substrates of enzymes within de novo purine synthesis are known to influence other processes within the cell, such as energy homeostasis. In humans, de novo purine synthesis disorders are rare, with around 100 people identified. These patients exhibit a range of phenotypes, with varying degrees of mental retardation, seizure activity, …

Rab39 And Klp98a Are Required For Furrow Formation During Early Drosophila Embryogenesis, Megan R. Millage

Electronic Theses and Dissertations

The formation of a plasma membrane furrow is an essential process during development. Furrow formation is necessary for successful cell division and cytokinesis in addition to the ability to create multicellular tissues. Here, I will explore the role of the Golgi-associated Rab protein Rab39 in furrow formation during early Drosophila embryogenesis. Rab39 is one of eight Rab proteins that has been shown to localize to discrete puncta by live imaging in early Drosophila embryos, but its function and pathway have not been well characterized. In this thesis will I show that Rab39 forms dynamic, tubular structures that colocalize with trans-Golgi …

Land Use Influences Along Elevation Gradient On Macroinvertebrate Communities, Brittany Sprout

Electronic Theses and Dissertations

Land use activities have caused disturbances that affect the quality of freshwater ecosystems worldwide. How the influences of land use along an environmental gradient and the associated environmental variables that may influence stream diversity and function is unclear. We address these issues by studying biodiversity, abundance, and functional diversity of macroinvertebrates across different land types along a gradient in Colorado, USA. We also address how diversity may change along an elevation gradient by analyzing previously published macroinvertebrate research. We found evidence that land use and disturbance are stronger explanations of changes in macroinvertebrate communities, rather than elevation. Functional trait patterns …

Ehbp1 And Ehd2 Regulate Dll4 Caveolin-Mediated Endocytosis During Blood Vessel Development, Amelia Margaret Webb

Electronic Theses and Dissertations

During angiogenesis, the development of new blood vessels from existing vessels, there must be tight regulation between a leading tip cell and trailing stalk cells during vascular patterning. Lateral inhibition produced by Notch signaling is central to the specification and maintenance of tip and stalk cells in growing angiogenic sprouts. The Notch pathway is activated by the ligand Delta at adherens junctions between tip and stalk cells. Despite the centrality of Delta/Notch signaling to angiogenesis, relatively little is known about the mechanism of this signaling event. It has been proposed that the force generated by the Notch bound Delta ligand …

Actin Regulation And Furrow Dynamics During Early Drosophila Embryogenesis, Yi Xie

Electronic Theses and Dissertations

Drosophila embryogenesis starts with a single nucleus undergo 13 rounds of nuclear divisions called syncytial cycles. Staring at cycle 10 when nuclei migrate to the surface of the embryo, massive and dynamic cortical actin structures and cleavage furrow formations occur. How actin regulators coordinate into an organized network directing three-dimension actin structures in the developing organisms is an unsolved question. Here, I present an in-depth characterization of actin cap dynamics: the actin caps go through expansion, stabilization, elongation and fragmentation phases in each cycle. Arp2/3 is the major contributor to actin cap formation. The functions of 7 different actin and …

Divergence Of Threespine Stickleback That Differ In Nuptial Coloration, Clara Sophie Jenck

Electronic Theses and Dissertations

Recent research has led to a much better understanding of the evolutionary processes that mold and structure variation within and among populations. How populations diverge at the genome-wide level and how polymorphism is maintained within a species, however, remains unclear. We address these questions with two freshwater color morphs, red and black, of the threespine stickleback fish (Gasterosteus aculeatus) from the northwest United States, in which a shift from red to black nuptial coloration occurred in several locations following glacial retreat. We measured phenotypic variation in a suite of traits and used next generation sequencing to characterize within-species and among-morph …

Targeted-Ion Mass Spectrometry For The Identification Of Forensically Relevant Biological Fluids And Samples From Sexual Assault Evidence, Heather Erin Mckiernan

Electronic Theses and Dissertations

Forensic practitioners have long sought efficient and reliable means for identifying those samples that are best suited for successful genetic profiling. Traditional serological screening methodologies rely upon enzyme activity and antibody-based serological tests. These tests can be consumptive, laborious and costly while reliance on antibody-based serological testing can be prone to error. Positive results resulting from non-target biological fluids, the potential for cross- reactivity and non-specific binding events yield merely presumptive results. This has led forensic biologists to omit serological testing, at least in the case of sexual assault kit samples, in favor of Y-Screen assays. While these Y-Screen approaches …