Life Sciences Commons^™

The Effects Of Propofol, Sodium Pentobarbital, And Ketamine Hydrochloride On In Vitro Mouse Embryonic Development, Tyler C. Balak

Theses and Dissertations in Biomedical Sciences

Every year more than 75,000 pregnant women are exposed to teratogenic medications or general anesthesia during non-obstetric surgery in the US,1-4 and embryonic effects of general anesthesia are of particular interest in laboratory research and veterinary medicine. The mouse system is used to screen potential toxic effects of anesthetics used in egg retrieval for in vitro fertilization (IVF), or those of pharmacologic agents which may come in contact with the egg or early embryo. Mouse preimplantation 2-cell embryos were exposed in vitro to incremental concentrations of common general anesthetics within and exceeding the normal clinical dosage range for mice (propofol …

Experimental And Computational Analysis Of The Synucleins, Agatha Munyanyi

Theses and Dissertations in Biomedical Sciences

The synuclein proteins α, β and γ which are located in the brain, have been a subject of intense research. Of particular interest is α-synuclein, which is found in misfolded forms in Lewy bodies that are associated with Parkinson's disease. Despite the efforts of researchers across the world, the physiological structure and function of the synucleins remains elusive. In recent years, highly controversial reports by some investigators indicate that in its natural form, α-synuclein exists as a tetramer instead of as an intrinsically unstructured monomer. This dissertation presents results of the experimental and computational analysis of the synucleins. First, we …

Mouse Oocyte Maturation And Embryo Development After Exposure To Vemurafenib (Plx4032), An Anti-Melanoma B-Raf V600e Inhibitor, Bo Liu

Theses and Dissertations in Biomedical Sciences

Vemurafenib is a selective B-Raf^V600E inhibitor in melanoma targeted therapy which also inhibits the wild type B- and C-Raf. In oocyte maturation, the C-Raf/MAPK pathway acts as an important self-enhancing and promoting system, w hereas in embryo development, the C-Raf/M APK pathway participates in pre- and post- implantation embryo proliferation and differentiation.

The hypothesis: Vemurafenib has detrimental effects on oocyte maturation and/or embryo development. Mouse oocytes and one cell (1C) mouse embryos were tested by ex vivo culturing with Vemurafenib in serial dilution. Oocytes were evaluated by cell cycle morphology, spindle formation and chromosomal alignment by im munofluorescence (IF) …

New Types Of Morpho-Physiological Changes In Cells Exposed To Nanosecond Pulsed Electric Field, Mikhail A. Rassokhin

Theses and Dissertations in Biomedical Sciences

Exposure of cells to a pulsed electric field (PEF) is the basis of multiple techniques and treatments. Nanosecond pulsed electric field (nsPEF) poses unique characteristics to induce subtle cellular effects while preserving cell integrity. Improving understanding of the mechanisms triggered by nsPEF in cells inspires new applications for the nanosecond pulse technology. Although many effects of nsPEF remain unknown, they can be inferred from morpho-physiologic changes, or cell reshaping, that accompany nsPEF exposure. During the exposure cells undergo reshaping that is manifested in swelling and diffuse blebbing. Recently we identified two new distinct forms of reshaping, pseudopod-like blebbing and microvesiculation, …

Attachment Ability And Melanoma Inhibitory Activity Mrna Expression Level Changes In Murine B16-F10 Melanoma Cells Post Nanosecond Electric Pulses, Hongxia Jia

Theses and Dissertations in Biomedical Sciences

The effects of high-voltage nanosecond electric pulses (nsEPs) on metastatic melanoma are still unclear. Hence, we applied one, two, three, and four 300 ns 40 kV/cm pulses to murine B16-F10 melanoma cells. Cell attachment ability was determined by comparing the number of floating cells and the percentage of attached cells. Melanoma inhibitory activity (MIA) is a secretory protein that is highly correlated with the malignancy and metastasis of malignant melanomas. We used MIA as our target to evaluate the effect of nsEPs on metastasis. Pulsed (experimental) and unpulsed (control) cells were incubated at 37°C under a 5% CO₂ atmosphere. …

The Dietary Isoprenoid Perillyl Alcohol Inhibits Telomerase Activity In Prostate Cancer Cells, Tabetha Sundin

Theses and Dissertations in Biomedical Sciences

This is the first evidence that a plant-derived compound–perillyl alcohol regulates telomerase activity via the mammalian target of rapamycin (mTOR) pathway in prostate cancer cells. Telomerase–the enzyme responsible for immortalizing cells through telomeric repeats addition–is de-repressed early in an aspiring cancer cell. We hypothesized that perillyl alcohol regulates hTERT (human telomerase reverse transcriptase) at the translational and post-translational levels via its effects on the mTOR pathway. A rapid suppression of telomerase activity was detected in prostate cancer cell lines (PC-3 and DU145) in response to biologically-relevant concentrations and short incubations of perillyl alcohol or the mTOR inhibitor—rapamycin.

Western blot analysis …

Nano- And Micro-Second Electrical Pulsing Of B16-F10 Mouse Melanoma Cells: Plasma Membrane And Sub-Cellular Organelle Changes, Yiling Chen

Theses and Dissertations in Biomedical Sciences

High electric field-treated cells are permeable to molecular dye through either opening of pores in the plasma membrane or other unknown processes which can disturb the membrane in an organized way. However, direct morphological evidence is lacking and responses of intracellular organelles are not clear. We used traditional chemical fixatives and biochemical techniques to capture cell membrane and organelle changes immediately after pulsing with high voltage electric field application. Different pulse durations, nanosecond (ns) and microsecond (µs), and field magnitudes, 60 kV/cm and 1.2 kV/cm, were applied to mouse melanoma B16-F10 cells. Two different ns durations (60 and 300 ns) …

Treatment Of Aortic Heart Valve Conduit With Glutamine And Heat Shock As A Means To Deter The Constituent Cellular Population From Becoming Apoptotic, Alyce Marie Linthurst Jones

Theses and Dissertations in Biomedical Sciences

Cryopreserved allograft heart valves represent the best solution for a patient with a failing heart valve. However, the constituent cells become apoptotic and within months of transplant the heart valve becomes acellular and the recipient's cells do not repopulate the allograft (3, 51). A strategy to prevent this situation would be to minimize or prevent apoptosis from occurring by strategically altering steps during heart valve processing. Recently it has been demonstrated that: 1) Heat shock protein 70 is a negative modulator of the apoptotic cascade; 2) Cells in culture exposed to hypothermic conditions produce heat shock protein 70 upon rewarming; …

Nanosecond Pulsed Electric Field Induction Of Programmed Cell Death Is Cell Type Dependent: An In Vitro Study, Wei Ren

Theses and Dissertations in Biomedical Sciences

Nanosecond pulsed electric fields (nsPEFs) present a novel and effective method for cancer ablation by eradicating the ubiquitous cancer hallmark of apoptosis evasion and enforcing cancer programmed cell death. To develop nsPEFs as an anticancer method, a comprehensive understanding of cell death mechanisms is required. The overall objective of this dissertation is to elucidate molecular mechanisms underlying effects of nsPEFs on E4 murine squamous cell carcinoma and human T-cell Jurkat clones that are wildtype, deficient in FADD (ΔFADD) and deficient in caspase-8 (ACas-8). The overall hypothesis is that nsPEFs eliminate cancer cells through activating caspase-dependent and caspase-independent programmed cell death …

Age-Related Hamster Mitochondrial Changes And Oocyte Changes Following Autologous Platelet Mitochondrial Microinjection, Fang Li

Theses and Dissertations in Biomedical Sciences

This study's objective was to verify age-related mitochondrial changes in oocytes from old hamsters compared with those from young hamsters. We used autologous platelet mitochondrial microinjection to improve the mitochondrial quality and quantity of aged oocytes to improve their pregnancy potential. Metaphase II oocytes were collected from super-ovulated female hamsters and prepared for biochemical and morphological analysis. Adenosine triphosphate (ATP) levels, mitochondrial DNA (mtDNA) number, reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were determined in individual oocytes from young and old hamsters. Transmission electron microscopy (TEM) allowed evaluation of oocyte ultra-structure. In oocytes from old hamsters, ATP …

Altering Nitric Oxide Bioavailability And Lipid Profiles In Endothelial Cells By Polycyclic Aromatic Hydrocarbons In Particulate Matter, Liang Yu

Theses and Dissertations in Biomedical Sciences

Epidemiologic studies have demonstrated a significant association between exposure to particulate matter (PM) and atherosclerosis. Polycyclic aromatic hydrocarbons (PAHs) present in particulate matter, are well known to induce oxidative stress and lipid peroxidation via generation of reactive oxygen species (ROS). Lipid peroxidation involves regulating endothelial nitric oxide synthase via inhibition of its activity, and as a result, mediates dilation of coronary arterioles is involved with the pathogenesis of atherosclerosis. However, data on assessment of oxidized lipid formation is limited due to low resolution of mass spectrometer methods. Taking the advantage of a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR-MS) …

Characterization Of Metastasis-Associated Cell Surface Glycoproteins In Prostate Cancer, Lifang Yang

Theses and Dissertations in Biomedical Sciences

Prostate cancer (PCa) is a major health problem in males in the United States. Its lethality is mostly attributed to the primary tumor metastasizing to distant sites that are highly resistant to conventional therapies. Serum Prostate Specific Antigen (PSA) is the only protein biomarker used in clinic for prediction of prostate cancer recurrence following local therapies. Nonetheless, PSA lacks the ability to predict the behavior of an individual tumor in an individual patient. Therefore, development of reliable biomarkers for detection of metastatic potential in primary tumors, as well as discovery of new therapeutic targets, is in a great need for …

Organization Of Human Sperm Chromosomes During Pronuclei Formation, Estella Jones

Theses and Dissertations in Biomedical Sciences

Chromosome organization is regarded as an important factor in regulating gene expression. In addition to the histone code, it is a part of the epigenetic mechanisms participating in fertilization and early embryonic development. Recent studies have demonstrated that chromosomes of human spermatozoa have well-defined spatial organization that includes preferred intranuclear positioning. The hypothesis of this dissertation is that sperm chromosome architecture participates in controlled transformation and activation of the paternal genome following fertilization.

Adequate model systems are necessary to study early nuclear events characteristic of human fertilization. This research focused on introduction and application of heterologous intracytoplasmic sperm injection (ICSI) …

Cellular Immunity In Mouse Models Of Viral Encephalitis, Christina Dawn Steel

Theses and Dissertations in Biomedical Sciences

Evidence is presented herein that intranasal application of vesicular stomatitis virus (VSV) caused acute infection of the murine central nervous system (CNS) with associated morbidity and significant mortality in mice. However, VSV encephalitis was not invariably fatal, suggesting that the CNS contained a professional antigen-presenting cell (APC) capable of inducing or propagating a protective antiviral immune response. To examine this possibility, we administered VSV via the intranasal route and then characterized the cellular elements that infiltrate the brain as well as the activation status of resident microglia, cells widely believed to represent the major APC population in the CNS. To …

Study Of The Dna Damage Complexes Within The Htlv-1 Tax Oncoprotein Interactome, Sidi Mehdi Belgnaoui

Theses and Dissertations in Biomedical Sciences

Human T-cell leukemia virus type 1 (HTLV-1) is a transforming retrovirus that can give rise to adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Tax is a virally encoded oncoprotein that is involved in HTLV-1 mediated cellular transformation. It has been hypothesized that Tax induces genomic instability via repression of the cellular DNA damage repair response. Our laboratory has previously shown that the interaction between Tax and various proteins involved in the DNA-damage response pathway impairs the ability of these proteins to mount an efficient repair response. As part of these observations, we proposed that Tax induces …

Nanosecond Pulsed Electric Fields Induce A Mitochondria-Independent Apoptosis In B16f10 Melanoma Cells In Vitro, Wentia Elissa Ford

Theses and Dissertations in Biomedical Sciences

Nanosecond pulsed electric fields (nsPEFs) are ultra-short pulses that induce direct electric field and biological effects that initiate apoptosis. Here the application of ten 300ns pulses ranging in electric fields from 12kV/cm-60kV/cm was administered to determine the effects on B16F10 melanoma cells evaluated by in vitro studies. Initial application of nsPEFs demonstrated apoptosis induction in an electric field- and pulse number-dependent manner measured by caspase activation that correlated with decrease in cell viability 24hr post pulse. In addition caspase activity was shown to be independent of calcium mobilization though ions may play a part in other aspects of apoptosis. The …

Gene Therapy Using Tet-Repressor System To Modulate Prostate Tumor Microenvironment, Nazita Yousefieh

Theses and Dissertations in Biomedical Sciences

Prostate cancer is the most commonly diagnosed malignancy in men in the United States and is projected to be the third most frequent cause of male cancer-related deaths in 2007 after lung and skin cancers. The initial treatment for prostate cancer at early stages is prostatectomy or radiation, which usually is curative. However, approximately 20% of patients are not cured by such treatments and their cancer recurs, sometimes with long latencies. In other patients prostate cancer is diagnosed only after the cancer has metastasized and there are no effective therapies at this stage. Therefore immunotherapy seems to be a promising …

Maldi Mass Spectrometry Imaging For The Discovery Of Prostate Carcinoma Biomarkers, Lisa Harris Cazares

Theses and Dissertations in Biomedical Sciences

The elucidation of new biological markers of prostate cancer (PCa) should aid in the detection, and prognosis of this disease. Diagnostic decision making by pathologists in prostate cancer is highly dependent on tissue morphology. The ability to localize disease-specific molecular changes in tissue would help improve this critical pathology decision making process. Direct profiling of proteins in tissue sections using MALDI imaging mass spectrometry (MALDI-IMS) has the power to link molecular detail to morphological and pathological changes, enhancing the ability to identify candidates for new specific biomarkers. However, critical questions remain regarding the integration of this technique with clinical decision …

Subcellular Localization Of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein, Kimberly Anne Fryrear

Theses and Dissertations in Biomedical Sciences

Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus that gives rise to Adult T-cell Leukemia (ATL) and a variety of other subneoplastic conditions such as HTLV-Associated Myelopathy/Tropical Spastic Paraperesis (HAM/TSP). In ATL, the transformation and immortalization of T-lymphocytes has been attributed to the expression and activity of a single HTLV-1 viral protein, namely the trans-activating protein Tax. Although the exact mechanism of Tax-mediated transformation is uncertain, current studies support a model in which Tax induces genomic instability in the host cell through interference with DNA repair mechanisms, dysregulation of cell cycle progression, transcriptional activation of cellular …

Down-Regulation Of Natural Killer Cell Activation In Response To Influenza Virus In Older Adults, Yu Jing

Theses and Dissertations in Biomedical Sciences

Immune senescence contributes to influenza-associated high mortality and morbidity and reduced vaccine efficacy in elderly people. Type I T cell (Thl)-mediated immunity plays a significant role in Immune responses to influenza infection and vaccination. Natural killer (NK) cells secrete significant amount of IFN-7 , a hallmark Thl cytokine, in response to influenza infection. How aging influences human NK cell IFN-7 production in response to influenza virus has not been well documented. In this study we employed human peripheral blood mononuclear cells (PBMC) and performed intracellular cytokine staining and flow cytometry primarily to investigate how aging influences NK cell activation with …

Cellular Mechanism Of Arglabin-Dimethylaminohydrochloride Cytotoxicity, Xiaofei Qin

Theses and Dissertations in Biomedical Sciences

Cancer is the second leading killer in the United States. Anticancer drug development is always based on the understanding of molecular and cellular mechanisms of carcinogenesis, as well as comprehensive knowledge of potential anticancer drugs. Arglabin-dimethylaminohydrochloride (arglabin-DMA) represents one of the new classes of anti-cancer agents that have shown promise in suppressing the growth of various tumor cells. However, the cellular mechanism of arglabin-DMA cytotoxic effects on tumor cells is still unclear. The current study was to determine the farnesyltransferase (FTase) inhibitory activity of arglabin-DMA and to investigate the effects of arglabin-DMA on three proteins: Ras, Rho and cyclin kinase …

Nanosecond Pulsed Electric Field Effects On Cell Cycle And Apoptosis, Emily H. Hall

Theses and Dissertations in Biomedical Sciences

Apoptosis, programmed cell death, is a highly regulated and complex pathway essential for embryonic development, immune-system function and maintenance of tissue homeostasis where cells induce their own cell death. Cells undergoing apoptosis exhibit a distinctive phenotype characterized by maintenance of membrane integrity, cell shrinkage, phosphatidylserine (PS) externalization at the plasma membrane, caspase protease activation, DNA fragmentation, release of cytochrome c from the mitochondrion, and membrane blebbing. An important regulatory protein in the apoptotic pathway is p53. The p53 protein functions to modulate the cell cycle by arresting cells in the G1 and G 2 phases to repair DNA damage, and/or …

The Study Of Nitric Oxide Synthase Expression, Function, And Regulation In The Renal Vasculature During Postnatal Renal Development, Brian Blake Ratliff

Theses and Dissertations in Biomedical Sciences

The newborn kidney is vulnerable to vasomotor acute renal failure (ARF) from adverse perinatal events or complications of prematurity. Nitric oxide (NO) vasodilation is vitally protective in this type of ARF, but its relationship with other vasoactive factors, such as angiotensin II (AII) has not been examined. In the immature kidney, nitric oxide synthase (NOS) isoforms, specifically eNOS and nNOS, are developmentally regulated, but their specific role and regulation are unknown.

The enhanced vasodilatory role of NO in the immature kidney was hypothesized to be attributed to regulatory, expressional, and functional differences in eNOS and nNOS isoforms from the adult. …

Modulation Of Tgfβ-Induced Pai -1 Expression By Changes In Actin Polymerization In Human Mesangial Cells, Keyur Patel

Theses and Dissertations in Biomedical Sciences

Chronic renal diseases show increased deposition of extracellular matrix (ECM) in the glomerulus (glomerulosclerosis). Glomerulosclerosis is associated with activation of normally quiescent glomerular mesangial cells into myofibroblast-like cells. The overall objective of this study is to delineate cellular mechanism/s of myofibroblast-differentiation in disease states. In cultured mesangial cells certain characteristics of myofibroblast differentiation (α-smooth muscle actin (α-SMA) and hypertrophy) are associated with an increase in polymeric actin microfilaments (stress fibers). It is likely that other genes are also regulated in an actin cytoskeleton-dependent manner during myofibroblast differentiation. In these studies, we therefore examined the hypothesis that changes in the actin …

Diverse Effects Of Nanosecond Pulsed Electric Fields On Mammalian Cells, Jody Anne White

Theses and Dissertations in Biomedical Sciences

The continuing effort to manipulate cell-signaling pathways for therapeutic benefit has lead to the exploration of electric field effects on cells. Current electric field applications include electroporation of the plasma membrane for introduction of drugs, genes, or other macromolecules into cells. Modeling of how these pulsed electric fields affect cells depicts the cell as an excitable circuit. In this model, the electric fields, administered in short pulses to a cell, charge the plasma and internal membranes, which act as dielectric layers, and between these the cytoplasm acts as a conductive medium. The pulse lengths of this treatment are traditionally in …

Isolation And Functional Mapping Of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Dna-Damage Complexes, Sarah Saionz Durkin

Theses and Dissertations in Biomedical Sciences

Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus which causes Adult T-cell Leukemia (ATL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Cellular transformation can be caused by a single viral trans-activating protein, Tax. Tax may contribute to transformation through interaction with components of the DNA damage response pathway, promoting cellular genomic instability. We examined cellular Tax complexes in an effort to elucidate potential protein-protein interactions that can model the Tax-induced molecular events.

We also investigated the role of post-translational modification in regulating Tax function. We employed a direct physical analysis of Tax complexes isolated from mammalian …

The Antitumor Agent, Arglabin-Dma, Preferentially Induces Apoptosis In Human Colon Tumor Cells, Sung Wook Kwon

Theses and Dissertations in Biomedical Sciences

Arglabin-DMA, an analog of farnesyl pyrophosphate (FPP), reportedly inhibits farnesyltransferase (FTase) directly by competitively blocking the binding of Ras protein and its posttranslational modification, as suggested in previous studies. But, the mechanisms by which Arglabin-DMA inhibits tumor growth in vivo and in vitro are still relatively poorly characterized. To determine the mechanism by which this drug inhibits tumor growth, the effects of Arglabin-DMA in two human colon tumor cell lines (mutant K-ras HCT 116 and wild-type ras HT-29) were explored on cell proliferation, apoptosis, and cell cycle kinetics in vitro. In cell viability studies, we showed that Arglabin-DMA …

Developing And Optimizing Conditions For Single Cell Genetic Analysis, Susan Adele Gitlin

Theses and Dissertations in Biomedical Sciences

Preimplantation Genetic Diagnosis (PGD) allows for couples to obtain genetic information at the embryo stage, therefore preventing lethal genetic diseases in their offspring. A recent multi-center study determined a 17% amplification failure rate overall and a 3.4% misdiagnosis rate (ESHRE PGD consortium steering committee, 2002). Experiments presented here were conducted to decrease those rates by optimizing single cell sensitivity and specificity of PGD techniques. Primer extension preamplification (PEP) was used after initial amplification failure, followed by gene specific PCR. Reamplification experiments yielded 54% amplification rates using blastomeres that previously failed amplification. In the clinical trial, four of six blastomeres were …

Regulation Of Sparc Gene Expression By The Activator Protein 1 Transcription Factor, Joseph William Briggs

Theses and Dissertations in Biomedical Sciences

Overexpression of the c-Jun proto-oncogene in MCF7 breast cancer cells results in a variety of phenotypic changes related to malignant progression including a shift to estrogen independent growth, increased cell motility and invasion. Concurrent with these phenotypic changes are alterations to cellular gene expression patterns. One gene that becomes highly upregulated is SPARC (secreted protein acidic and rich in cysteine). Increased SPARC expression is associated with malignant progression in a variety of different cancers, although little is known regarding the mechanisms of SPARC gene regulation. Therefore, the objectives of this study were: (1) to determine the mechanisms by which c-Jun …

The Ultrastructure Of The Olfactory System In Two Species Of Short-Tailed Shrews, Blarina Brevicauda And Blarina Carolinensis, Lisa Johnson Byrum

Theses and Dissertations in Biomedical Sciences

Several studies of the fine structure of the olfactory system of rodents have been conducted, but very little research has been done on members of the Insectivora. The olfactory systems of the northern short-tailed shrew, Blarina brevicauda, and the southern short-tailed shrew, Blarina carolinensis, were examined by light and electron microscopy. These shrews were live trapped in the vicinity of Norfolk, Virginia throughout all months of the year. Olfactory tissues were processed following standard transmission and scanning electron microscopy protocols. The olfactory system structures investigated included the olfactory epitheliumlmucosa (OEM), main olfactory bulb (MOB), accessory olfactory bulb (AOB), …