Medical Sciences Commons^™

Patterned Cell Cultures For High Throughput Studies Of Cell Electrophysiology And Drug Screening Applications, Anupama Natarajan

Electronic Theses and Dissertations

Over the last decade, the field of tissue and bio-engineering has seen an increase in the development of in vitro high-throughput hybrid systems that can be used to understand cell function and behavior at the cellular and tissue levels. These tools would have a wide array of applications including for implants, drug discovery, and toxicology, as well as for studying cell developmental behavior and as disease models. Currently, there are a limited number of efficient, functional drug screening assays in the pharmacology industry and studies of cell-surface interactions are complicated and invasive. Most cell physiology studies are performed using conventional …

Studies On A Novel Human Cardiospecific Transcription Factor And Its Involvement In Omi/Htra2 Mediated Cell Death, Meenakshi Puthucode Balakrishnan

Electronic Theses and Dissertations

Omi/HtrA2 is a mitochondrial serine protease that is known to translocate to the cytoplasm upon induction of apoptosis and to activate caspase-dependent and caspase-independent cell death. The molecular mechanism of Omi/HtrA2's function is not clear but involves degradation of specific substrates. These substrates include cytoplasmic, mitochondrial, as well as nuclear proteins. We have isolated a new Omi/HtrA2 interactor, the THAP5 protein. THAP5 is a fifth member of a large family of transcription factors that are involved in cell proliferation, apoptosis, cell cycle control, chromosome segregation, chromatin modification and transcriptional regulation. THAP5 is an approximately 50kDa nuclear protein, with a restricted …

Study Of The Egfr, Src And Stat3 Pathway In Pancreatic Cancer, Soumya Jaganathan

Electronic Theses and Dissertations

Cancer is associated with many molecular aberrations that support the malignant phenotype. In that regard, aberrant activation of epidermal growth factor receptor (EGFR), Src, and signal transducer and activator of transcription 3 (Stat3) occur concurrently in pancreatic cancer and are implicated in the disease phenotype. Notwithstanding, increasing evidence indicates that therapies that target only EGFR or Src are rather ineffective in modulating the cancer phenotype. The poor therapeutic outcome of the monotherapies targeting EGFR or Src may in part be due to the increased incidence of signaling cross-talks among aberrant signaling pathways in cancer. Molecular details of the signaling integration …

In-Frame Mutagenesis Of Genes Encoding A Selenium-Dependent Molybdenum Hydroxylase And Putative Accessory Proteins In Enterococcus Faecalis, Christopher J. Mallard

Electronic Theses and Dissertations

Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative seleniumdependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the …

The Cytokine, Interleukin-7, Transcriptionally Regulates The Gene Expression Of The Hexokinase Ii To Mediate Glucose Utilization, Mounir Chehtane

Electronic Theses and Dissertations

The cytokine, interleukin-7 (IL-7), has essential growth activities that maintain the homeostatic balance of the immune system. Little is known of the mechanism by which IL-7 signaling regulates metabolic activity in support of its vital function in lymphocytes. We observed that IL-7 deprivation caused a rapid decline in ATP levels that were attributable to loss of intracellular glucose retention. To identify the transducer of the IL-7 metabolic signal, we examined the expression of three important regulators of glucose metabolism, the glucose transporter, GLUT-1, and two glycolytic enzymes, Hexokinase II (HXKII) and phosphofructokinase-1 (PFK1), using an IL-7-dependent T-cell line and primary …

Interleukin-7 Differentially Regulates The Activation, Proliferation, And Homing Of T-Cells: Implications For Immunotherapy, Christina Kittipatarin

Electronic Theses and Dissertations

Interleukin-7 (IL-7) is an essential lymphocyte growth factor required for the survival and proliferation of mature T-cells. As a therapeutic agent, IL-7 has the potential to restore T-cell numbers following immune depletion and to promote immunity against cancers. While the survival function of IL-7 is well established, less is known about how it supports T-cell expansion, a critical feature of the immune response. To study the biological effects of IL-7 on T-cell growth, we developed an in vitro culture technique to expand T-cells ex vivo. A significant finding from our studies is that IL-7 did not induce the expansion of …

Magnetic Nanosensors For Multiplexed Bacterial Pathogenesis Identification, Charalambos Kaittanis

Electronic Theses and Dissertations

Developing diagnostic modalities that utilize nanomaterials and miniaturized detectors can have an impact in point-of-care diagnostics. Diagnostic systems that (i) are sensitive, robust, and portable, (ii) allow detection in clinical samples, (iii) require minimal sample preparation yielding results quickly, and (iv) can simultaneously quantify multiple targets, would have a great potential in biomedical research and public healthcare. Bacterial infections still cause pathogenesis throughout the world (Chapter I). The emergence of multi-drug resistant strains, the potential appearance of bacterial pandemics, the increased occurrence of bacterial nosocomial infections, the wide-scale food poisoning incidents and the use of bacteria in biowarfare highlight the …

Electroconvulsive Shock Ameliorates Disease Processes And Extends Survival In Huntington Mutant Mice, Akanksha Baharani

Electronic Theses and Dissertations

Huntington's disease (HD) is a devastating autosomal dominantly inherited neurological disorder caused by an abnormal expansion of CAG trinucleotide repeats in the gene coding for the Nterminal region of the huntingtin (Htt) protein, which leads to the formation of a polyglutamine stretch. The greater the CAG repeats, the earlier the onset of the disease. The polyglutamine stretch destabilizes the Htt protein leading to misfolding, abnormal processing, aggregation, and inclusion formation. Mutant Htt protein is believed to damage and kill neurons in the striatum by a mechanism involving increased oxidative and metabolic stress, and impaired adaptive cellular stress responses. A large …

Transplantation Of Ips Cells Reduces Apoptosis And Fibrosis And Improves Cardiac Function In Streptozotocin-Induced Diabetic Rats, Sarah Elizabeth Neel

Electronic Theses and Dissertations

Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a propancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg …

Cellular And Molecular Mechanisms Of Toxin Resistance For Endoplasmic Reticulum Translocating Toxins, Christopher Massey

Electronic Theses and Dissertations

The endoplasmic reticulum (ER) is the site of co- and post-translational modification for secretory proteins. In order to prevent vesicular transport and secretion of misfolded or misassembled proteins, a highly regulated mechanism called ER-associated degradation (ERAD) is employed. This pathway recognizes misfolded proteins in the ER lumen and targets them to the cytosol for ubiquitination and subsequent degradation via the 26S proteasome. Sec61 and Derlin-1 are ER pores through which export occurs. AB-type protein toxins such as cholera toxin (CT), Shiga toxin (ST), exotoxin A (ETA), and ricin have evolved means of exploiting the ERAD pathway in order to reach …

Bacterial Selenoproteins: A Role In Pathogenesis And Targets For Antimicrobial Development, Sarah Rosario

Electronic Theses and Dissertations

Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of …

Epigenetic Control Mechanisms In Somatic Cells Mediated By Dna Methyltransferase 1, Bongyong Lee

Electronic Theses and Dissertations

DNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications in somatic cells. The mechanistic details that explain how DNMT1 catalytic action is directed in a chromatin setting are not well understood. We hypothesize that post translational modifications and a variety of protein-protein interactions processes are key regulatory elements that set the methylation of CpG elements essential for normal growth behavior in somatic cells. These fundamental …

Tissue Engineering The Motoneuron To Muscle Segment Of The Stretch Reflex Arc Circuit Utilizing Micro-Fabrication, Interface Design And Defined Medium Formulation, Mainak Das

Electronic Theses and Dissertations

The stretch reflex circuit is one of the most primitive circuits of mammalian system and serves mainly to control the length of the muscle. It consists of four elements: the stretch sensor (muscle spindle/ intrafusal fiber lie parallel between extrafusal, contractile musculature), extrafusal muscle fiber, sensory neuron and motoneuron. The basic principle of the stretch reflex arc circuit is as follows: whenever there is a sudden stretch in a muscle, it needs to compensate back to its original length so as to prevent any kind of injury. It performs this compensation process using a simple negative feed back circuit called …

A Study Of Clinical Outcomes Using Serum Albumin And Percentage Of Weight Loss Following Nutritional Intervention In Post-Operative Bariatric Patients., Jennifer Michelle Angus

Electronic Theses and Dissertations

The purpose of this study was to determine if post-operative serum albumin and percentage of weight loss improved in patients who received formalized pre-operative nutrition counseling. Nutrition intervention was measured quantitatively. A retrospective review of records was conducted on 77 RYGB patients (68 female subjects and 9 male subjects), ages 21-64, during January 2001 through January 2006.

The results indicated that patients who received pre-operative nutrition intervention had better clinical outcomes of serum albumin than those with no nutrition intervention from a registered dietitian. However, outcomes regarding percentage of weight loss varied. Both pre-operatively and at the 3 month post-operative …

The Effect Of Caffeine And Ethanol On Flatworm Regeneration., Erica Leighanne Collins

Electronic Theses and Dissertations

Flatworms, or planarian, have a high potential for regeneration and have been used as a model to investigate regeneration and stem cell biology for over a century. Chemicals, temperature, and seasonal factors can influence planarian regeneration. Caffeine and ethanol are two widely used drugs and their effect on flatworm regeneration was evaluated in this experiment. Non-toxic levels of caffeine, a stimulant, and ethanol, a depressant, were determined. The tails of the flatworms were removed and the regeneration stage was analyzed every 3 days for 15 days to see the effect of these drugs alone and in combination on regeneration. For …

Studies On A 50s Ribosomal Precursor Particle As A Substrate For Erm E Methyltransferase Enzyme In Staphylococcus Aureus ., Indira Pokkunuri

Electronic Theses and Dissertations

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. We are interested in investigating the true substrate for methylation because it is known from our work and the work of others that fully assembled 50S subunits are not substrates for methylation. We have published a model for 50S ribosomal subunit formation where, the precursor particle that accumulates in erythromycin treated cells is a target for methyl transferase activity. Current studies are aimed …

Physical Characteristics Of An Individual: The Identification Of Biomarkers For Biological Age Determination, Michelle Alvarez

Electronic Theses and Dissertations

It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information …

Dectin-1 Expression Is Altered By Fungal Infection, Polymicrobial Sepsis, And Glucan Administration., Tammy Regena Ozment-Skelton

Electronic Theses and Dissertations

Glucans are fungal cell wall PAMPs that promote survival in polymicrobial and candidal sepsis. Dectin-1 is the primary PRR for glucans. The goals of the present study were to characterize 1) the effects of fungal infection on Dectin-1; 2) the effects of polymicrobial sepsis in the presence and absence of glucan on Dectin-1; 3) the effects of systemic administration of glucans on Dectin-1; and 4) the intracellular trafficking of glucans. Mice were either systemically infected with Candida albicans, or made septic by CLP with and without glucan phosphate (GP) injection, or injected with GP. Flow cytometry was performed to …

Role Of Zmpste24 In Prelamin A Maturation., Douglas Paul Corrigan

Electronic Theses and Dissertations

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. Following the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 as a candidate to carry out one or both of these proteolytic reactions. In this body of work, the CAAX endopeptidase activity of recombinant, membrane reconstituted, Zmpste24 is demonstrated using …

Angiogenesis And Myogenesis In A Chronic Ischemic Heart., Esha Ibrahim

Electronic Theses and Dissertations

Miniswine underwent procedures to evaluate treating chronic ischemia with the implantation of autologous satellite cells and laser transmyocardial revascularization (TMR). The objective was to combine two therapies to restore cardiac function. This experiment involved three surgical procedures: (1) placing a constrictor on the coronary artery; (2) producing channels and implanting cells; (3) obtaining samples. The swine were divided into groups: Group 1, Ischemia; Group 2, Ischemia + Laser TMR; Group 3, Ischemia + Laser TMR+ Cells; Group 4, Ischemia + Cells. Sonomicrometry and Millar pressure transducers were used to determine contractility, left ventricular pressure, and pressure-volume loops. There were no …

Role Of Macrophage Apoptosis In Atherosclerosis., June Liu

Electronic Theses and Dissertations

The presence of apoptotic cells in atherosclerotic lesions has been broadly reported in the past ten years. The majority of these apoptotic cells are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains to be elucidated. Elevated expression of Bax, one of the pivotal pro-apoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol treated mouse macrophages. In this study, we evaluated the influence of Bax deficiency on apoptosis in macrophage-like P388D1 cells by using small interfering RNA (siRNA) to suppress Bax …

Degradation Of 23s Rrna In Azithromycin-Treated Ribonuclease Mutants Of Escherichia Coli., Jessica A. Silvers

Electronic Theses and Dissertations

Azithromycin, a macrolide antibiotic, specifically binds to the 50S ribosomal subunit of bacterial ribosomes and inhibits translation. Azithromycin also prevents 50S ribosomal subunit assembly by binding to a 50S ribosomal subunit precursor particle. When exposed to azithromycin, several ribonucleases in wild-type Escherichia coli cells degrade antibiotic-bound 50S precursor particles. Presumably, cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound precursor less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonucleaseûdeficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability, growth rates, and protein synthesis rates were measured. Degradation …

Dose-Dependent Effects Of Salmon Calcitonin On Bone Turnover In Ovariectomized Rats., Beatrice H. Owens

Electronic Theses and Dissertations

In the United States, osteoporosis results in about 1.5 million annual fractures, costing approximately $15 billion. Calcitonin is safe and effective in slowing osteoporotic bone loss, but its effect is transient. The current studies were designed to explore the dose-dependent effects of salmon calcitonin on bone turnover in ovariectomized rats and to determine if the decrease in therapeutic effectiveness of calcitonin demonstrated over time with higher doses is due to oversuppression of bone turnover. Doses of 5, 15, & 50 IU/kg BW/day of calcitonin were compared to placebo in 12-week-old ovariectomized and sham-ovariectomized Sprague-Dawley rats for 24 weeks. The spinal …

Papain: A Novel Urine Adulterant., David Burrows

Electronic Theses and Dissertations

The estimated number of employees in the United Stated screened annually for illicit drugs is approximately 20 million, with marijuana being the most frequently abused drug. Urine adulterants provide an opportunity for illicit drug users to obtain a false negative result on commonly used primary drug screening methods such as the Fluorescence Polarized Immunoassay (FPIA) technique. Typical chemical adulterants such as nitrites are easily detected or render the urine specimen invalid as defined in the proposed federal guidelines for specimen validity testing based on creatinine, specific gravity and pH. Papain is a cysteine protease with intrinsic ester hydrolysis capability. The …

Nongenomic Effects Of Estrogens On Epithelial Chloride Secretion., Sabyasachi Moulik

Electronic Theses and Dissertations

The human colonic cell line T84, a model for studying epithelial chloride secretion and cystic fibrosis chloride channel (CFTR) function, was used to investigate the regulatory role of estrogens in transepithelial ion transport. Estrogens and other steroid hormones do not stimulate chloride secretion by themselves. However, 17 β-estradiol (17β-E2) rapidly (within seconds to minutes) potentiates carbachol- and thapsigargin-stimulated chloride secretion measured as short circuit current in voltage-clamped T84 monolayer cultures. The cholinergic agonist carbachol and the SR Ca²⁺ ATPase inhibitor thapsigargin stimulate chloride secretion by elevating intracellular calcium. 17α-estradiol, a stereoisomer that does not activate nuclear estrogen receptors, is …

The Role Of Erp57 In Hras Intracellular Trafficking And Function., Jaime Lyn Parman

Electronic Theses and Dissertations

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led …

Flavone: The Molecular And Mechanistic Study Of How A Simple Flavonoid Protects Dna From Oxidative Damage., Jennifer Dawn Dean

Electronic Theses and Dissertations

Dietary flavonoids are ubiquitous and are marketed as supplements. Characterized as antioxidants, they offer protection against a number of degenerative diseases. Flavonoid mechanics involve free radical scavenging, metal chelation, and substrate association. The skeletal structure of flavonoids is a fused ring system modified by hydroxyl, sugar, and carbohydrate additions. Flavone is a structurally simple flavonoid. Quercetin and its glycosidic analog rutin are complex structures. Using a DNA oxidation/cleavage assay, flavone reduces DNA nicking by 91%. Depending on the solvent system used, quercetin can either increase or decrease DNA oxidation. Rutin exhibits neither pro- nor antioxidant activity. The molecular interactions responsible …

Characterization Of Sympathetic Ganglion Sensitivity To Substance P In A Genetic And A Non-Genetic Rat Model Of Hypertension., John Daniel Tompkins

Electronic Theses and Dissertations

Intravenous injection of substance P (SP) stimulates sympathetic ganglia to evoke a greater increase in renal sympathetic nerve activity, heart rate (HR) and blood pressure (BP) in hypertensive than normotensive rats due to upregulation of the NK₁ receptor. These experiments were designed to determine the cellular basis for the enhanced ganglionic responsiveness to NK₁ agonists in spontaneously hypertensive rats (SHR) in comparison to their normotensive counterparts, Wistar-Kyoto rats (WKY). Studies were also conducted to determine whether the increased ganglion responsiveness to SP in SHR is causally related to the increased BP or is a unique characteristic of this …

Protection Of Pifithrin-Α And Melatonin Against Doxorubicin-Induced Cardiotoxicity., Xuwan Liu

Electronic Theses and Dissertations

The current studies were designed to explore the protective effects of pifithrin-α and melatonin against doxorubicin-induced cardiotoxicity. Doxorubicin was injected at a dose of 22.5 mg/kg (i.p.) in mice to induce cardiotoxic effects. Meanwhile, doxorubicin caused a significant increase of cardiac cell apoptosis following injection (14.2 ± 1.1% for doxorubicin-5 d vs. 1.8 ± 0.12% for control, P < 0.01). Ribonuclease protection assays and Western blot analyses revealed that doxorubicin upregulated the p53-dependent genes Bax, BclxL, and MDM2 at least 2-fold. p53 was phosphorylated at Ser 15 in mouse hearts 1 h following doxorubicin injection, and p38 and ERK1/2 MAPKs mediated the phosphorylation of p53. In addition, caspases-3 and -9 were activated 24 h after doxorubicin injection. A p53 inhibitor, pifithrin-α, inhibited doxorubicin-induced apoptosis when administered at a dose of 2.2 mg/kg. Pifithrin-α abolished p53 transactivation activity, but did not influence doxorubicin-induced phosphorylation at Ser 15. By effectively inhibiting the expression of p53-dependent genes, pifithrin-α blocked doxorubicin-induced activation of caspases-3 and -9, thereby preventing cardiac apoptosis. In addition, pifithrin-α attenuated doxorubicin-induced structural and functional damages, without diminishing its anti-tumor efficacy on p53-null PC-3 cancer cells. The protective effects of melatonin and its metabolite 6-hydroxymelatonin on doxorubicin-induced cardiac dysfunction were evaluated in an isolated perfused mouse hearts and in vivo doxorubicin-treated mice. While perfusion of mouse hearts with 5 μM doxorubicin for 60 min resulted in a 50% suppression of HRxLVDP and a 50% reduction of coronary flow, pre-exposure of hearts to 1 μM melatonin or 6-hydroxymelatonin eased the cardiac …

Activation Of Ca2+-Activated K+ Channels And Cell Migration By Hepatocyte Growth Factor/Scatter Factor In Madin-Darby Canine Kidney Cells., Min Jin

Electronic Theses and Dissertations

Hepatocyte Growth Factor/Scatter Factor (HGF/SF) stimulates migration of various cells and has been linked via Met tyrosine kinase signaling to transformation and the metastatic phenotype. HGF/SF-Met signaling also plays a role in malignancy. Migration of transformed MDCK-F cells depends on activation of a charybdotoxin (ChTX)-sensitive, volume-activated membrane K⁺ current. Patch-clamp electrophysiology and transwell migration assays were used to study the effects of HGF/SF on membrane K⁺ currents and cell migration in MDCK II cells. HGF/SF activated membrane K⁺ currents that increased over 24 hr, and these could be modulated by altering intracellular free calcium concentration [Ca^{2+ …}