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Mechanism (S) Of Metal-Induced Apoptosis In Saccharomyces Cerevisiae, Amrita Mohan Nargund

Biology Dissertations

Heavy metals, such as copper and cadmium have been linked to a number of cellular dysfunctions in single and multicellular organisms that are associated with apoptosis. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cu and Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in YCA1∆ mutant. Other apoptotic markers, including sub-G1 DNA fragmentation …

Mechanisms Of Controlled Proteolysis During Drosophila Spermatogenesis: Coordinate Action Of Apoptotic Caspases And The Ubiquitin-Proteasome System, Maya Bader

Student Theses and Dissertations

The regulation of apoptosis, or programmed cell death has been the subject of a vast body of research because of its implication in normal development, tissue homeostasis and a wide range of diseases. The major point of focus for understanding apoptosis regulation is the activation of its primary executioner: the caspase. This family of proteolytic enzymes has been shown to be tightly controlled, as many different proteins govern their transcription, stability, activation, and activity. Consequently, caspase regulation is extremely complex, and is further complicated by a discrepancy between different cell types and paradigms. Caspases have also been shown to have …

Cell Death Mechanisms In Drosophila Differentiated Photoreceptor Neurons, Alexis Robert Gambis

Student Theses and Dissertations

Apoptosis, or programmed cell death, is a form of physiological cell death that is essential for normal development and homeostasis. At the end of pupal development of the Drosophila retina, cell death terminates and photoreceptor neurons complete their differentiation process. We use these terminally differentiated photoreceptor neurons as a system to study neurodegeneration. We first adapt and develop fluorescent tools for photoreceptor visualization in vivo. These tools enable a recessive genetic screen to search for genes required for the survival of differentiated photoreceptors. Many redox and mitochondrial genes were found to protect photoreceptors from late cell death. Here, we focus …

Bartonella Henselae Inhibits Cellular Apoptotic Regulators To Ensure Survival, Jeffery Todd Parker

Biology Dissertations

Human pathogens survive anti-pathogen host immune assault by either circumventing or evading the host immune response. Bartonella henselae, an intracellular pathogen previously shown to disrupt intrinsic apoptotic messengers to enhance its survival, exploits multiple facets of the cellular apoptotic mechanisms. Cellular pathways affected by apoptotic processes were assessed using real-time reverse-transcriptase-polymerase-chain-reaction (rRT-PCR) to measure the effect of B. henselae on cell regulator gene expression (TRADD, FADD, caspase-8 and caspase-3), caspase activity, DNA cell cycle analysis, cell regulator protein expression and overall cell viability and morphology. The presence of B. henselae suppresses overall gene expression for TRADD and FADD and it …

Structural Basis Of Caspase-3 Substrate Specificity Revealed By Crystallography, Enzyme Kinetics, And Computational Modeling, Bin Fang

Biology Dissertations

Caspase-3 is a cysteine protease that hydrolyzes diverse intracellular proteins during programmed cell death (known as apoptosis). It has been a popular target for drug design against abnormal cell death for more than a decade. No approved caspase based drug, however, is available so far. Therefore, structural insights about the substrate recognition of caspase-3 are needed for the future development of caspase-3 based inhibitors and drugs. In this study, crystal structures of recombinant caspase-3 in complex with seven substrate analog inhibitors, including acetyl (Ac)-DEVD-aldehyde (Cho), Ac-DMQD-Cho, Ac-IEPD-Cho, Ac-YVAD-Cho, Ac-WEHD-Cho, Ac-VDVAD-Cho, and tert-butoxycarbonyl (Boc)-D-fluoromethylketone (Fmk), have been analyzed in combination with …

Generation Of Macrophage Chemotactic Factors By Spla2, Andria Chambers

Theses and Dissertations

Removal of apoptotic cells by macrophages is required in order to prevent autoimmune responses. Previous studies have reported that group IIA secretory phospholipase A2 (sPLA2) preferentially binds to apoptotic cells and induces a chemotactic factor which promotes the migration of macrophages to apoptotic cells. Based on these observations, we hypothesized that the pool of sPLA2 trapped on apoptotic cells produces chemoattractant lipids that recruit macrophages to phagocytose the apoptotic cells. To test this hypothesis, Jurkat cells were cultured under normal conditions and induced to undergo early apoptosis through treatment with D,L-threo-dihydrosphingosine. Live and early apoptotic cells were incubated with catalytically …

Regulation Of Apoptosis By The Muscle Regulatory Transcription Factor Myod, Terri J. Harford

ETD Archive

We have previously reported that the level of MyoD expression correlates with the level of apoptosis that occurs in a subpopulation of skeletal myoblasts induced to differentiate by serum withdrawal. Herein we document that MyoD expression dictates the apoptotic threshold in myoblasts and fibroblasts in response to a variety of apoptotic stimuli. Specifically, re-expression of MyoD in skeletal myoblasts rendered defective for both differentiation and apoptosis by the expression of oncogenic Ras restores their ability to undergo both differentiation and apoptosis in response to serum withdrawal. Further, using a fibroblast cell line expressing an estrogen receptor:MyoD fusion protein, we have …

Myf5 Does Not Induce Apoptosis In Skeletal Myoblasts But Is Regulated By Oncogenic Ras Expression, Alexander Phillip Talarico

ETD Archive

Skeletal myogenesis is initiated by a family of muscle regulatory factors (MRFs) composed of the transcription factors MyoD, Myf5, MRF4, and myogenin. Of these, MyoD was the first discovered, is the most studied, and plays a well-characterized role in skeletal myoblast determination and differentiation. Our laboratory has also discovered that MyoD plays an important role in the apoptotic process that occurs in a subpopulation of myoblasts induced to differentiate. Expression of oncogenic Ras is known to inhibit myoblast differentiation and to decrease the expression of MyoD mRNA and protein. Our laboratory was the first to report that expression of oncogenic …

Effects Of Aging On Regulators Of Muscle Apoptosis In The Female F344bn Rat, Murali K. Gadde

Theses, Dissertations and Capstones

Age-related muscle atrophy is a consequence of normal aging characterized by decreases in muscle mass and strength. The mechanism(s) underlying the loss of muscle mass with increasing age is not fully understood, however recent data has suggested that muscle cell apoptosis may be involved. Here we investigate how aging affects the regulation of muscle apoptosis in the extensor digitorum longus (EDL) and soleus muscles of young (6-month), aged (26-month), and very aged (30-month) female Fischer 344/NNiaHSD X Brown Norway / BiNia (F344BN) rats. EDL and soleus muscle mass/body weight ratios were lower in aged animals but not different between 26- …

Effects Of Direct Mechanical Ventricular Actuation On The Apoptotic Signaling Of A Failing Heart, Scott Kerns

Browse all Theses and Dissertations

Cardiovascular disease accounts for more than 40% of all deaths in the United States (AHA-2004 report). A non blood contacting ventricular assist device (VAD) can be used to treat heart failure without the complications that arise from blood contacting VADs. This study used cellular markers of heart failure as indicators of heart function in an attempt to assess if direct mechanical ventricular actuation (DMVA) support lessened the impact of heart failure in rabbits. Cell signaling proteins were monitored using enzyme activity measurements and quantitative immunoblotting with antibodies against intrinsic and extrinsic apoptotic pathways during heart failure with and without DMVA …

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 …

The C-Phycocyanin/Beta Protein Inhibits Cancer Cell Proliferation, Haizhen Wang

Biology Theses

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-cancer activities. In this study, the beta-subunit of C-PC (ref to as C-PC/beta) was expressed and purified from bacteria E. coli BL-21. The recombinant C-PC/beta has been demonstrated to have anticancer properties. Under the treatment of 5 microM of the recombinant C-PC/beta, four different cancer cell lines accrued a high proliferation inhibition and apoptotic induction. The C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found. Under the treatment of the C-PC/beta, depolymerization of microtubulin and actin-filament was observed. The cells underwent …

Mechanisms By Which Apoptotic Membranes Become Susceptible To Secretory Phospholipase A2, Rachel Williams Bailey

Theses and Dissertations

During apoptosis, changes occur in T-lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A2 (sPLA2). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was first applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: initial hydrolysis rate was elevated, total reaction product was increased four-fold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by substrate availability rather than enzyme adsorption. Fluorescence experiments identified three membrane alterations that might affect substrate access to the …

The Effect Of Febrile Temperature On Plasmodium Falciparum, Heidi Sue Porter

Theses and Dissertations

Previously it has been shown that cultures of Plasmodium falciparum died following exposure to a febrile temperature of 40°C, as demonstrated by a decrease in parasitemia of the following generation. In the current study, the effect of 40°C treatment on culture media, erythrocytes, and parasite glucose consumption, were ruled out as possible influences on parasite death, demonstrating that 40°C impacted the parasites directly. Metabolic profiling of DNA synthesis, protein synthesis, and glucose utilization during exposure to 40°C clearly indicated that febrile temperatures had direct effect on major metabolic pathways and parasite development, beginning 20-24 hr after erythrocyte invasion. The ring …

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 …

Phospholipase A2 Induced Monocyte Chemotaxis To Apoptotic Cells, Kwasi Karikari

Theses and Dissertations

Apoptosis is a form of programmed cell death that is essential in such processes as organ and tissue remodeling and maturation of hematopoietic cells. The clearance of apoptotic cells is essential to prevent autoimmune responses to sequestered antigens. This process is mediated by phagocytes of the monocyte lineage. Before phagocytosis can occur, macrophages must be recruited to the apoptotic cells through chemotaxis. Products of the reaction catalyzed by the phospholipases A2 (PLA2) have been shown to induce monocyte chemotaxis either directly or indirectly. Some investigators have implicated a cytosolic calcium-independent PLA2 (iPLA2) in the production of these products during apoptosis. …

Novel Cancer Therapeutics, The Generation Of Ros, And Cell Survival, Clint Mitchell

Theses and Dissertations

The impact of Ad.mda-7 on the survival of renal cell carcinoma lines (RCC), primary renal epithelial cells, glioblastoma multiforme lines (GBM), and primary rodent astrocytes is unknown. The present studies examine whether the GST fusion protein, GST-MDA-7, and the adenovirus, Ad.mda-7, altered the growth and survival of the A498 and UOK121N RCC lines or radiosensitized GBM, respectively. Due to previous findings that the RCC lines, but not primary renal epithelial cells, were resistant to type 5 adenoviral infection, we used purified GST-MDA-7 protein to show that GST-MDA-7, but not GST, caused a dose-dependent reduction in A498 and UOK121N proliferation but …

Assessment Of Cell Death Parameters In Bovine Parvovirus-Infected Ebtr Cells, Lubna Salah Eldin Abdel Latif

Theses and Dissertations

Bovine parvovirus (BPV) is a helper-independent parvovirus. It has a small icosahedral capsid with a single stranded DNA genome. It is a highly stable virus with a narrow host range. It causes acute gastroenteritis in calves. It is considered to be a cytolytic virus because it kills the host cells. However, the mechanism by which the virus causes cell death is not known. The work described in this thesis assessed different parameters of cell death in BPV infected embryonic bovine tracheal (EBTr) cells. There are several ways for viruses to induce cell death. Viruses can induce apoptosis in the infected …

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 …

Identification Of The Rna Cis-Elements That Interact With Srp30a To Regulate The Alternative Splicing Of Caspase 9 Pre-Mrna, Prabhat Mukerjee

Theses and Dissertations

Studies have shown that the alternative splicing of caspase 9 and the phospho-status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and de novo ceramide via the action of protein phosphatase-1 (PP1). Two RNA splice variants are derived from the caspase 9 gene, pro-apoptotic caspase 9a and anti-apoptotic caspase 9b, via alternative splicing by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. In this study, the link between SR proteins and the alternative splicing of caspase 9 was established. Sequence analysis of the exon 3, 4, 5, and 6 …

Type-5 Phosphodiesterase Inhibition In The Prevention Of Doxorubicin Cardiomyopathy, Patrick William Fisher

Theses and Dissertations

Prior studies have demonstrated the effect of diazoxide in protecting against apoptosis via mitochondrial KATP channel opening in vitro. The current investigations are designed to determine if sildenafil, a phosphodiesterase-5 inhibitor and known mitochondrial KATP channel opener, would protect against chronic doxorubicin cardiomyopathy both in vivo and in vitro.Male ICR mice were randomized to 1 of 4 treatments: saline, sildenafil (0.7 mg/kg IP), doxorubicin (5 mg/kg IP), and sildenafil (0.7 mg/kg IP)+doxorubicin. Apoptosis was determined using the terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling and in situ oligo ligation methods. Desmin distribution was determined via immunofluorescence. Bcl-2 was analyzed by …

Role Of Iron In The Mechanism Of Asbestos-Induced Apoptosis In Human Lung And Pleural Target Cells, Aleksander Baldys

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Occupational exposure to asbestos has been associated with increased incidence of pulmonary interstitial fibrosis, mesothelioma of the pleura, and bronchogenic carcinoma. Although the mechanism by which asbestos causes cancer remains unknown, iron associated with asbestos is thought to play a role in the pathogenic effects of fibers.

The aim of this research was to examine and compare the asbestos-induced signaling phenomena in relevant human lung and pleural target cells, and to determine the role of iron from asbestos fibers in these events. Exposure of human airway epithelial (A549) cells, human pleural mesothelial (MET5A) cells, and normal human small airway epithelial …

Apoptosis Pathways: Presence And Significance In Ejaculated Human Spermatozoa, Steven Lewis Taylor

Theses and Dissertations in Biomedical Sciences

Ejaculated sperm display markers that are indicative of apoptosis in somatic cells. The question remains as to whether sperm have operative apoptosis mechanisms. The aim of this research was to test the hypothesis that apoptosis markers in sperm and somatic cells are different.

Ejaculated human sperm from patients and donors were separated into high and low motility fractions using Percoll™ gradients. Contaminating cells were removed using anti-CD45 conjugated paramagnetic beads. Fractions were divided into groups: staurosporine, anti-Fas antibody, and hydrogen peroxide treated and control. Direct enzymatic measurement of caspase activity, flow cytometric evaluation of phosphatidylserine translocation, immunoblots, and immunocytochemistry were …

The Effects Of R-Flurbiprofen In Reducing Tumors In A Multiple Intestinal Neoplasia Mouse Model, David Douglas Quiggle

Theses Digitization Project

The design of the proposed study was to administer R-FB to 72-day old Min/+ mice for up to 42 days. In order to capture the process of tumor reduction, animals were necropsied at various time points. At each time point animals were evaluated for tumor loads and presence of apoptotic cells along the small intestine. Studies have shown that when R-flurbiprofen (R-FB) is administered in the Min/+ mouse model it can cause the prevention and regression on intestinal tumors.

Induction Of Apoptosis In Human Prostate Cancer Cells By Resveratrol, Gary Zulfikar Morris

Chemistry & Biochemistry Theses & Dissertations

Recently attention has been brought to trans-resveratrol's {TR) anticancer activity, as determined through a number of cultured cancer cell models. This activity was attributed to TR behaving as an estrogen, and the orientation of TR' s hydroxyl groups. Based on this work it was of interest to determine whether TR would also be toxic in prostate cancer cells; if toxic, did TR induce necrosis or apoptosis in the cells; was it toxic through hormone mediated pathways; and were TR's hydroxyl groups responsible for its biological activity. To this end, cellular viability was assessed in two different prostate cancer cell …

Cd40-Mediated Signaling Of Interleukin-1(Beta) Synthesis And Rescue From Apoptosis In Monocytes: Modulation By Il-4 And Il-10, Jonathan C. Poe

Electronic Theses and Dissertations

To date, the cellular mechanisms involved in the progression of diseases characterized by chronic inflammation, such as rheumatoid arthritis (RA), remain largely unknown. However, cell-to-cell contact interactions between CD4+ helper T (Th) cells and monocytes have been implicated in the induction and maintenance of pro-inflammatory cytokine synthesis that is characteristic to the pathogenesis of RA. One such cytokine produced during monocyte-Th cell contact is interleukin (IL)-1 β, a mediator directly involved in the characteristic tissue destruction that occurs in the synovia of individuals with RA. Previous studies in our laboratories have shown that ligation of CD40 on monocytes with CD40 …

The Role Of Fas And Fas Ligand In Apoptosis During Regression Of The Corpus Luteum, Sharon Roughton

Theses : Honours

Apoptosis, a form of physiological cell death, has been found to occur during regression of the corpus luteum (Juengel etal, 1993; Dharmarajan etal, 1994). The pathways involved in this process, however, have yet to be specified. One possible mediator of corpus luteum regression is the Fas (or AP0-1 or CD95) receptor, a transmembrane protein which induces apoptosis in the cell when ligated. In order to further confirm this hypolhesis, the present study establishes and quantitates the presence and regulation of Fas receptor and Fas ligand (Fasl) in the rat corpus luteum during pregnancy and post-partum. The animals used were sexually …