Department: Medicine : Pathobiology and Molecular Medicine ![[clear]](close-x.png "Remove this limiter")
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1.
Accurso, Charity Einhaus.
Fibrinogen-Coated Droplets of Olive Oil for the Targeted Delivery of Docetaxel to Fibrin(ogen)-Rich Tumors: Evaluation of Efficacy and Mechanism.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2004, University of Cincinnati
► Fibrinogen, a protein associated with all malignant tumors, binds rapidly and near…
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▼ Fibrinogen, a protein associated with all malignant tumors, binds rapidly and near irreversibly to hydrophobic surfaces, including droplets of olive oil. When administered to a mouse, fibrinogen-coated oil droplets will accumulate at fibrin(ogen)-rich sites, i.e., inflammatory regions and tumors. For my thesis research I show that fibrinogen-coated oil droplets can be used effectively to target lipophilic drugs to fibrin(ogen)-rich tumors. I use the lipophilic anticancer taxane docetaxel for my experiments. I demonstrated, first, that packaging docetaxel into fibrinogen-coated oil droplets does not limit the drug’s cytotoxicity. Preliminary survival studies demonstrate that the formulation prolongs the survival of B16F10 melanoma-bearing mice by 330% when compared to survival conferred by the clinical formulation, Taxotere®. Using the TA3/St ascites tumor model, I then show that the formulation increases the lifespan of tumor-bearing mice 211% as compared to 53% for Taxotere®. I demonstrate next that thrombin activity has a role in the retention of droplets, and that inhibition of thrombin activity reduces the efficacy of the formulation to that of Taxotere®. Although mice treated with fibrinogen-coated oil droplets develop significant levels of anti-fibrinogen antibodies, those antibodies do not elicit an overt coagulopathy, they do not prolong the activated partial thromboplastin time, and they do not appear to have an effect on the therapeutic efficacy of the oil droplets. Taken together, my results and observations indicate that fibrinogen-coated oil droplets markedly improve the therapeutic efficacy of docetaxel for the treatment of a mammary tumor grown in ascites form, a consequence of thrombin-mediated retention of the drug-loaded droplets within the tumor microenvironment. I performed additional studies to achieve mechanistic understanding of fibrinogen binding to hydrophobic surfaces, and to identify a naturally-occurring system for the potential delivery/targeting of lipophilic drugs. Using proteolytic and radiologic methods, I demonstrate that fibrinogen binds to hydrophobic surfaces via its d2Dγ region. The protein and its binding orientation of fibrinogen is concentration dependent. I also demonstrate that fibrinogen binds to, and remains functional on, chylomicrons in the gastrointestinal lymph, a phenomenon that I believe may be exploited for drug delivery.
Advisors/Committee Members: Retzinger, Dr. Gregory S.
Subjects: Health Sciences, Pathology
Keywords: fibrinogen; drug delivery; emulsion; ascites; docetaxel; olive oil
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2.
Barber, Latorya Arnold.
The Activity of Lipid Transport Proteins in Normal and Sickle Red Blood Cells.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2009, University of Cincinnati
► Phosphatidylserine (PS) externalization occurs in sickle red blood cells (RBC) and is…
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▼ Phosphatidylserine (PS) externalization occurs in sickle red blood cells (RBC) and is thought to contribute to (1) increased sickle RBC-endothelial cell adhesion, (2) increased thrombogenesis and (3) decreased sickle RBC survival. Normal phospholipid (PL) asymmetry is maintained through a normally active ATP-dependent aminophospholipid translocase (APLT) and a normally inactive phospholipid scramblase (PLSCR). When this balance is disturbed, via PLSCR activation in the presence or absence of active APLT, PS externalization occurs. This body of work addresses relative APLT and PLSCR activities in control RBC without external phosphatidylserine (PS-) and in PS-exposing sickle RBC. We also address potential mechanisms of PS externalization via APLT inhibition, PLSCR activation, or both. Our studies show dehydration is one mechanism of APLT inhibition, and this most likely contributes to the high levels of PS externalization observed in dense sickle RBC. Our studies also provide evidence that protein kinase C (PKC) activation is a mechanism of PS externalization through activation of PLSCR. Finally, we show that APLT activity decreases as normal human RBC age in vivo. In accordance with the generally held but previously unsubstantiated view, the organization of phospholipids in the mature RBC membrane depends on the relative activities of APLT and PLSCR. Contrary to conventional wisdom, however, we show that PS externalization can occur in the presence of normal levels of APLT activity when PLSCR is sufficiently activated. These studies indicate that that preventing or reversing PLSCR activity may be an effective way to prevent or reverse PS externalization in sickle RBC.
Advisors/Committee Members: Franco, Robert.
Subjects: Molecular biology
Keywords: Sickle Cell Disease; Red Blood Cells; Phosphatidylserine
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3.
Bhabhra, Ruchi.
The role of the nucleolar protein CgrA in thermotolerant growth, ribosome biogenesis and virulence of Aspergillus fumigatus.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2007, University of Cincinnati
► The opportunistic fungal pathogen Aspergillus fumigatus has become a serious obstacle to…
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▼ The opportunistic fungal pathogen Aspergillus fumigatus has become a serious obstacle to the effective treatment of transplant and cancer patients. The fungus is among the fastest growing species of its genus, and is notoriously thermotolerant, allowing it to cause disseminated infection despite the use of available therapy. A. fumigatus infections continue to be associated with a poor clinical outcome, underscoring the need for increased understanding of pathways that support rapid growth in the host. The purpose of this thesis was to gain insight into the contribution of ribosome biogenesis to the growth and virulence of A. fumigatus, focusing on the conserved fungal nucleoar protein CgrA. Results from Chapter 1 demonstrated that CgrA is required for virulence in a mouse model of aspergillosis at 37°C, but is dispensable for virulence in an insect model at 25°C. This was shown to be a direct consequence of the effect of CgrA on thermotolerant growth; CgrA was dispensable for growth below 25°C, but was necessary for the rapid growth rate associated with 37°C. Results shown in Chapter 2 revealed that CgrA redistributes between the nucleolus and the cytoplasm in a temperature-dependent manner, indicating that CgrA function is thermally regulated at the level of intracellular localization. CgrA was shown to facilitate the accumulation of another protein in the nucleolus, NopA, a function that was also shown to be temperature-dependent. Finally, in Chapter 3, polysome profiling was used to determine the contribution of CgrA to ribosome biogenesis. The findings demonstrated that CgrA is essential to maintain normal 60S and 40S subunit stoichiometry, and that CgrA function is more necessary for ribosome biogenesis at 37°C than it is at 22°C. A. fumigatus was shown to respond to a loss of CgrA function by upregulating the expression of a subset of the translational machinery, in addition to increasing the number of nuclei during germination. These experiments define a role for CgrA in ribosome synthesis and highlight potential compensatory mechanisms to loss of CgrA function. Taken together, these findings provide new insights into how A. fumigatus meets the challenge of ribosome assembly and growth at host temperature, which may ultimately lead to the development of novel therapeutic approaches.
Advisors/Committee Members: Askew, Dr. David S.
Keywords: Aspergillus fumigatus; Thermotolerance; Ribosome biogenesis
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4.
Chapman, Jamie M.
CARBOXYL ESTER LIPASE (CEL) IS A MAJOR ENZYME RESPONSIBLE FOR THE HYDROLYSIS OF CHOLESTERYL ESTERS IN THE SELECTIVE UPTAKE PATHWAY OF THE LIVER.
Degree: MS, Medicine : Pathobiology and Molecular Medicine, 2000, University of Cincinnati
► Carboxyl ester lipase (CEL) is primarily a pancreatic enzyme that is also…
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▼ Carboxyl ester lipase (CEL) is primarily a pancreatic enzyme that is also expressed in other tissues, including the mammary gland. Its expression and activity in the liver has been debated for many years. Our primary goal has been to link this enzyme's activity with the selective uptake pathway in the liver. Cholesterol esters that are accepted into cholesterol rich microdomains, termed caveolae, are preferentially shunted to the bile though a series of loosely defined endosomes and vesicles. These cholesterol esters must be hydrolyzed into free cholesterol before they are excreted into the bile. The site of this hydrolysis, and the enzyme responsible have not been identified. Our data indicate that CEL has activity in the liver, and that it is associated with caveolae. Mice deficient in this gene, have a mild increase (~20%) in cholesterol, both in the liver and in the serum, suggesting that cholesterol metabolism is altered in some way. Data also show that the hydrolysis of cholesterol esters at the level of caveolae is markedly decreased (> 2 fold) in the knockout animal, however, the liver's ability to take up cholesterol through this pathway does not appear to be altered. Excretion of cholesterol into the bile is increased two fold in the CEL knockout animals, and a statistically significant increase in phospholipid concentration and the bile acid, taurocholate, is also seen. Histological sectioning of CEL knockout liver shows a marked increase in the amount of neutral lipid in the liver. These data taken together indicate that CEL serves an important role in the metabolism of selectively internalized cholesterol esters. The data also show that this enzyme has its activity at the level of caveolae where the uptake of cholesterol esters through the selective pathway initiates.
Advisors/Committee Members: Howles, Philip N.
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5.
Deem, Tracy L.
VCAM-1 Signaling in Endothelial Cells for Lymphocyte Migration.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2004, University of Cincinnati
► Leukocyte migration is important for (patho-) physiological processes. The role of the…
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▼ Leukocyte migration is important for (patho-) physiological processes. The role of the leukocyte during migration has been well characterized; however, the function of the endothelium during lymphocyte migration is relatively unknown. Adhesion molecules on the endothelial cells are known to play a role in leukocyte rolling and capture. Recently, it has been demonstrated that these adhesion molecules mediate endothelial cell signals upon leukocyte binding. Our laboratory has shown that lymphocyte binding to vascular cell adhesion molecule-1 (VCAM-1) stimulates the activation of NADPH oxidase for the catalysis of low levels of reactive oxygen species (ROS). Activation of this enzyme complex is required for lymphocyte migration. This thesis describes a functional role for the VCAM-1-stimulated release of ROS in mediating actin structural changes within the endothelial cell necessary for lymphocyte migration. Furthermore, we have shown a mechanism for how these ROS modulate endothelial cell shape changes. VCAM-1-dependent release of ROS rapidly activates within minutes endothelial cell MMP-2 and MMP-9. Even though we demonstrate that VCAM-1-dependent ROS can activate lymphocyte MMP-9 hours after lymphocyte binding, it is the endothelial cell MMPs that are required for lymphocyte migration. This is the first report to examine endothelial cell MMP activity during lymphocyte migration. We further demonstrate that VCAM-1-dependent ROS can modulate endothelial cell shape changes through indirect activation of PTP1B. Activation of PTP1B is required for lymphocyte migration likely through modulation of endothelial cell junction proteins. In fact, we demonstrate that VCAM-1-dependent PTP1B activity causes increased serine phosphorylation of the tight junction protein zonula occludens-1 (ZO-1). Increased serine phosphorylation of ZO-1 increases epithelial cell permeability. This is the first report to determine a mechanism for how adhesion molecule signaling modulates endothelial cell junctions for lymphocyte migration. Taken together, these data further advance our knowledge of VCAM-1-mediated endothelial cell function during migration. It is important to understand VCAM-1 signaling for potential intervention of diseases such as atherosclerosis and tumor metastasis.
Advisors/Committee Members: Cook-Mills, Dr. Joan M.
Keywords: VCAM-1; adhesion molecule; migration; signaling; endothelial
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6.
Fortwendel, Jarrod R.
Aspergillus Fumigatus Ras Homologs Regulate Vegetative Growth, Development and Virulence.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2005, University of Cincinnati
► Conidial germination and growth by apical extension are two processes required for…
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▼ Conidial germination and growth by apical extension are two processes required for the initiation and progression of disease by the opportunistic pathogen Aspergillus fumigatus. The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. Therefore, we sought to determine the requirements for Ras proteins in conidial germination and hyphal develolpment of A. fumigatus. Previously, only one homolog, rasA, had been identified, but nothing is known of its role in growth and development. We have identified a second homolog, rasB, which characterizes a new subclass of Ras genes, found only in fungi that undergo hyphal growth. Unique to the rasB homologs is a conserved amino acid domain found just upstream of the third GTP/GDP binding region. To gain insight into the roles played by RasB in A. fumigatus, dominant active (DA), dominant negative (DN) and deletion mutations were constructed for both Ras homologs. DArasB expression led to reduced conidiation. Expression of DNrasB slightly delayed the initiation of germination and caused the development of conidiophores in submerged culture, an abnormal event in A. fumigatus. Deletion of A. fumigatus rasB caused decreased germination and growth rates but no significant change in total biomass after 24 hours of growth. Deletion of rasB also created an irregular hyphal morphology characterized by increased branching. Expression of rasBΔ113-135, a mutant lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype. Virulence of the rasB deletion strain was also reduced, as mice infected with this strain exhibited ~70% survival compared to ~10% with wild type and reconstituted strains. In contrast, DArasA expression led to reduced conidiation, malformed conidiophores, and altered mitotic progression. Expression of DNrasA caused a significant reduction in the rate of conidial germination, without further altering development. However, complete deletion of rasA caused slowed germination, nearly absent radial outgrowth, and decreased hyphal mass. Although conidiation was reduced and delayed, the conidial viability was near wild type levels. The ΔrasA mutant was also resistant to protoplast preparation using normal levels of cell wall digestion enzymes. Normal mitotic events in the mutant were also disturbed, as staining of nuclear material revealed many small nuclei that were irregularly positioned throughout the hyphal compartments. RasA and RasB appear to play distinct, and overlapping, roles in the vegetative growth and asexual development of A. fumigatus. The data in this study, along with the fact that rasB homologs are highly conserved among fungi that undergo hyphal growth, also support the hypothesis that rasB may control signaling modules important to the directional growth of fungal hyphae.
Advisors/Committee Members: Rhodes, Dr. Judith C.
Keywords: Aspergillus; Ras; Fungus; Branching; Germination; Development; Virulence
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7.
GANDHI, MANOJ SURESH.
ROLE OF NUCLEAR ORGANIZATION, GENE TOPOLOGY AND CHROMATIN ARCHITECTURE IN GENE REARRANGEMENTS.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2006, University of Cincinnati
► Chromosomal rearrangements are common in human cancer. Some genes are preferentially involved…
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▼ Chromosomal rearrangements are common in human cancer. Some genes are preferentially involved in interchromosomal rearrangement, where the genes involved are located on different chromosomes, while other genes recombine mainly with genes on the same chromosome. While spatial proximity of recombinogenic partners has been established as one of the important factors in this process, little information exists to explain why certain genes are in close proximity and why they preferentially participate in intrachromosomal or interchromosomal rearrangement. The following aims address the hypothesis that specific nuclear organization of genes within the interphase nucleus predisposes them to recombine with particular partners. Aim 1: To determine the interphase structure of the 10q11.2-q21 chromosomal region that is frequently involved in RET/PTC rearrangements in thyroid cancer Previously, it has been found that spatial proximity exists between RET/PTC1 partners, RET and H4. Using a novel ex-vivo technique of nuclear preparation, we performed 3D FISH on interphase thyroid nuclei and established spatial proximity between RET/PTC3 partners, RET and NCOA4. Spatial coordinates of several chromosomal loci in this region were determined, allowing manual reconstruction and mathematical modeling of chromosomal shape in the 10q11.2-q21 region. This approach suggested that this region is folded in the form of an irregular helix with ~8Mb turns, and provides a structural basis for the spatial proximity between the recombinogenic loci. Aim 2: To determine whether the positioning of genes with respect to their chromosome territories plays a role in their participation in intrachromosomal or interchromosomal rearrangement. It is known that chromosomes occupy distinct sub-compartments within the interphase nucleus known as chromosome territories (CT). It is possible that gene position within a CT may predispose a gene to recombine with loci, while positioning on the periphery of a CT may facilitate recombination with loci on different chromosomes. To address these possibilities, we studied the intraterritorial positions of genes that are known to be involved in either intrachromosomal or interchromosomal rearrangements in thyroid cells. There was a correlation between peripheral CT positioning and participation in interchromosomal rearrangement, while genes that are positioned away from their CT edge were preferentially involved in intrachromosomal rearrangements. We also examined expression of these genes in thyroid cells. Genes with higher expression levels were preferentially positioned towards the CT periphery as compared to genes that had lower expression levels, which were positioned closer to the territory center. These findings provide evidence that nuclear architecture plays a role in determining the types of carcinogenic chromosomal rearrangements that occur in human cells. The results of this study allow better understanding of radiation carcinogenesis in human cells and, in the long-term, will facilitate developing strategies for diminishing the risk of radiation-induced thyroid cancer in human populations.
Advisors/Committee Members: Nikiforov, Dr. Yuri E.
Subjects: Health Sciences, Pathology
Keywords: Chromosomal Rearrangements, Nuclear Architecture, Chromosomal Territories, FISH
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8.
Hess, Krista Lynn.
High Endothelial Venule Cell Phagocytosis of Apoptotic Leukocytes.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2000, University of Cincinnati
► High endothelial venule (HEV) cells are specialized endothelial cells found in post…
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▼ High endothelial venule (HEV) cells are specialized endothelial cells found in post capillary venules within the paracortical areas of secondary lymphoid tissues, such as lymph nodes, tonsils, and Peyer’s patches (1). These cuboidal-shaped endothelial cells are most prominently known for their role in promoting naive lymphocyte migration from the blood into these peripheral lymphoid tissues (2). However, we have demonstrated that HEV cells also perform the additional function of recognizing and phagocytosing apoptotic leukocytes (3), (Chapter 3). By performing both functions, HEV cells regulate leukocyte trafficking by removing apoptotic cells prior to membrane integrity loss and thus authorize the migration of only functional lymphocytes into lymphoid tissues. Apoptosis and the clearance of apoptotic cells via phagocytosis is a widespread, biological process that occurs during development, tissue maintenance, and disease states (4). External and internal signals induce cells to progress through the apoptotic pathway resulting in cell death. Current literature suggests that cells acquire various apoptotic characteristics while undergoing apoptosis. The reported order for acquisition of these characteristics is that membrane changes such as increases in membrane fluidity and externalization of phosphatidylserine precede DNA fragmentation which then precedes membrane permeability to propidium iodide (5, 6). In contrast, we have determined that the order in which four types of leukocytes obtain apoptotic characteristics was dependent on both the cell type and the method of inducing apoptosis (Chapter 4). For example, IL-3-deprived 32D cells undergo DNA fragmentation prior to obtaining an increase in membrane fluidity (Chapter 4). Secondly, WEHI-231 cells that have been induced to undergo apoptosis by two different methods have two different orders in which they acquire apoptotic membrane and nuclear changes (Chapter 4). This implies that in vivo, different modes of inducing apoptosis stimulate the acquisition of multiple apoptotic characteristics in different cell-specific orders. This further suggests that recognition of temporally expressed apoptotic characteristics is completed by different phagocytes present within a microenvironment or by a specific phagocytic cell type which has been given multiple opportunities to internalize its apoptotic target cell. Removal of apoptotic cells is pivotal for tissue protection against toxic cellular debris, such as noxious enzymes and auto antigens. Multiple types of phagocytes, including endothelial cells, are capable of recognizing and ingesting apoptotic cells. Therefore, the employment of multiple types of phagocytes insures that this clearance process is successful throughout the body (7). Phagocytes such as macrophages (8) and fibroblasts (9) are currently used as model systems to examine the mechanisms involved in this removal process. However, current methods used to quantitate phagocytosis include cumbersome cell counts by light microscopy. Therefore, we developed a flow cytometry-based assay to quantitate HEV cell phagocytosis (10), (Chapter 2). Our assay differentiates between HEV cells with internalized cells versus cells that are merely bound to the HEV cells’ surface. This differentiation is crucial because not all cells bound to HEV cells are designated for phagocytosis; viable leukocytes that bind to HEV cells via adhesion molecules migrate into lymphoid tissues. In Chapter 5, we examined the phagocytic receptors utilized by mHEV cells to ingest apoptotic leukocytes and correlated receptor usage with acquisition of apoptotic characteristics by apoptotic leukocytes as determined in Chapter 4. Using our phagocytic assay (Chapter 2), we ascertained that the mHEV cells utilized fucoidin and mannan-specific lectin receptors to internalize apoptotic cells, prior to apoptotic cell membrane and nuclear changes that were examined. In addition, phagocytosis did not interfere with mHEV cell promotion of lymphocyte migration. The results contained herein confirms that mHEV cells are able to phagocytose apoptotic cells via fucoidin/mannan-specific lectin receptors while promoting lymphocyte transendothelial migration. This suggests that in vivo, HEV cells have the ability to regulate lymphocyte trafficking successfully and maintain tissue homeostasis in the surrounding microvasculature and lymphoid tissues. Moreover, these results imply that HEV cells remove apoptotic cells to insure that only viable lymphocytes migrate into lymphoid tissues to encounter sequestered antigen and become stimulated to confront pathological agents at sites of infection and inflammation.
Advisors/Committee Members: Cook-Mills, Dr. Joan.
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9.
ICENHOUR, CRYSTAL RENEE PERRY.
EXPERIMENTAL EVIDENCE FOR COMPETITIVE COEXISTENCE OF TWO SPECIES OF PNEUMOCYSTIS WITHIN RAT LUNGS.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2002, University of Cincinnati
► Pneumocystis burden were associated with Pneumocystis fluctuations, suggesting that the competition was…
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▼ Pneumocystis burden were associated with Pneumocystis fluctuations, suggesting that the competition was mediated by environmental factors. Microscopic analysis of rat lung sections showed that both species could exist in close apposition within the same alveoli, excluding habitat heterogeneity as a mechanism for coexistence. The immediate environment of the rat colony was surveyed for the presence of both species to find reservoirs of Pneumocystis, resulting in their identification from walls, floor, air vents, bedding, fur, and feces. Putative infective forms were isolated from air vents and oral cavities with P. carinii-specific antibody coated magnetic beads. These findings indicate that the immediate environment may harbor viable Pneumocystis. Differences between acquisition/transmission of both species were evaluated using targeted PCR of DNA from oral swabs, an ante mortem method developed to monitor P. carinii and P. ratti within the same rat. This technique could predict P. carinii infection outcome, but not P. ratti. Application of this technique showed that P. carinii could be acquired by neonatal rats within the first hour of life, but there was no evidence for vertical transmission of P. carinii by PCR analysis of fetal tissues. These studies suggested that Pneumocystis was acquired early in life. When P. carinii and P. ratti were present in the same lung, a competitive relationship occurred. Neither species was eliminated from the colony, suggesting coexistence. The competition interaction of the two species was likely influenced by environmental factors, suggesting such extrinsic conditions had an influence on the life cycle of these organisms.
Advisors/Committee Members: Cushion, Dr. Melanie T.
Keywords: pneumocystis; infectious disease; medical mycology
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10.
Joffrion, Tiffany Michelle.
Sterol biosynthesis and sterol uptake in the fungal pathogen Pneumocystis carinii.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2010, University of Cincinnati
► Fungi in the genus Pneumocystis are the cause of a potentially life…
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▼ Fungi in the genus Pneumocystis are the cause of a potentially life threatening pneumonia, Pneumocystis pneumonia (PCP). The understanding of the lifecycle, metabolism, and drug development has been hindered due to a lack of a long term in vitro culture system. Unlike most other fungi, members of the genus Pneumocystis do not appear to synthesize the major fungal sterol, ergosterol. However, genome scans and in vitro assays suggest the presence of functional genes involved in a sterol pathway. One of the goals of this work was to characterize the P. carinii sterol enzyme, lanosterol synthase (Erg7p), an essential enzyme of the sterol pathway. The activity of P. carinii Erg7p was assessed by heterologous expression of P. carinii Erg7p in a Saccharomyces cerevisiae Erg7p null mutant. Growth rates and lanosterol production were similar between S. cerevisiae expressing the P. carinii enzyme and S. cerevisiae expressing its own Erg7p under the same conditions, indicating that not only does P. carinii produce a functional Erg7p, but also that the enzyme functionally complements the S. cerevisiae enzyme. Western blotting and fluorescent localization studies revealed that P. carinii Erg7p localizes to lipid particles in S. cerevisiae as does S. cerevisiae Erg7p. A novel finding of these studies, was that P. carinii contains lipid particles, and that P. carinii Erg7p localizes to lipid particles in P. carinii. These studies indicate that P. carinii Erg7p functions similar to the S. cerevisiae enzyme, and may perform a similar function in P. carinii. Biochemical analyses of sterols within the membranes of P. carinii have shown that it utilizes cholesterol rather than ergosterol as its bulk sterol. However, P. carinii does not appear to synthesize cholesterol from a de novo pathway, but rather scavenges exogenous sterols from its mammalian host. S. cerevisiae is induced to undergo sterol scavenging under anaerobic conditions. Consequently, another goal of this work was to provide information on the effect of O2 on sterol biosynthesis and sterol scavenging by P. carinii. ATP measurements revealed that the viability of P. carinii is severely decreased when maintained under hypoxic conditions, and this decrease correlated with an increase in drug susceptibility. We show that uptake of exogenous cholesterol by P. carinii occurred under normal O2 tensions, indicating that sterol scavenging is not limited to anaerobic conditions. Microarray analysis indicated that hypoxic maintenance of P. carinii resulted in decreased transcription of several genes involved in sterol and lipid biosynthesis suggesting that while hypoxic conditions down-regulated genes involved in sterol biosynthesis, down-regulation of sterol biosynthesis is not a requirement for sterol scavenging in P. carinii. The ability of P. carinii to scavenge exogenous sterols under normal O2 tensions at which the sterol pathway is unaffected provides evidence that sterol scavenging may be the primary means that P. carinii utilizes to obtain its sterols.
Advisors/Committee Members: Cushion, Melanie.
Subjects: Microbiology
Keywords: Pneumocystis carinii; Sterol Biosynthesis; Sterol Uptake; Lanosterol synthase; Hypoxia
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11.
Keshavan, Pavitra.
Bilirubin: Physiological Role in Cancer and Inflammation.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2006, University of Cincinnati
► Bilirubin is the principal end-product of heme catabolism. While bilirubin is traditionally…
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▼ Bilirubin is the principal end-product of heme catabolism. While bilirubin is traditionally considered a metabolic waste product, recent epidemiological analyses support an inverse association between serum bilirubin levels and cancer-related mortality; hence, we postulated that bilirubin plays a role in the suppression of tumorigenesis. To evaluate the effect of bilirubin on intestinal tumorigenesis, we administered bilirubin orally to Min mice, which develop intestinal adenomas due to an inherited mutation in the Apc gene. Bilirubin treatment resulted in a ~70% reduction in both macroscopic and microscopic tumor burden compared to untreated animals; while tumor size remained unaffected. Bilirubin did not alter the nuclear translocation of β-catenin (a hallmark of Apc lack) in intestinal crypts, suggesting that the inhibitory effect of bilirubin is exerted subsequent to tumor initiation. Furthermore, bilirubin, at physiologic concentrations, causes apoptosis of cultured colon adenocarcinoma cells by a mechanism involving mitochondrial depolarization. On the other hand, we did not observe an increase in apoptosis in the intestines of Min mice treated with bilirubin, possibly due to efficient phagocytosis of apoptotic nuclei. Leukocyte transmigration across vascular endothelia is mediated by vascular cell adhesion molecule-1 (VCAM-1) via a mechanism involving generation of reactive oxygen species (ROS) by NADPH oxidase. As bilirubin is the most potent endogenous antioxidant, we postulated that bilirubin inhibits inflammatory responses by blocking VCAM-1 signaling through scavenging of ROS. In an in vitro model of VCAM-1-mediated lymphocyte migration, bilirubin significantly inhibited lymphocyte movement across endothelial monolayers and also VCAM-1-stimulated activation of matrix metalloproteinases-2 and -9, potentially preventing NADPH oxidase-dependent ROS production. In a mouse model of ovalbumin-induced asthma, intraperitoneal bilirubin administration blocked the pulmonary accumulation of eosinophils and lymphocytes, which is VCAM-1-dependent. As bilirubin did not alter pulmonary VCAM-1 expression or cytokine profiles, we speculate that the suppressive effect of bilirubin on lung inflammation is exerted due to the scavenging of the ROS generated by VCAM-1 activation. Taken together, these data support that bilirubin exerts an inhibitory effect on colonic tumor formation and inflammation. We speculate that bilirubin may play a vital role in the suppression of chronic inflammation-associated cancers and also modulate other ROS-regulated processes.
Advisors/Committee Members: Zucker, Stephen D.
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12.
Kindel, Tammy Lyn.
The Effects of Duodenal-jejunal Bypass on Glucose Homeostasis.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2010, University of Cincinnati
► Roux-en-y gastric bypass (RYGB) is a bariatric surgery used for the treatment…
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▼ Roux-en-y gastric bypass (RYGB) is a bariatric surgery used for the treatment of morbid obesity that involves the creation of a small gastric pouch with partial gastric and proximal small bowel exclusion and expedited distal small bowel nutrient delivery. Evidence exists that RYGB is among the most effective bariatric procedures in treating type 2 diabetes with the rate of diabetes resolution well exceeding that which can be explained by weight loss. Although the mechanisms for the weight-independent restoration of euglycemia have yet to be established, the surgical diversion of nutrients away from the duodenum appears to play an important role. The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), are secreted from the gastrointestinal tract in response to enteral nutrients and augment glucose-mediated insulin secretion from the pancreas. We hypothesized in this thesis that an important aspect of the resolution of type 2 diabetes after RYGB involves altered incretin secretion after duodenal-jejunal bypass (DJB) with enhanced GLP-1 and decreased GIP secretion. To test this hypothesis, we first needed to determine if DJB decreases gastrointestinal GIP secretion. We used lymphatic sampling to study nutrient-induced incretin secretion two weeks after DJB or Sham surgery in Wistar rats. We found that DJB did not improve glucose tolerance or alter meal-stimulated GIP secretion. The second aim tested the hypothesis that DJB increases GLP-1 secretion and thus improves glucose tolerance in type 2 diabetes. We first characterized the lymphatic incretin response to different nutrients in type 2 diabetic, Goto-Kakizaki (GK) rats. We found that GK rats have a defect in glucose-mediated secretion of both incretins to a glucose-containing meal. We subsequently compared DJB and ileal interposition (IT) in GK rats to determine if duodenal bypass offers an independent mechanism to improve glucose tolerance. We found that both DJB and IT equally and modestly improved oral glucose tolerance suggesting a mechanism mediated by enhanced distal small bowel nutrient stimulation. DJB increased GLP-1 secretion, and systemic GLP-1 receptor antagonism reversed the small improvement noted in glucose tolerance. This study was the first to our knowledge to document a cause and effect relationship between duodenal bypass, an enhancement in GLP-1 receptor signaling, and the improvement in glucose tolerance. Finally, we tested the hypothesis that DJB improves insulin resistance independent of weight loss by performing DJB or Sham surgery in high-fat fed Wistar and Long-Evans rats. We found that DJB did not affect body weight or oral glucose tolerance. Formal tests of insulin sensitivity via a hyperinsulinemic-euglycemic clamp found no improvement in insulin resistance with DJB. In summary, the magnificent reversal of glucose intolerance and insulin sensitivity seen in type 2 diabetic patients after RYGB can not be solely explained by duodenal bypass. This work supports that gastric exclusion of the neuro-endocrine stomach may be the most important contributing component beyond weight loss mediating a significant metabolic improvement after bariatric surgery. Further, animal and clinical studies are needed to determine how gastric exclusion or resection alters post-prandial glucose homeostasis.
Advisors/Committee Members: Tso, Patrick P.W.
Subjects: Surgery
Keywords: duodenal-jejunal bypass; gastric bypass; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; ileal interposition; type 2 diabetes mellitus
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13.
Klustaitis, Kori M.
Activation of the central nervous system by circulating Glucagon-Like Peptide-1.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2009, University of Cincinnati
► The aim of this dissertation research was to investigate how Glucagon-Like Peptide-1…
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▼ The aim of this dissertation research was to investigate how Glucagon-Like Peptide-1 activates the central nervous system to illicit changes in blood glucose. Understanding how the body regulates blood glucose levels is important for not only understanding the disease diabetes, but also in developing treatments for the disease. Though numerous studies have looked the actions and mechanisms of GLP-1 action, there still is not a concise and agreed upon mechanism of action. Our hypothesis is that it acts in a neural manner by activating the central nervous system to stimulate efferent pathways to regulate glucose homeostasis. Two likely places for this signaling to take place are the portal vein and the small intestine. By using retrograde tracing and immunohistochemistry techniques, we were able to identify sensory afferent nerves that were capable of accessing circulation that expressed the GLP-1r. After a visiting student demonstrated that when GLP-1 was infused into the portal vein no resulting increase in insulin was observed, we decided to investigate the GLP-1/GLP-1r signaling system in the small intestine. We were able to identify a population of enterocytes that express the GLP-1r in the duodenum, jejunum and ileum. We attempted to identify which enterocytes were expressing the GLP-1r, but were only able to rule out expression on L-cells, D-cells, and enterochromaffin cells. Additionally, we were able to identify GLP-1r on synaptophysin positive cells, in both the enterocytes and myenteric plexi. Further work is required to identify this unique population of enterocytes, and whether or not GLP-1r are present on vagal nerve elements in the small intestine. In conclusion, we identified that sensory neurons in the portal vein can access circulation, that there is a population of enterocytes in the small intestine that express the GLP-1r and that the GLP-1r is can also be found on some neural elements in the small intestine. These findings are important for the understanding of GLP-1's multiple effects and sites of action to elicit changes in glucose homeostasis, particularly concerning pharmacological manipulation of the GLP-1 system for treatment of diabetes. Further work will continue to expand on our knowledge of this important global glucose homeostasis hormone.
Advisors/Committee Members: D'Alessio, David.
Subjects: Biology
Keywords: GLP-1; GLP-1r; vagal; vagal afferents; afferent; Tracer; intestine
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14.
Laskowski, Meggan C.
Uncovering molecular mechanisms that regulate mating in Histoplasma capsulatum.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2009, University of Cincinnati
► Genetic techniques requiring recombination in the pathogenic fungus, Histoplasma capsulatum, are currently…
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▼ Genetic techniques requiring recombination in the pathogenic fungus, Histoplasma capsulatum, are currently limited because the organism rapidly loses mating ability in culture. The studies presented in this body of work sought to determine molecular mechanisms that regulate mating in H. capsulatum, as a step toward preventing or reversing the loss of mating ability in cultured strains. Fungal mating loci are characterized by the presence of transcription factors that regulate mating, and some mating loci contain multiple genes involved in the mating response. We identified the mating locus of H. capsulatum and showed that the transcription factors MAT1-1-1 and MAT1-2-1, present at the MAT1-1 and MAT1-2 idiomorphs of the mating locus, respectively, are transcriptionally responsive to mating conditions. To identify additional components of mating regulation in H. capsulatum, we used syntenic analysis to identify a putative alpha pheromone. The alpha pheromone was found to be produced by MAT1-2 mating type organisms under mating conditions, and it was determined that organisms of MAT1-1 mating type respond to the alpha pheromone. UC1, an H. capsulatum strain that gained the ability to form empty cleistothecia with a mating partner after insertional mutagenesis, was used to determine additional molecular mechanisms that contribute to cleistothecia formation. Silencing HMK1, a predicted MAP kinase involved in the pheromone response MAP kinase pathway, had no effect on cleistothecia formation by UC1; however, Pkc1 activity was linked with pheromone production in this strain. These studies showed that the H. capsulatum mating locus transcription factors and the alpha pheromone play a role in mating, similar to that of other fungi. Pkc1 activity, however, is only indirectly linked to mating ability in other fungi, opening up areas for future studies determining the role Pkc1 plays in regulation of mating and mating competency in H. capsulatum.
Advisors/Committee Members: Smulian, George.
Subjects: Biology
Keywords: Histoplasma capsulatum; fungi; mating; pheromone; mating locus
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15.
MOORE, ZACHARY W. Q.
APOLIPOPROTEIN E MODULATION OF VASCULAR SMOOTH MUSCLE CELL RESPONSE TO INJURY.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2005, University of Cincinnati
► Apolipoprotein E (apoE) has been shown to exert anti-proliferative and anti-migratory effects…
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▼ Apolipoprotein E (apoE) has been shown to exert anti-proliferative and anti-migratory effects on smooth muscle cells in vitro and protection against neointimal hyperplasia in vivo. In this study, apoE was detected in the medial layers of the carotid artery following vascular injury. The presence of a liver-specific human apoE transgene confirmed the recruitment from circulation, although detection of native apoE message by in situ hybridization indicated that local synthesis also occurs. Although the neointima-resistant C57BL/6 strain showed a transient apoE presence, the susceptible FVB/N strain showed a continuous presence. Interestingly, detection of iNOS expression was positive only in the C57BL/6 wildtype strain. Upon addition of the overexpressed apoE transgene, the FVB/N strain recovered expression of iNOS, while loss of the apoE gene in the C57BL/6 strain resulted in a concurrent loss of iNOS expression. ApoE and iNOS colocalization was demonstrated in the medial layer of mice that are resistant to injury-induced neointimal hyperplasia. NOS2-/- deficient C57BL/6 mice showed no difference in neointimal hyperplasia compared to wildtype, but a significant increase in medial thickness and area was observed. These data documented that injury-induced activation of iNOS requires apoE recruitment, both apoE and iNOS are necessary for suppression of cell proliferation, and apoE recruitment without iNOS expression resulted in medial smooth muscle hyperplasia without their migration to the intima. In addition, the apoE-dependent inhibition of smooth muscle cell migration is dependent on LRP-1 function in the smooth muscle cell. Interestingly, apoE accumulation in apoE transgenic mice followed a layer-specific pattern, and was inversely associated with smooth muscle alpha-actin expression. The vascular accumulation of apoE after endothelial denudation, and its association with alpha-actin-depleted smooth muscle cells, suggests that apoE inhibition of injury-induced neointimal hyperplasia is not due to the inhibition of injury-induced smooth muscle cell de-differentiation, but is likely a direct effect of apoE on smooth muscle cell migration and proliferation. Examination of A7R5 cells in culture indicated that the addition of apoE prior to PDGF mitogenic stimulation resulted in a loss of alpha-actin signal as seen my immunocytofluorescence. Specific inhibition of kinase mediators of the alpha-actin polymerization pathway showed that both PKC and PKA mediate the apoE/PDGF effect. Examination of the RhoA activation state showed that apoE pretreatment lowers active levels of RhoA, causing a depolymerization of the alpha-actin cytoskeleton. We conclude there to be a significant apoE-dependent effect on the smooth muscle cell component of the vessel wall in response to vascular injury, including direct recruitment, iNOS-dependent inhibition of proliferation, LRP-dependent inhibition of migration, and modulation of the cytoskeleton.
Advisors/Committee Members: Hui, David Y.
Keywords: Apolipoprotein E, LRP-1, iNOS, RhoA, Vascular Injury, PDGF, Mouse
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16.
Nauli, Andromeda Margono.
Intestinal Lipid Uptake and Secretion of VLDL and Chylomicron.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2005, University of Cincinnati
► Despite decades of research, our understanding of intestinal lipid absorption is limited.…
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▼ Despite decades of research, our understanding of intestinal lipid absorption is limited. In this Ph.D. thesis, I have dealt with two main aspects of intestinal lipid absorption, namely the uptake of lipids and the formation and secretion of triacylglycerol-rich lipoproteins (very low density lipoproteins [VLDL] and chylomicrons). In terms of uptake, CD36 is one of the plasma membrane proteins implicated in mediating lipid uptake by the intestine. In order to test this hypothesis, we utilized the CD36 knockout mouse model equipped with intraduodenal and lymph cannulas. Our studies showed that the disruption of the CD36 gene led to a significant decrease in the uptake of cholesterol but not of fatty acids. Interestingly, the role of CD36 was not limited to uptake but also appeared to affect the formation and secretion of chylomicrons, the major lipoproteins carrying the absorbed dietary fat from the gut (Chapter 2). It was first proposed by Tso et al. (202) that the small intestine secretes both VLDL and chylomicrons. Previous work by Vahouny et al. (212) suggested that female rats produced more VLDL than male rats. In addition, personal communication with Dr. Renee LeBoeuf leads us to believe that the female C57BL/6 mice may absorb lipids less efficiently than the male mice. We therefore studied the formation and secretion of lipoproteins in male and female C57BL/6 mice. Our data agree with those of Vahouny in that the female mice had a slightly higher ratio of VLDL to chylomicron secretion relative to that of the male mice. In addition, we also found that the intestinal lymphatic lipid transport of the C57BL/6 female mice segregated into two groups, a phenomenon that was absent in the male mice. In summary, our work suggests that CD36 is involved not only in intestinal cholesterol uptake, but also in regulating the formation and secretion of chylomicrons. In addition to the regulation by CD36, our studies also show that the regulation of the formation and secretion of chylomicrons are potentially different between male and female animals.
Advisors/Committee Members: Tso, Patrick.
Subjects: Biology, Animal Physiology
Keywords: sex; CD36; lymph; absorption; fat
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17.
Nicolaou, Stella A.
K+ Channel Trafficking in the Immunological Synapse of Human T Cells in Health and Autoimmunity.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2007, University of Cincinnati
► T cell receptor (TCR) engagement by an antigen presenting cell (APC) results…
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▼ T cell receptor (TCR) engagement by an antigen presenting cell (APC) results in reorganization of intracellular and membrane molecules at the T/APC interface, forming a “signalosome”, the immunological synapse (IS). An early event associated with T/APC interaction is Ca2+ influx. K+ channels, Kv1.3 and KCa3.1, modulate Ca2+ signaling in human T cells. Resting and activated human T cells express both channels, albeit to different degrees. Kv1.3 channels modulate Ca2+ in resting, while KCa3.1 channels do so in activated, T cells. Although these channels play such an important role in Ca2+ homeostasis, very little is known about their localization in the IS. Furthermore, aberrant T cell responses in IS formation and Ca2+ influx have been documented in T cells from patients with systemic lupus erythematosus (SLE). The potential involvement of K+ channels in the etiology and progression of SLE remains unknown. Herein we determined K+ channel membrane distribution in resting and activated human T cells following TCR engagement and performed comparative studies with SLE T cells to decipher the role of K+ channels in the Ca2+ response anomaly. Our data show that in SLE T cells, Kv1.3 channels constitute the dominant channel and are functionally identical to their normal counterparts. We also found that resting SLE T cells show faster Kv1.3 kinetics out of the IS as compared to healthy T cells and comparable to healthy pre-activated T cells. However, normal pre-activated T cells recruit and maintain KCa3.1 channels in the IS after Kv1.3 channels leave, while SLE T cells do not express the appropriate KCa3.1 channel number to support this activated phenotype. This Kv1.3 mobility defect appears to be specific to SLE and not other autoimmune diseases, as it was not observed in rheumatoid arthritis (RA) patients. Further, transcription factor activation and gene expression relies on the shape of the Ca2+ response. Although SLE T cells demonstrate abnormal transcription factor regulation and gene expression, the potential contribution of the Ca2+ responses to these abnormalities remains unknown. We performed single T cell Ca2+ response analysis to better define the Ca2+ shape in SLE T cells. Our data suggest that there is an increase in cells with more sustained Ca2+ response in SLE as compared to normal and RA T cells, while a transient, short duration, Ca2+ response is more pronounced in normal T cells. We also found that during and upon termination of the Ca2+ response, Kv1.3 channels are retained in the IS in healthy T cells, implying a role for Kv1.3 in the termination of the Ca2+ response. We conclude that altered localization of Kv1.3 channel in the IS of SLE T cells is at least in part responsible for more sustained Ca2+ response in SLE T cells. This phenotype, in turn, supports T cell hyperactivity in SLE T cells. Therefore we propose that Kv1.3 channels may offer novel therapeutic targets for SLE.
Advisors/Committee Members: Conforti, Dr. Laura.
Subjects: Biology, Molecular
Keywords: Human T cells; Potassium channels; Immunological Synapse; Systemic lupus erythematosus
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18.
OLIVER, BRIAN G.
MOLECULAR CLONING AND IN VITRO CHARACTERIZATION OF THE ASPERGILLUS FUMIGATUS CAMP-DEPENDENT PROTEIN KINASE.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2001, University of Cincinnati
► Cyclic AMP signaling has been shown to be essential for growth, morphology…
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▼ Cyclic AMP signaling has been shown to be essential for growth, morphology and virulence in fungal pathogens of plants and animals. However, the function of this pathway is unknown in the opportunistic pathogen, Aspergillus fumigatus. As a first step toward elucidating the function of this pathway in A. fumigatus the genes encoding both the regulatory and catalytic subunits were cloned and sequenced and the response to exogenous cAMP was characterized. The availability of pkaR and pkaC sequences was used to construct vectors, facilitating future studies into the function of these genes. The regulatory subunit gene, pkaR, is expressed from an intronless coding sequence and the predicted translation product shares 79% sequence identity with the regulatory subunit of Aspergillus nidulans, both of which are defined as a type II regulatory subunits by the presence of a conserved autoinhibition site and two cAMP-binding domains. The gene encoding the A. fumigatus catalytic subunit, pkaC, is interrupted by three introns. The predicted PkaC protein shares 83% sequence identify with A. nidulans PkaC, and contains domains that are characteristic of PkaC proteins. Both pkaR and pkaC mRNAs are expressed throughout the asexual lifecycle of A. fumigatus. In regard to the effects of exogenous cAMP, both cAMP and phosphodiesterase inhibitors markedly reduced radial growth rate of A. niger, which has previously been demonstrated to respond exogenous cAMP, after 48 hours on minimal media with glucose as the carbon source, whereas growth of A. fumigatus was not affected. However, when glycerol, which does not initiate carbon catabolite repression, was used as a carbon source, cAMP inhibited the radial growth rate of only A. fumigatus (p<0.05). The addition of cAMP to glycerol-minimal medium resulted in a two-fold increase in protein kinase A activity in A. fumigatus cell extracts when compared with negative controls. The PKA activity in A. fumigatus cell extracts from cultures grown in glucose did not change significantly with the addition of cAMP. This document reports the first analyses of the PKA pathway in A. fumigatus and provides the foundation for future analysis of PKA function in the growth, and potentially the virulence, of A. fumigatus.
Advisors/Committee Members: Rhodes, Dr. Judith.
Subjects: Biology, Microbiology
Keywords: Aspergillus; cAMP; PKA; Fungus
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19.
O'Malley, Jennifer A.
Improving Therapeutics for Parkinson's Disease.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2009, University of Cincinnati
► The following results are from studies designed with the overall goal of…
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▼ The following results are from studies designed with the overall goal of elucidating means for improving therapeutics for Parkinson’s disease. There is still much to be understood regarding the cellular and molecular mechanisms underlying the development of side effects to therapies for Parkinson’s disease such as levodopa-induced dyskinesias. Additionally, therapeutic intervention is further complicated when one considers the role of the functionally waning host environment, especially in the context of its responsivity to therapeutic agents. The work in this thesis was designed to focus on two aspects of Parkinson’s disease therapeutics, (1) improving graft efficacy by contributing additional information on how the host environment in regards to age impacts graft function in Parkinson’s disease, and (2) determining whether specific morphological changes of the nigral target neurons within the striatum impact development or severity of levodopa-induced dyskinesias. The first study demonstrates that while the aging striatum can, under specific conditions, support the survival of large numbers of grafted embryonic dopamine neurons, there is limited functional benefit even with robust cell survival. This realization is an important contribution to the field as it newly suggests the aged striatum is capable of supporting grafted dopamine neurons, but the limited efficacy of these grafts demonstrates the importance of the intricate balance between grafted cells and the aged host environment. The second study demonstrate that maintaining appropriate synaptic contacts that are presumably maintained when striatal medium spiny neuron cytoarchitecture is preserved, resulting in reduced drug-induced side-effects in parkinsonian rats. Future experiments are needed to explore the specific requirements of the aging striatum for functional recovery and how to implement synaptic stability of medium spiny neurons in clinical practice to potentially decrease side-effects of dopamine replacement pharmacotherapy.
Advisors/Committee Members: Steece-Collier, Kathy.
Subjects: Surgery
Keywords: Parkinson; dopamine; dyskinesia; levodopa; dendritic spine; medium spiny neuron
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20.
PANEPINTO, JOHN CARLO.
The role of the Aspergillus fumigatus rheb homologue, rhbA, in nitrogen sensing and the pathogenesis of invasive pulmonary aspergillosis.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2001, University of Cincinnati
► A gene encoding a ras protein with homology to the rheb family…
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▼ A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. The rhbA gene contains four exons separated by three introns, and is predicted to encode a protein of 187 amino acids. Although conserved ras domains are present, the RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. Although the levels of rhbA mRNA remained similar throughout the A. fumigatus asexual developmental cycle, they accumulated over five-fold in response to nitrogen starvation. The rhbA gene was able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Δrhb1 mutants, suggesting that the two proteins share overlapping function. To determine the function of the A. fumigatus rheb homologue, rhbA, in growth and pathogenesis, a strain of A. fumigatus lacking rhbA was constructed. Three of the four coding exons of the rhbA gene were replaced with a hygromycin resistance cassette. In vitro analysis of the growth rate of the rhbA mutant compared with that of the wild type and complemented strain revealed a significant reduction in growth of the mutant on medium containing poor nitrogen sources (p<0.05), while maintaining wild type growth rate on ammonium containing medium. The ΔrhbA strain exhbited hypersensitivity to the TOR kinase inhibitor rapamycin, but was hyposensitive to the histidine analog 3-amino triazole. Survival of mice inoculated with the ΔrhbA strain was significantly increased when compared to those groups inoculated with the wild type or reconstituted strain (p<0.05). Image analysis of lung sections from mice infected with the ΔrhbA strain revealed a smaller average fungal lesion area compared to the lesions from mice infected with the wild type or reconstituted strain at 4 and 7 days post-inoculation (p<0.05). These data suggest that rhbA is involved in nitrogen discrimination and contributes to the virulence of A. fumigatus.
Advisors/Committee Members: Rhodes, Dr. Judith C.
Subjects: Biology, Microbiology
Keywords: Aspergillus; rheb; Rapamycin; Virulence
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21.
PEARSON, KEVIN JOSEPH.
THE STRUCTURE AND FUNCTION OF APOLIPOPROTEIN A-IV.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2005, University of Cincinnati
► Apolipoprotein (apo) A-IV is a protein synthesized by the small intestine in…
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▼ Apolipoprotein (apo) A-IV is a protein synthesized by the small intestine in response to lipid absorption. It has been proposed to play a role in cholesterol efflux, lipoprotein metabolism and food intake. Unfortunately, little information on its structure/function relationship is known. Therefore, it was important to establish a recombinant expression system for apoA-IV so deletion mutagenesis could be performed to achieve the specific aims. The following aims address the hypothesis that specific regions of apoA-IV are involved in its ability to interact with lipid and inhibit food intake. Aim 1: Determine the region of apoA-IV that is responsible for its ability to bind lipid as well as to identify the generalities of its structure. Initially, it was found that removing the C-terminal 44 amino acids from human apoA-IV caused a significant increase in lipid binding ability as compared to WT. Eventually, a smaller region from amino acid 333-343 was established as the ‘inhibitory’ region of apoA-IV at the C-terminus. Using the Δ333-343 as a template, the N-terminus was also mutated and it was found that the N-terminal amino acids from 11-20 were required for the mutants to be fast lipid binders. Therefore, we propose there is an interaction between the ‘inhibitory’ region from 333-343 and the N-terminus of the protein that does not allow the proper conformation for accelerated lipid binding. Aim 2: Determine the region of apoA-IV that is responsible for its role in the inhibition of food intake. Originally, several C-terminal deletion mutants were made in rat apoA-IV. The mutants were injected into the 3rd ventricle of rat brains and their food intake was determined. It was found that the food intake region was located in the N-terminal 116 amino acids. Next a 3rd mutant was made that removed the N-terminal 61 amino acids. This mutant did not inhibit food intake as compared to WT suggesting the region involved in food intake is located in the first 61 amino acids. In separate studies, a 14-residue peptide from amino acid 17-30 also inhibited food intake in rats suggesting this is the active region.
Advisors/Committee Members: Davidson, Dr. William Sean.
Keywords: Apolipoprotein A-IV; Lipid Binding; Recombinant
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22.
Richie, Daryl Lynn.
The contribution of apoptosis, autophagy, and the unfolded protein response to the growth and virulence of Aspergillus fumigatus.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2009, University of Cincinnati
► Aspergillus fumigatus is a ubiquitous saprophytic fungus and the leading mold pathogen…
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▼ Aspergillus fumigatus is a ubiquitous saprophytic fungus and the leading mold pathogen in bone marrow⁄solid organ transplantation units in developed countries. Due to the continuing increase in immunocompromised individuals, combined with the ineffectiveness and adverse side effects associated with current antifungal agents, there is a need for additional research to identify new targets for antifungal therapy. In order to inhabit the environmental niche of compost, A. fumigatus must be capable of adapting to a diverse array of environmental stressors. Since many of the stresses found in the environment may also be encountered in the host, it is hypothesized that some of the attributes that allow A. fumigatus to thrive in the competitive environment of compost also enable the fungus to be a successful opportunistic pathogen. This dissertation focuses on three stress response pathways that have been largely unexplored in A. fumigatus. They include: the metacaspase-dependent stress response, the autophagy-dependent stress response, and the endoplasmic reticulum stress response. The overarching hypothesis of this dissertation is that each of these stress response pathways provides a function that contributes to the virulence of A. fumigatus. To test this, loss-of-function mutants were generated by targeting key regulatory genes within the different stress response pathways. Upon the inhibition of the pathways, the ability of the mutant strains to grow in the presence of various environmental stressors and to cause invasive disease was determined. The data revealed an unexpected pro-survival function for the metacaspases during endoplasmic reticulum stress and identified a novel role for autophagy during metal ion deficiency. However, both the metacaspase-dependent and the autophagy-dependent stress response pathways were found to be dispensable for in vivo growth. In contrast, severe disruption of the major ER-stress response pathway, termed the unfolded protein response (UPR), resulted in attenuation of virulence, hypersensitivity to cell wall stress and a decrease in growth on complex substrates. It is the long-term goal of this study to provide information that can be used to develop more effective drug interventions in the future.
Advisors/Committee Members: Askew, David.
Subjects: Cellular biology; Microbiology; Pathology
Keywords: Aspergillus, unfolded protein response, autophagy, apoptosis
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23.
Richie, Nicole.
The Retinoblastoma Tumor Supressor Protein is a Critical Regulator of Lung Epithelial Repair after Injury.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2008, University of Cincinnati
► Airway remodeling is associated with the vast majority of lung diseases including…
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▼ Airway remodeling is associated with the vast majority of lung diseases including chronic obstructive pulmonary disease, asthma, and lung cancer. Epithelial regeneration is a key component in airway remodeling after injury. Accordingly, deregulated epithelial cell proliferation, survival, and differentiation play a prominent role in the pathogenesis of chronic lung disease. The lung epithelium is composed of specialized cell types that result from coordinate regulation of progenitor/stem cell proliferation and differentiation. The retinoblastoma gene product (Rb) regulates both proliferation and differentiation, and is inactivated in nearly all cases of lung cancer strongly implicating Rb as a critical regulator in the lung epithelium. The objective of this dissertation project was to test the hypothesis that Rb is essential for proper lung epithelial repair after injury. Rb ablation was targeted to the lung epithelium using a tetracycline regulated Cre/LoxP system, and epithelial injury was induced with naphthalene to mimic human lung disease. These studies demonstrate that although Rb is not required for establishing and maintaining epithelial quiescence during homeostasis, Rb is essential for establishing quiescence during epithelial repair after injury. Rb ablation during development and in the postnatal lung had similar effects providing evidence that timing of Rb loss was not critical to the phenotypic outcomes, and that the injury induced phenotype was not secondary to compensatory alterations occurring during development. After establishing this critical role for Rb in epithelial remodeling after a single episode of injury, Rb function was assessed in a chronic injury model to more closely mimic human lung disease. These studies led to the discovery of previously unknown effects of the highly utilized naphthalene injury model; namely naphthalene injury results in altered epithelial composition and subsequent inflammation. Importantly, Rb dependent sustained epithelial proliferation enhanced progenitor cell restoration after injury without resulting in tumor formation. Altogether, this dissertation project identifies a unique critical role for Rb in the context of epithelial remodeling after injury, and provides evidence that precise regulation of Rb function is important for balancing progenitor cell regeneration, and thus tissue renewal capacity, against the risk of developing cancer.
Advisors/Committee Members: Wikenheiser-Brokamp, Kathryn.
Subjects: Molecular biology; Oncology; Pathology
Keywords: Retinoblastoma (Rb), Cre-LoxP, Lung injury, Lung progenitor and stem cells
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24.
SCHMID, KARA E.
ENDOGENOUS AND EXOGENOUS SOURCES OF CHOLESTEROL DURING FETAL DEVELOPMENT.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2003, University of Cincinnati
► Cholesterol is essential for proper fetal development. Defects in fetal cholesterol metabolism…
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▼ Cholesterol is essential for proper fetal development. Defects in fetal cholesterol metabolism can cause developmental abnormalities, including mental retardation and stunted growth as demonstrated by patients with Smith-Lemli-Optiz syndrome. Understanding fetal cholesterol metabolism could lead to the development of a method to prevent or lessen cholesterol deficiency related birth defects. Therefore, the goal of this dissertation was to examine cholesterol metabolism in the developing fetus. Understanding cholesterol metabolism requires the examination of both sources of cholesterol: cholesterol synthesized de novo and cholesterol provided exogenously. De novo cholesterol synthesis in the adult can be regulated by dietary polyunsaturated fatty acids. If fetal cholesterol synthesis rates are regulated as in the adult, then the consumption of polyunsaturated fatty acids during pregnancy may have an impact on fetal development. Therefore, the regulation of cholesterol synthesis in the fetus was examined. Pregnant hamsters were fed various fatty acids throughout gestation and sterol synthesis rates in the fetus were determined. Although the rate of sterol synthesis in the adult hamster was decreased by dietary polyunsaturated fatty acids, sterol synthesis rates in the fetus were unaffected. In addition, the adult could be made to become unresponsive to dietary polyunsaturated fatty acids by inducing a negative sterol balance across the liver. These data demonstrated that sterol synthesis is regulated differently in fetal tissues as compared to adults, possibly due to a negative sterol balance in the fetus. The placenta regulates the transport of exogenous nutrients from the maternal circulation to the fetal circulation. The placental transport of glucose and fatty acids is well established, however the transport of cholesterol remains undefined. Therefore, a human choriocarcinoma cell line, BeWo, was utilized to model the transport of cholesterol across a placental monolayer. It was demonstrated that cholesterol could be transported from the apical surface of the placental cell to the basolateral surface for efflux to components of fetal human serum, fetal HDL and phospholipid vesicles but not apolipoprotein A-I. BeWo cells demonstrated a mass movement of cholesterol from the apical to basolateral chamber that could be manipulated by increasing the cellular cholesterol concentration. These were the first studies to utilize an in vitro model to demonstrate the transport of cholesterol from the maternal circulation (apical side) to the fetal circulation (basolateral side). Understanding and dissecting this pathway may aid in the development of in utero treatments for fetuses with defects in cholesterol biosynthesis.
Advisors/Committee Members: Woollett, Dr. Laura A.
Subjects: Biophysics, Medical
Keywords: fetal cholesterol metabolism; placental transport; cholesterol synthesis; Smith-Lemli-Optiz
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25.
Thacker, Robert I.
Modulation of Human Dendritic Cell Activity by Adsorbed Fibrin(ogen).
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2008, University of Cincinnati
► Fibrinogen, a plasma protein central to clot formation, has long been considered…
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▼ Fibrinogen, a plasma protein central to clot formation, has long been considered to play a role in inflammation and immunity. Fibrin(ogen) interactions with various immune cells have been heavily investigated; lacking is an understanding of the protein's influence on dendritic cell activity. Results from Chapter 2 demonstrate that fibrinogen initiates human dendritic cell production of inflammatory cytokines and chemokines. Adsorbed fibrin(ogen) has increased stimulatory capacity over fibrin(ogen) free in solution, indicating the bound protein, acting through the ß2-integrins, is the active species. Adsorbed fibrin(ogen) also stimulates the focal accumulation of dendritic cells. This is likely due to ß2-integrin-mediated chemotaxis of dendritic cells toward released chemokines and fibrin(ogen) degradation fragments. Because studies suggested adsorbed fibrinogen might be exploited to enhance vaccine adjuvanticity, fibrinogen-coated olive oil droplets were investigated as vaccine adjuvant. Results from Chapter 3 demonstrate the importance of surface in adjuvant activity, and the possible use of olive oil droplets as a safe and effective vaccine adjuvant. Having investigated the interactions between fibrinogen-coated particles and human dendritic cells, Chapter 4 describes the existence of a not yet recorded phenomenon: extracellular transport of cell-sized particles by dendritic cells. Results presented in that chapter not only demonstrate particles can be carried extracellularly by dendritic cells, but also that the process can be directed. Adsorbed fibrin(ogen) appears to enhance particle/dendritic cell interactions mediated through the ß2-integrins. The influence of adsorbed fibrin(ogen) on dendritic cells provides new knowledge into the protein's involvement in initiating inflammatory and immune responses, knowledge that may be applied to the development of new therapeutics to treat and prevent disease.
Advisors/Committee Members: Retzinger, Gregory.
Subjects: Immunology; Pathology
Keywords: fibrinogen; surfaces; inflammation; CD18; vaccine adjuvants; oil emulsion; dendritic cell; extracellular translocation; cancer metastasis; dendritic cell migration; olive oil; integrins; cytokine; chemokines
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26.
THULLEN, TIMOTHY DAVID.
ANALYZING THE HOST IMMUNE RESPONSE TO PNEUMOCYSTIS UTILIZING TWO RAT MODELS.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2003, University of Cincinnati
► Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in patients with…
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▼ Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in patients with HIV+, cancer patients, organ transplant patients, and other immunocompromised individuals. Although information about Pneumocystis pneumonia has increased due to its propensity to infect AIDS patients, there is much that needs to be understood about the interaction between the host and Pneumocystis, and the host’s ability to defend against the organism. Mouse models of Pneumocystis pneumonia have revealed valuable information about host defense, yet the corticosteroid- treated Pneumocystis pneumonia rat model shows similarities to HIV+ patients infected with the organism. Two approaches were made to study host defense to Pneumocystis in the rat model. The first was the adoptive transfer of immune donor splenocytes sensitized to the Pneumocystis antigen, major surface glycoprotein, to corticosteroid- treated rats with Pneumocystis pneumonia. This study was designed to study the cytokines that were involved in the clearance of Pneumocystis pneumonia. Splenocyte adoptive transfer was successful in these studies, resulting in significant Pneumocystis reduction. However, some rats developed a hyperinflammatory reaction following adoptive transfer, and this was associated with increases in the proinflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6), Th1 cytokine IFN-gamma, and Th2 cytokines (IL-5 and IL-10). These studies also revealed a differential effect of splenocyte adoptive transfer on the Pneumocystis morphological forms. The second approach to study host defenses was the development of a CD4- depleted rat model of Pneumocystis pneumonia. The non- depleting W3/25 antibody and depleting OX-38 antibody were both capable of reducing CD4+ expressing T lymphocytes and rendering rats susceptible to Pneumocystis infection. This rat model avoids the immunosuppressive effects of corticosteroids and serves as an additional rat model to test host defenses to the organism and other host/ Pneumocystis interactions. The two different approaches to Pneumocystis host defense in the rat presented in this dissertation have both strengthened the hypothesis that the CD4+ T lymphocyte is the principal effector cell in defense and demonstrated cytokines that are important for organism clearance and possibly detrimental to the host. These two Pneumocystis pneumonia rat models will allow further insight into important immune factors of humans infected with Pneumocystis.
Advisors/Committee Members: Walzer, Dr. Peter D.
Keywords: pneumocystis; adoptive transfer; CD4-depletion; rats; cytokines
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27.
TUBB, MATTHEW ROBERT.
Apolipoprotein A-IV Structural Models and Functional Implications.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2008, University of Cincinnati
► Human apolipoprotein (apo)A-IV is a 46 kDa exchangeable plasma protein produced mainly…
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▼ Human apolipoprotein (apo)A-IV is a 46 kDa exchangeable plasma protein produced mainly in the small intestine in response to lipid meals with numerous proposed functions ranging from forward and reverse cholesterol transport to anti-inflammation, anti-oxidation, and satiation. Unfortunately, there is very little structural basis for these functions largely due to the lack of a three-dimensional model of the protein. The following aims were constructed in order to answer specific questions about apoA-IV structure and function.AIM 1: Determine the specific sequences in apoA-IV that modulate its ability to bind to lipid. We have used site-directed mutagenesis to introduce point mutations into apoA-IV in order to discover what regions influence lipid binding. Once these regions were identified we performed experiments to conclusively demonstrate that the two regions, distant in terms of the primary sequence, were quite near each other in the native lipid-free conformation. We propose that there is a direct intramolecular interaction within apoA-IV that inhibits its inherent lipid binding ability. AIM 2: Create a three dimensional model of apoA-IV, both lipid-free and lipid-bound, using mass spectrometry and computer modeling. Since both lipid-free and lipid-bound apoA-IV likely play distinct physiological roles, we set out to develop models of apoA-IV structure in both states. First, we developed a method to produce highly homogeneous apoA-IV recombinant HDL particles. We then used cross-linking chemistry and high resolution mass spectrometry to create "proximity maps" of lipid-free and lipid-bound apoA-IV. This was followed by sequence threading and computer modeling to generate the first ever all-atom molecular models of apoA-IV. AIM 3: Identify an apoA-IV binding protein or receptor. A novel apoA-IV receptor has yet to be identified and, although many groups have attempted, discovering it would provide tremendous information about the way in which apoA-IV is able to carry out its diverse functions in vivo. We employed various experimental methods to attempt to identify a novel protein partner for apoA-IV. Among the techniques used were affinity chromatography, far western blotting, yeast two hybrid and label transfer. Certain results remain promising, yet identification of a specific protein has eluded us.
Advisors/Committee Members: Davidson, W. Sean.
Subjects: Biochemistry
Keywords: apolipoprotein; cholesterol; mass spectrometry; cross-linking; protein structure; amphipathic
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28.
Wheat, Valerie Jo.
MECHANISM OF BICARBONATE SECRETION ACROSS THE TRACHEAL EPITHELIUM: ABERRANT REGULATION BY CFTR.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2001, University of Cincinnati
► Cystic Fibrosis is the most common lethal inherited disease in the Caucasian…
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▼ Cystic Fibrosis is the most common lethal inherited disease in the Caucasian population. It is caused by autosomal recessive inheritance of mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Although CFTR is a chloride channel, the disease process of CF seems to involve not only defective chloride secretion, but defective bicarbonate secretion as well. The purpose of these studies was to determine the mechanism of bicarbonate secretion in the trachea, and to explore its regulation by CFTR. These studies utilize a tissue culture model system of CFT-1 cells derived from a patient with the homozygous ΔF508 mutation in CFTR, and CFT-1 cells retrovirally infected with functional CFTR (CFT-WT cells). Functional presence of a Cl - /HCO 3 - exchanger is shown, with decreased activity in CFT-1 cells. This decreased activity is attributed to absence of expression of the apical Cl - /HCO 3 - exchanger DRA/CLD. To confirm the role of CFTR in inducing DRA expression, adenoviral infection of the CFT-1 cells with wild-type CFTR was performed. In the monolayers infected with Ad-CFTR (20 pfu/cell) for 24, 48, and 72 hours, DRA expression was detected. Control cells did not show DRA expression, nor did the monolayers infected for 7 days. These studies demonstrate the absence of CFTR expression, as in the CF cells, causes absence of expression of DRA, an apical Cl - /HCO 3 - exchanger. This lack of DRA expression could account for a portion of the bicarbonate secretion defect in CF. Next, the basolateral uptake and accumulation of bicarbonate within the cell was examined. These studies identify the Na+/H+ exchanger (NHE) and Na + :HCO 3 - cotransporter (NBC) isoforms expressed in tracheal epithelial cells. Tracheal epithelial cells express the NHE-1 isoform, and exhibit the presence of functional Na+/H+ exchanger with properties consistent with the classical NHE. NHE functional activity is inhibited by the addition of amiloride. These cell lines express four NBC isoforms, and NBC-2 and NBC-3 isoforms show increased expression in the presence of functional CFTR. Both cell lines show Na+-dependent HCO 3 - uptake that is sensitive to DIDS, characteristic of NBC functional activity. These studies demonstrate that CFTR expression can also regulate expression of other acid-base transporters, and that aberrant regulation of transporter isoform expression by CFTR may contribute to the bicarbonate secretion defect of CF. The mechanism of CFTR expression affecting expression of other acid-base transporters is hypothetical, and will involve further investigation.
Advisors/Committee Members: Soleimani, Manoocher.
Keywords: CFTR; BICARBONATE SECRETION; SODIUM-BICARBONATE COTRANSPORTER; CYSTIC FIBROSIS
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29.
Witting, Scott R.
The Role of Sphingolipids in Cholesterol Efflux Mediated by ATP-Binding Cassette Transporter AI (ABCAI).
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2004, University of Cincinnati
► Cardiovascular disease, including stroke and atherosclerosis, remains a major cause of morbidity…
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▼ Cardiovascular disease, including stroke and atherosclerosis, remains a major cause of morbidity and mortality in the United States due to consumption of a high-fat “Western” diet. A major indication of these disease states is excess lipid deposition, particularly cholesterol, in the arterial walls. It has been well documented that high levels of circulating high-density lipoprotein (HDL) and its main protein component apolipoprotein AI (apoA-I) are associated with lowered risk of cardiovascular disease. The protective effects of HDL are thought to be mediated by a process called reverse cholesterol transport - in which HDL takes up excess cholesterol from peripheral tissues and transports it to the liver for excretion in the bile. It is now widely accepted that the interaction of apoA-I with the cell membrane protein ATP-binding cassette transporter AI (ABCAI) is critical for the formation of nascent HDL particles. Since sphingomyelin maintains a preferential interaction with cholesterol in membranes, the breakdown of sphingomyelin may regulate the availability of cellular cholesterol utilized by ABCAI. Furthermore, the catabolite of sphingomyelin, ceramide, is a potent signaling molecule and may play an important role in ABCAI regulation or function. The following study examines the potential contribution of sphingolipids in ABCAI-mediated cholesterol removal from the cell. It was discovered that treatment with C2-ceramide enhances cholesterol release to apoA-I. This effect appeared to becaused by an increase in cellular ABCAI content with enrichment at the cell surface. These findings may lead to new ways to increase cellular ABCAI and further promote cholesterol removal from regions of excess cholesterol such as the atherosclerotic lesion.
Advisors/Committee Members: Davidson, Dr. William Sean.
Keywords: Cholesterol; Sphingolipids; Atherosclerosis; ABC Transporter; Apolipoprotein AI; High Density Lipoprotein
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30.
ZHANG, MEI.
GENETICALLY MANIPULATED MOUSE MODELS FOR THE STUDY OF INSULIN-LIKE GROWTH FACTOR I IN BONE.
Degree: PhD, Medicine : Pathobiology and Molecular Medicine, 2002, University of Cincinnati
► Insulin-like growth factor I (IGF-I) exerts anabolic effects on bone and is…
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▼ Insulin-like growth factor I (IGF-I) exerts anabolic effects on bone and is thought to amplify other osteogenic signals. However, the cellular and molecular mechanisms that mediate the anabolic actions of IGF-I have been difficult to address experimentally primarily due to the complexity of the IGF system. To study the local actions of IGF-I and its regulation by binding protein 4 (IGFBP-4) in skeletal tissue in a physiological context, I have developed mouse models with either overexpression of IGFBP-4 in osteoblasts or conditional disruption of the gene encoding the type I IGF receptor, Igf1r gene, in osteoblasts. Mice with overexpression of IGFBP-4 in bone osteoblasts exhibited reduced bone formation and turnover and severely impaired skeletal growth with global growth retardation. These effects were attributed to the sequestration of IGF-I by IGFBP-4 with consequent impairment of IGF-I actions. Osteoblast-specific disruption of the Igf1r gene reduced trabecular bone volume and severely impaired mineralization of bone matrix. Deficient IGF-I signaling led to a compensatory state of accelerated trabecular bone turnover. These studies established valuable models to further investigate the role of IGF-I signaling in bone.
Advisors/Committee Members: Clemens, Dr. Thomas.
Subjects: Biology, Molecular
Keywords: insulin-like growth factor (IFG-I); bone osteoblast; transgenic
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