Department: College of Medicine ![[clear]](close-x.png "Remove this limiter")
98 matches in the database.
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1.
Alam, Goleeta N.
Role of the Lineage Gene Phox2B in Neuroblastoma Development.
Degree: PhD, College of Medicine, 2009, University of Toledo Health Science Campus
► The oncogene MYCN is amplified in a subset of unfavorable neuroblastomas thatconsist…
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▼ The oncogene MYCN is amplified in a subset of unfavorable neuroblastomas thatconsist of predominantly undifferentiated neuroblasts. We used the MYCN transgenic mice - an animal model for the human disease, to examine the cellular basis of neuroblastoma development. These mice develop overt neuroblastomas preceded by a pre-neoplastic stage characterized by development of hyperplastic lesions in the sympathetic ganglia during the first week after birth. We show that both hyperplastic lesions as well as primary tumors are composed of highly proliferating cells that express Phox2B. MYCN promotes the proliferation of these Phox2B+ neuronal progenitors and arrests their differentiation, thereby leading to an expansion of this population. A minor population of undifferentiated cells expressing the neural stem cell marker, nestin, was also identified in both the hyperplastic lesions and primary tumors implicating these cells as possible precursors to the proliferating Phox2B+ neuronal progenitors. Immunoblot analysis of a panel of human neuroblastoma cells showed Phox2B expression in a majority of cells examined which was downregulated significantly upon retinoic acid-induced neuronal differentiation, implicating a role for Phox2B in maintenance of the neuroblastic phenotype. Interestingly, Phox2B knockdown in the bipotent BE(2)-C neuroblastoma cell line resulted in shift towards a glial fate while in the SK-N-AS cells Phox2B downregulation resulted in decreased survival. Given together, these data support the notion that Phox2B may function as a lineage-survival oncogene in an aberrant developmental context, leading to neuroblastoma development.
Advisors/Committee Members: Ding, Han-Fei.
Subjects: Neurology; Oncology
Keywords: neuroblastoma; MYCN; Arrested Differentiation; hyperplasia; Phox2B; BE(2)-C Cells
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2.
Angevine, Kristine R.
Menin Regulates Oxidative Stress Through Heme Oxygenase-1 and the p38 MAPK Pathway.
Degree: PhD, College of Medicine, 2012, University of Toledo Health Science Campus
► Oxidative stress is caused by an increase in reactive oxygen species (ROS)…
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▼ Oxidative stress is caused by an increase in reactive oxygen species (ROS) with a resultant down-regulation of antioxidants. Two known regulators of oxidative stress response are menin and heme oxygenase-1 (HO-1). Currently, little is known about the mechanism by which menin is modulated during stress response, as well as the role of HO-1 in the interaction between adipocytes and endothelial cells. In these studies, we evaluated whether changes in menin expression mediate oxidative stress in correlation with HO-1 induction. Two different cell lines, human dermal microvascular endothelial cells (HMECs) and adipocytes derived from mesenchymal stem cells, were cultured in either growth media or endothelial cell conditioned media (CM) to determine factors produced in response to oxidative stress and HO-1 regulation. Using angiotensin II (Ang II) to mimic oxidative stress conditions and cobalt protoporphyrin IX (CoPP) to induce HO-1, we measured cytokines produced and the maturation potential of adipocytes. Western blot analysis and RT-PCR were used to determine expression of protein and mRNA. Cytokines secreted by adipocytes and endothelial cells were determined using ELISA. Furthermore, we generated mice harboring a liver-specific hemizygous (HET) and homozygous (KO) deletion of the Men 1 gene. They were fasted overnight and then refed for 7 hours. We measured various cytokines via RT-PCR and proteins via western blot to determine stress response. To determine the mechanism involved, wild type (WT) and menin KO mice embryonic fibroblasts (mefs) were used. Basal expression of pp38 and HO-1 were measured via western blot. We also treated both cell lines with CoPP and another inducer of HO-1, MG132, to determine changes in the signaling pathways. We discovered that menin was significantly up-regulated with treatment of Ang II, but was rescued by the induction of HO-1 via pre-treatment with CoPP prior to Ang II treatment. Furthermore, culturing pre-adipocytes in endothelial cell CM treated with CoPP, resulted in complete ablation of menin combined with increased maturation of adipocytes. These change were not detected in adipocytes directly treated with CoPP alone. Additionally, the liver-specific hemizygous and homozygous menin deletion resulted in increased oxidative stress in both the liver and kidney. This was combined with a significant increase in the expression of HO-1. Furthermore, we discovered that menin KO mefs have a higher basal level of HO-1 and pp38 compared to the WT mefs. When treated with the HO-1 inducers CoPP and MG132, HO-1 was only induced in the KO mefs, indicating menin’s ability to inhibit the induction of HO-1. Together, these results indicate that menin regulates stress response by modulating HO-1 levels through pp38.
Advisors/Committee Members: Mensah-Osman, Edith.
Subjects: Biology; Biomedical Research; Cellular Biology
Keywords: Oxidative Stress; Menin; HO-1; p38 MAPK
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3.
Arunachalam, Sasi.
The Role of store operated calcium channels in human carcinoid cell lines.
Degree: PhD, College of Medicine, 2010, University of Toledo Health Science Campus
► Carcinoid tumors are a heterogeneous set of uncommon slow-growing gastroenteropancreatic neuroendocrine cancers…
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▼ Carcinoid tumors are a heterogeneous set of uncommon slow-growing gastroenteropancreatic neuroendocrine cancers originating primarily from amine or peptide secreting enteroendocrine cells of gastrointestinal tract. Here, we show that carcinoid cell lines express an array of store operated calcium entry (SOCE) channels. Ca2+ entry following ER Ca2+ depletion and store-operated Ca2+ channel activation can regulate proliferation, migration and apoptosis in some cancer cells including those of neuroendocrine phenotype. Consistent with these observations, SOCE was activated in carcinoid cell lines following depletion of ER Ca2+ stores by artificial or physiological agonist. Moreover, treatment with pharmacological inhibitors of SOCE generally reduced Ca2+ entry. SOCE in carcinoid cell lines evoked by GPCR activation suggested that Ca2+ entry may be important for mediating neural, paracrine or autocrine signals in the gut. Promising molecular candidates artificially or physiologically activated for Ca2+ entry pathways are the recently discovered plasma membrane channel protein Orai and ER calcium sensor protein STIM. Molecular profiling by RT-PCR indicated that STIM 1 and Orai1-Orai3 were robustly expressed in GEPNET cell lines derived from foregut and midgut tumors. Thus to gain insight into the roles of STIM1 and Orai1 in foregut and midgut cancers we used targeted gene silencing and over expression techniques in combination with live cell imaging of Ca2+ entry. These studies demonstrated significant reductions in Ca2+ entry following store depletion in cells where STIM1 was reduced by targeted knocked down compared to controls. Conversely, overexpression of STIM1 and Orai1 or Orai3 dramatically enhanced Ca2+ entry that was largely abolished by coexpression with its corresponding dominant negative/ mutant Orai proteins. This study points to a dominant role for STIM1 and Orai1 in mediating SOCE in foregut and midgut carcinoid cells. Furthermore, STIM1 and Orai1 have been proven critical for breast cancer migration and invasion. Hence to study the role of these proteins in carcinoid tumor genesis we developed a novel ex vivo xenograft organotypic slice culture technique. This provided the model system that resembled the three-dimensional multicellular tumor microenvironment. Using organotypic slice culture, we observed that stable BON cells over expressing shRNA for Orai1 showed altered cell movement characteristics and their ability to form tumorlets were greatly reduced in contrast to control cells. Broadly, this work contributes to the search for strategies to inhibit metastasis of carcinoid tumors.
Advisors/Committee Members: Giovannucci, David.
Subjects: Biomedical research
Keywords: SOCE; STIM1; Orai1; Carcinoid tumor; organotypic liver slice culture method; tumorgenesis; migration; amoeboid; mesenchymal migration; multiphoton imaging; Fura-2AM; live cell imaging
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4.
Banasavadi-Siddegowda, Yeshavanth Kumar.
Functional Dissection of FKBP38 in the Biogenesis of Cystic Fibrosis Transmembrane Conductance Regulator in the Endoplasmic Reticulum.
Degree: PhD, College of Medicine, 2011, University of Toledo Health Science Campus
► FK506-binding protein 38 (FKBP38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, associates with…
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▼ FK506-binding protein 38 (FKBP38), a membrane-anchored, tetratricopeptide repeat (TPR)-containing immunophilin, associates with nascent ion channels such as the cystic fibrosis transmembrane conductance regulator (CFTR) and the HERG potassium channel in the endoplasmic reticulum (ER). It promotes the maturation of the HERG channel and maintains the steady state level of CFTR but the underlying mechanisms remain unclear. We show that FKBP38 regulates CFTR biogenesis in a dose dependent manner. It inhibits protein synthesis but simultaneously enhances the post-translational folding of CFTR, leading to reduced ER-associated degradation (ERAD) and improved maturation. The membrane anchorage of FKBP38 is required for the regulation of protein synthesis but not for CFTR post-translational folding or maturation. In contrast, the peptidylprolyl cis/trans isomerase (PPIase) activity of FKBP38 is required for the regulation of CFTR maturation but not for the regulation of its synthesis. Uncoupling FKBP38 from Hsp90 by K308A substitution in the TPR domain modestly enhances CFTR maturation, further reduces its synthesis, but has little effect on its post-translational ERAD. Removing the N-terminal glutamic acid rich domain (ERD) modestly enhances CFTR synthesis but reduces its maturation, suggesting a role for ERD in FKBP38 biological activities. Depletion of FKBP38 or deletion of the transmembrane (TM) domain decreases the association of DF508 CFTR with FKBP38 and Hsp90. This implies that FKBP38 recruits a subpopulation of Hsp90 to CFTR in the ER and its TM domain plays a vital part in this recruitment. Since the deletion of TM domain does not affect CFTR stability or maturation efficiency, such Hsp90 recruitment by FKBP38 might not play a major role in the CFTR biogenesis. Our data support a threefold role for FKBP38 in regulating CFTR synthesis, post-translational ERAD and maturation. In contrast to earlier prediction but consistent with the in vitro enzymological studies, FKBP38 serves as a multifunctional folding PPIase on the cytoplasmic side of the ER membrane during membrane protein biogenesis, whose activity is negatively regulated by Hsp90 through the TPR domain.
Advisors/Committee Members: Wang, Xiaodong.
Subjects: Biomedical Research
Keywords: CFTR; FKBP38; Hsp90
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5.
Beil, Elizabeth.
Identification of fcγRIIA STAT6 Interaction and the Subsequent Effects.
Degree: MS, College of Medicine, 2012, University of Toledo Health Science Campus
► FcγRIIA, a transmembrane receptor for immunoglobulin G (IgG), triggers phagocytosis, endocytosis and…
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▼ FcγRIIA, a transmembrane receptor for immunoglobulin G (IgG), triggers phagocytosis, endocytosis and oxidative burst in leukocytes. Signal transduction via FcγRIIA leading to these outcomes has been relatively well studied. To identify additional signaling partners, a yeast two-hybrid analysis employing the cytoplasmic domain of FcγRIIA was performed. Interestingly, the SH2 domain of signal transducer and activator of transcription 6 (STAT6) appeared in our analysis. STAT6 traditionally interacts with interleukin-4 (IL-4) receptors to induce expression of IL-4 and IL-13 in T cells. Macrophages have also been observed to produce IL-4 in response to pharmaceutical and pathogenic stimuli and to respond to IL-4. However, production of IL-4 or IL-13 subsequent to FcγR ligation has not been observed. Therefore, to better define the specific interaction of FcγRIIA with STAT6, we engineered Chinese hamster ovary cells to express FcγRIIA and RFP-STAT6 to determine if specific ligation of FcγRIIA induces STAT6 nuclear translocation. This resulted in lack of consistent expression of the RFP-STAT6 causing us to perform our subsequent experiments in the human macrophage-like cell line, THP-1. To further elucidate this interaction, FcγRIIA immunoprecipitation followed by western blotting of FcγRIIA and the whole cell lysates from THP-1 macrophages, was performed. We observed the constitutive interaction of non-phosphorylated STAT6 with FcγRIIA. We subsequently sought to examine STAT6 activation after stimulation of THP-1 macrophages with various FcγR ligands. Using IL-4 as a positive control, we observed heat-aggregated IgG induces significant amounts of STAT6 translocation compared to untreated cells (p<0.05). We then sought to determine if IL-4 was produced from THP-1 macrophages after FcγR ligation. We were unable to detect IL-4 from THP-1 macrophages, but were able to detect IL-4 from Jurkat cells, a human T cell line, that were stimulated with ionomycin and phorbol myristal acetate. We then examined the effects of IL-4 on FcγR-mediated activities. We observed that transient exposure of THP-1 macrophages to IL-4 had no significant effect on endocytosis or phagocytosis. Our current observations indicate an interaction exists between FcγRIIA and STAT6 through the SH2 domain of STAT6 producing nuclear translocation of non-phosphorylated STAT6 without induction of IL-4 gene expression. This observance may signal an important avenue of research in understanding the mechanisms of phosphorylation of STAT6 and STAT6 induced gene expression.
Advisors/Committee Members: Worth, Randall.
Subjects: Immunology
Keywords: FcγRIIA; immunoglobulin; phagocytosis; endocytosis; oxidative burst
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6.
Bevington, Joyce M.
Cellular Response to Adenovirus and Adeno- Associated Virus Coinfection.
Degree: PhD, College of Medicine, 2009, University of Toledo Health Science Campus
► Adeno-associated virus (AAV) is a small single-stranded linear DNA virus thatrequires a…
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▼ Adeno-associated virus (AAV) is a small single-stranded linear DNA virus thatrequires a helper-virus to efficiently replicate inside a host cell. The cellular effects of an AAV coinfection with its helper-virus, adenovirus (Ad), were assessed. Affinity column chromatography and crosslinking experiments identified an interaction between the cellular nucleolar protein, nucleophosmin (NPM, B23), with the AAV replicative proteins, Rep68 and Rep78. Increased substrate binding and nicking by Rep68/78 in the presence of NPM was observed using Rep protein functional assays with bacterially expressed proteins. Immunoflourescence studies demonstrated extensive co-localization between NPM and AAV capsid proteins at nucleoli and at small foci within the nucleus. Co-localization was minimal between Rep proteins and NPM during early infection occurring mostly in and around the edge of the nucleoli. Capsid proteins were pulled out with Rep and NPM proteins in crosslinking experiments indicating either a direct or indirect interaction between capsid proteins and NPM. With AAV and Ad having potentially antagonizing effects on the cell cycle, the cellular effects of an AAV and Ad coinfection were examined. The preliminary results indicated that there were no statistically significant changes in protein levels for the cell cycle and cell cycle-related proteins that were examined when comparing Ad infection to Ad and AAV coinfections. There was an increase in the amount of phosphorylated Sp1 in an Ad and AAV coinfection compared to Ad infection. Whether this is related to Sp1 activation of AAV promoters is unknown. With AAV infection affecting expression of Ad proteins involved in disrupting the cellular DSB response, the effects of an AAV and Ad coinfection on the DSB response were examined. Increased amounts of the active phosphorylated form of the DSB response proteins, NBS1, ATM, and Chk2, were observed in an AAV and Ad coinfection compared to Ad infection. This activation was induced by the presence of intact AAV virus and not due to increased viral DNA. Concatamerization of the Ad viral DNA was not detected during an AAV and Ad coinfection, which although unexpected does not preclude the possibility that AAV activation of the DSB response contributes to decreased Ad viral replication.
Advisors/Committee Members: Trempe, James.
Subjects: Virology
Keywords: Adeno-Associated Virus; Nucleophosmin; Cell cycle; Adenovirus; DNA Damage Repair Response; Host-Virus Interaction
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7.
Bhanot, Haymanti.
Mechanisms Underlying Ras-Induced Methuosis in Human Glioblastoma Cells.
Degree: PhD, College of Medicine, 2011, University of Toledo Health Science Campus
► Methuosis is a unique form of non-apoptotic cell death that is characterized…
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▼ Methuosis is a unique form of non-apoptotic cell death that is characterized by dysfunction in the trafficking of clathrin-independent endosomes. This leads to extensive cellular vacuolization and ultimately death of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively activated H-Ras. This study identifies the signaling pathways that govern H-Ras-induced methuosis in human glioblastoma cells. Our studies establish that the small GTPases, Rac1 and Arf6, and the Arf6 GAP, GIT1, act as key downstream components of the signaling pathway underlying H-Ras-induced methuosis. Activated H-Ras-induced methuosis depends upon Rac1 activation and the consequent interaction of active Rac1 with GIT1 to inactivate Arf6 and prevent the recycling of the clathrin-independent endosomes back to the plasma membrane. Studies to test the effect of a closely related Ras GTPase, R-Ras, in glioblastoma cells reveal that it induces cellular vacuolation but does not lead to cell death. Expression of constitutively active R-Ras in glioblastoma cells induces cytoplasmic vacuolation dependent upon Rac1 activation and subsequent inactivation of Arf6. The latter might prevent recycling of vacuoles back to the plasma membrane. However, R-Ras-induced vacuoles do not expand progressively like H-Ras-induced vacuoles, and the cells do not undergo methuosis. H-Ras(G12V) expression in the cells leads to an increase in the levels of active Rab7. Vacuoles in cells expressing H-Ras(G12V) contain H-Ras(G12V) and Rab7(Q67L) on their membranes. These vacuoles fail to fuse with lysosomes. Interestingly, R-Ras(G38V) does not localize to vacuoles. Unlike the cells expressing H-Ras(G12V), cells expressing R-Ras(G38V) are not metabolically compromised. We postulate that though the expression of active R-Ras in glioblastoma cells induces cellular vacuolation, the induced vacuoles might be competent to fuse with the lysosomes, thus preventing methuosis. Further studies to identify the different signaling pathways underlying the effects of H-Ras and R-Ras on vesicular trafficking would help to identify key regulators of the initial steps that lead to methuosis. This might help identify potential therapeutic targets that can be manipulated to induce methuosis in cancers that are refractory to apoptosis.
Advisors/Committee Members: Maltese, William.
Subjects: Biology; Cellular Biology; Molecular Biology
Keywords: Ras; methuosis; glioblastoma; Arf6; Rac1; R-Ras; cell death
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8.
Bhattacharya, Sumit.
Contribution of Purinergic Receptors to Calcium Signaling in Salivary Gland.
Degree: PhD, College of Medicine, 2012, University of Toledo Health Science Campus
► There is emerging consensus that P2X4 and P2X7 ionotropic purinoceptors (P2X4R and…
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▼ There is emerging consensus that P2X4 and P2X7 ionotropic purinoceptors (P2X4R and P2X7R) are critical players in regulating [Ca2+]i dynamics and fluid secretion in the salivary gland. In contrast, details regarding their compartmentalization and selective activation, contributions to the spatiotemporal properties of intracellular signals and roles in regulating protein exocytosis and ion channel activity have remained largely undefined. Moreover, information regarding modulation of P2X class of receptors by cAMP, the major purinergic cell signaling pathway for protein secretion has been limited. To address these gaps in our understanding, we profiled mouse parotid acinar cells using live-cell imaging to follow the spatial and temporal features of ATP-evoked Ca2+ dynamics and exocytotic activity. Selective activation of P2X7Rs revealed an apical-to-basal [Ca2+]i signal that initiated at the sub-luminal border and propagated with a wave speed estimated at 17.3 ± 4.3 μm/s (n = 6). The evoked Ca2+ spike consisted of Ca2+ influx and Ca2+-induced Ca2+ release from intracellular Ca2+ channels. In contrast, selective activation of P2X4Rs induced a Ca2+ signal that initiated basally and propagated toward the lumen with a wave speed of 4.3 ± 0.2 μm/s (n = 8) that was largely independent of intracellular Ca2+ channel blockade. Consistent with these observations, P2X7R expression was enriched in the sub-luminal regions of acinar cells while P2X4R appeared localized to basal areas. We also predict that P2X7R enriching the apical regions of acinar cells are activated by luminal ATP. We also showed that P2X4R and P2X7R activation evokes exocytosis in parotid acinar cells. Our studies also demonstrate that the P2X4R-mediated [Ca2+]i rise and subsequent protein exocytosis was enhanced by ivermectin (IVR) thereby making it a potential target treatment of salivary hypofunction diseases. Additionally, experiments designed to assess crosstalk between purinoceptors (P2X4R and P2X7R) and cAMP signaling pathways showed that [Ca2+]i levels were enhanced upon β-adrenergic receptor stimulation or during rises in intracellular cAMP levels by synthetic cAMP agonists. The current study supports the notion that PKA dependent crosstalk between purinoceptors evoked Ca2+ signals and cAMP pathway might be critical in regulation of both fluid and protein secretion in parotid acinar cells. Thus crosstalk between the two signaling pathways can also be exploited for construction of therapies associated with salivary hypofunction.
Advisors/Committee Members: Giovannucci, David.
Subjects: Biology; Neurobiology; Neurology; Neurosciences; Pharmacology; Physiology
Keywords: ATP; purinergic; exocytosis; calcium; p2x4; p2x7; saliva; parotid; acinar cells; cAMP
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9.
Bhrigu, Gargi.
Sorting Signals, Domain Conformation and Interdomain Interactions in CFTR Misprocessing and Rescue.
Degree: PhD, College of Medicine, 2010, University of Toledo Health Science Campus
► Cystic fibrosis (CF) is among the most common lethal inherited diseases in…
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▼ Cystic fibrosis (CF) is among the most common lethal inherited diseases in the US. Thedeletion of phenylalanine at residue 508 (6F508) within cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF causing mutation. 6F508 mutant is unable to exit the endoplasmic reticulum (ER). The precise mechanism of the defective ER-to-Golgi export of 6F508 CFTR is not known. A di-acidic ER exit code (DAD) has been identified within the first nucleotide binding domain (NBD1) of CFTR. Disruption of this motif leads to the defective coupling of CFTR to the COPII machinery and the subsequent impaired export of CFTR from the ER. We performed a systematic analysis of the sorting signals, domain conformation and inter-domain interactions of CFTR in presence of 6F508 mutation and during its rescue by low temperature or second site mutation (R555K). We found that rescue of 6F508 CFTR depends on the DAD motif within the NBD1. Disruption of DAD (DAA) reduces the Sec24 association of 6F508 CFTR whereas rescue mutation R555K increases it. Using in situ limited proteolysis we identified conformational defects in N-terminal region and NBD1 in 6F508 CFTR. 6F508 rescue is accompanied by global conformational reversion. Further domaindomain interactions within CFTR play an important role in both 6F508 CFTR misprocessing and rescue. Our data firmly establish that while DAA is an exit code mutant 6F508 CFTR has widespread conformational defect which compromises its export and stability. Restoration of its global conformation leads to restoration of its export as well as function.
Advisors/Committee Members: Wang, Xiaodong Robert.
Subjects: Cellular biology; Molecular biology; Physiological psychology
Keywords: CFTR; DF508; Protein trafficking; Endoplasmic Reticulum; conformation; exit code
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10.
Bhrigu, Vipul.
Replication of Adeno-Associated Virus in Murine Fibroblasts with Mouse Adenovirus Provided Helper Functions.
Degree: PhD, College of Medicine, 2009, University of Toledo Health Science Campus
► The adeno-associated virus (AAV) replicates to high titer when host cellare coinfected…
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▼ The adeno-associated virus (AAV) replicates to high titer when host cellare coinfected with a helper virus. Here we analyzed the coinfection of AAV and mouse adenovirus (MAV-1) in murine fibroblasts. We observed that MAV-1 coinfected 3T3 cells produced approximately 10-40 fold less AAV DNAse resistant particles (DRP) than Hela cells. Levels of AAV DNA replication were approximately 30 fold less in 3T3 cells as compared to Hela cells coinfected with human adenovirus (Ad-5). A study of these lower levels of infection in 3T3 cells compared to Hela cells revealed that receptor binding and internalization of AAV in 3T3 and Hela cells was comparable. However, AAV did not enter into the nucleus of 3T3 cells as efficiently as it does in Hela cells. Furthermore, viral DNA replication levels of AAV DNA were found to be lower in 3T3 cells than Hela cells even after transfection of proviral plasmid indicating a defect in support for the AAV DNA replication as well in 3T3 cells as compared to Hela cells.
Advisors/Committee Members: Trempe, James.
Subjects: Molecular biology; Virology
Keywords: Adeno-Associated Virus; Adenovirus; Murine Fibroblasts; Hela; Mouse; AAV genome copy
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11.
Bismack, Brian James.
Implementation of the Dosimetry Check Software Package in Computing 3D Patient Exit Dose Through Generation of a Deconvolution Kernel to be Used for Patients’ IMRT Treatment Plan QA.
Degree: MS, College of Medicine, 2010, University of Toledo Health Science Campus
► Using the Dosimetry Check IMRT QA package a deconvolution kernel to be…
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▼ Using the Dosimetry Check IMRT QA package a deconvolution kernel to be used in exit image dose calculations was created. This kernel modeled Electronic Portal Imaging Device response by incorporating the various machine characteristics along with the variant patient thickness and composition. To properly achieve this, Dosimetry Check first had to accurately model the beams for the machines being used. This was done by taking a series of in air and in water measurements including central axis depth dose values at various field sizes and in-air off center ratios to model beam flatness and symmetry. A deconvolution kernel for patient CT dose computation using pre-treatment (in-air) EPID images was created. This baseline helped establish the necessary measurements for the exit image kernel. The necessary measurements for the exit image kernel included EPID images of various field sizes with various thicknesses of water in the beam as well as a characterization of off axis narrow beam transmission. The fit was performed and a report generated for its variance. The kernel was then successfully used in the evaluation of an IMRT prostate plan that was created for an anthropomorphic phantom and compared to a baseline evaluation of the same plan using in air EPID images. Volumetric, planar, and point dose comparison between measured and computed dose distributions agreed favorably indicating the validity of technique used for IMRT QA.
Advisors/Committee Members: Parsai, E. Ishmael.
Subjects: Physics; Radiation
Keywords: exit image; transit image; 3D dose reconstruction; IMRT patient QA
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12.
Blomquist, Thomas M.
Development of Bimodal Gene Expression Analysis and Allele-Specific Competitive PCR for Investigation of Complex Genetic Traits, Lung Cancer Risk.
Degree: PhD, College of Medicine, 2010, University of Toledo Health Science Campus
► The majority of common traits (e.g. cancer predisposition) reside within the paradigm…
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▼ The majority of common traits (e.g. cancer predisposition) reside within the paradigm of complex genetic systems. Yet at present, no more than 5% of phenotypic variation in complex genetic traits have been explained. Difficulties in linking individual genetic changes to complex phenotypic traits may be due, in part, to limitations and challenges intrinsic to approaches and methodologies currently employed to study complex genetic phenomena (e.g. genome-wide association studies). The goal of the dissertation presented here was to develop new approaches and methodologies to untangle the intricate web of heritable mechanisms underlying complex genetic traits, using lung cancer predisposition as a model system. The guiding philosophy for the development of new approaches and methods was to investigate: a) novel aspects of the involvement of intermediate phenotypes (e.g. transcript expression) in explaining complex genetic trait penetrance, as well as b) the genetic basis of intermediate phenotypes (e.g. allele-specific expression). Utilization of intermediate phenotypes minimizes the effect of confounding variables when attempting to abstract the effect size (i.e. penetrance) of genetic variation on a complex genetic trait. During the course of my studies I developed bimodal and dispersion-based gene expression analysis, allele-specific competitive PCR methodology as well as an interpretive framework for allele-specific expression signal analysis. In these studies, I demonstrate that bimodal and dispersion gene expression patterns are an important classifying characteristic (intermediate phenotype) for genes with a high degree of likelihood of involvement in lung carcinogenesis. Moreover, using allele-specific competitive PCR and allele-specific expression signal theory I demonstrate the identification of cis-acting genetic variations which alter transcript expression of ERCC5 and CEBPG genes (both important in the progression of lung carcinogenesis) using various in vivo approaches.
Advisors/Committee Members: Willey, James.
Subjects: Bioinformatics; Biology; Biomedical research; Biostatistics; Cellular biology; Computer science; Epidemiology; Experiments; Genetics; Health; Health care; Molecular biology; Oncology; Pathology; Pharmacology; Philosophy; Public health
Keywords: allele-specific expression; lung cancer; complex genetic traits; normal bronchial airway epithelial epithelium cells; dispersion; bimodal; kurtosis; variance
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13.
Bowman, Thomas A.
Hepatic CEACAM1 Protects Against Metabolic Abnormalities Associated with Metabolic Syndrome.
Degree: PhD, College of Medicine, 2010, University of Toledo Health Science Campus
► Impaired hepatic insulin clearance causes hyperinsulinemia and secondary insulin resistance, which may…
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▼ Impaired hepatic insulin clearance causes hyperinsulinemia and secondary insulin resistance, which may progress to involve various components of metabolic syndrome. The carcinoembryonic-related cell adhesion molecule 1 (CEACAM1) has been shown to promote insulin clearance, downregulate the mitogenic action of insulin, and limit lipogenesis in the early hours of refeeding. Mice with liver-specific Ceacam1 inactivation (L-SACC1) or with global null mutation (Cc1–/–), exhibit impairment of insulin clearance and hyperinsulinemia, which causes insulin resistance. Since the lack of CEACAM1 correlates with insulin resistance, and regulates insulin action in liver, an important diet-responsive organ, we proposed that reduction of CEACAM1 is implicated in the pathogenesis of diet-induced insulin resistance; and that increasing hepatic levels of CEACAM1 would be protective against metabolic abnormalities associated with metabolic syndrome. Therefore we examined CEACAM1 levels in an animal model of metabolic syndrome and non-alcoholic steatohepatitis (NASH), the low aerobic capacity runner (LCR) rats, in comparison to high aerobic capacity runner (HCR) rats. We found that in response to caloric restriction by 30% over a period of 2-3 months profound improvements in insulin sensitivity and reversal of hepatic inflammation, oxidative stress and fibrosis. Caloric restriction exerts these effects along with increases in fasting levels of CEACAM1 in liver. Additionally we examined the effect of high-fat diet on wild-type mice and on a transgenic mouse with liver-specific overexpression of rat CEACAM1 (L-CC1). We found that an early event associated with high-fat feeding is repression of CEACAM1 by a PPARalpha-mediated mechanism, and that this leads to impaired insulin clearance prior to the development of a pro-inflammatory state. Transgenic overexpression of CEACAM1 in liver prevents hyperinsulinemia and insulin resistance, and limits visceral obesity and the metabolic response to high-fat intake.
Advisors/Committee Members: Najjar, Sonia.
Subjects: Biomedical research; Molecular biology
Keywords: CEACAM1; insulin; Hepatic; insulin resistance; PPARα; hyperinsulinemia; insulin clearance
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14.
Brown, Colin.
A Comprehensive Noise Characterization in a High School.
Degree: MS, College of Medicine, 2010, University of Toledo Health Science Campus
► This study characterized the noise levels in a high-school to assess the…
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▼ This study characterized the noise levels in a high-school to assess the exposure of students and school personnel by measuring one-minute averages of dBA and dBC for a few weeks during area sampling in almost all functional spaces of the high-school using noise dosimeters (Larson Davis SparkTM Model Number 705+). The highest level of noise was observed during pep rally with the levels ranging 59-107 dBA and a mean (SD) and median of 88 (16) and 97 dBA, respectively. Lunch periods showed noise levels ranging 61-90 dBA with a mean (SD) and median of 79 (4) and 79 dBA, respectively. Gym sports had noise levels ranging 58-113 dBA with a mean (SD) and median of 75 (8) and 78 dBA. The noise levels in the metals shop ranged 55-99 dBA with mean (SD) and median of 77 (4.6) and 77 dBA, respectively. The noise levels in the classrooms, hallways and other locations were, on average, below 71 dBA. Overall the levels of noise at school ranged 48-113 dBA with a mean (SD) and median of 66 (9) and 64 dBA, respectively. The peak noise levels ranged 105-153 dBC. The results of this study indicate that there are potentially harmful noise exposures within the high school.
Advisors/Committee Members: Akbar, Farhang.
Subjects: Occupational Health; Occupational Safety; Public Health
Keywords: noise characterization; noise dosimeters
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15.
Campbell, Sara J.
Mechanisms of Moraxella catarrhalis Induced Immune Signaling in the Pulmonary Epithelium.
Degree: PhD, College of Medicine, 2010, University of Toledo Health Science Campus
► Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause ofdeath in…
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▼ Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause ofdeath in the United States and is becoming increasingly more common. It has a global prevalence of 10% of the general population. COPD is a condition of irreversible airflow limitation due to airway obstruction and lung destruction, making breathing difficult. Exacerbations of COPD are characterized by an increase in symptoms, a worsening of lung function, and an increase of inflammation. The most common causes of acute exacerbations are infection and air pollution. Moraxella catarrhalis is one of the leading causes of acute exacerbations, responsible for approximately 10%. There are currently no treatments that can stop the progression of COPD or suppress the inflammation. A better understanding of the mechanism behind the inflammation is needed to identify new therapeutic targets. Little is known about how M. catarrhalis interacts with the host epithelium to lead to exacerbations. The airway epithelium recognizes pathogens via receptors such as the toll-like receptors (TLR). Many pathogens signal through TLRs to activate NF-κB which translocates to the nucleus and activates transcription of inflammatory mediators. It has been shown that Haemophilus influenzae, another cause of acute exacerbations, can induce the production of inflammatory mediators from the bronchial epithelium through the p38 MAPK (mitogen activated protein kinase) and ERK1/2 (extracellular signal-regulated kinase) pathways. PI3K (phosphoinositide-3 kinase) has also been implicated in airway inflammation, though the literature is conflicting about whether it functions as pro- or anti-inflammatory. We have found that M. catarrhalis can induce the production of the proinflammatory chemokines IL-8 and MCP-1. TLR2 is at least partly responsible for the chemokine expression. Three kinases, PI3K, p38 MAPK, and ERK are all activated in response to M. catarrhalis. PI3K is activated downstream of TLR2 and shows differential regulation of MCP-1 and IL-8. PI3K has a suppressive effect on p38 MAPK activation but has no effect on ERK. P38 MAPK positively regulates the expression of MCP-1 in response to M. catarrhalis, but has no effect on IL-8. ERK is a positive regulator of M. catarrhalis-induced MCP-1 and IL-8 production. We are currently working to elucidate these pathways. Our work aims to understand the lung epithelium’s response to infection by M. catarrhalis and the signaling pathways involved in that response in hopes of being able to better treat or prevent exacerbations and improve the quality of life for people with COPD.
Advisors/Committee Members: Pan, Z. Kevin.
Subjects: Immunology; Molecular biology
Keywords: Toll-like Receptor (TLR); phosphatidylinositol 3-kinase (P13k); p38 MAPK; Raf/MEK/ERK pathway; airway inflammation; Signal Transduction
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16.
Chang, Karin.
Platelets and Serotonin in Migraine.
Degree: MS, College of Medicine, 2010, University of Toledo Health Science Campus
► Migraine headache is a disabling chronic condition, especially in women of childbearing…
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▼ Migraine headache is a disabling chronic condition, especially in women of childbearing age. Its etiology is currently unknown. The efficacy of serotonin receptor (5-HT1B/1D) agonists, triptans, in relieving attacks of migraine headache suggests a role for serotonin in the course of the headache. Platelets contain the largest reserve of serotonin stored in the body and thus, platelets may play a role in the etiology of migraine attacks. The literature is equivocal about the role of the platelet in migraine. This study has identified evidence of a bleeding diathesis, including menorrhagia, gum bleeding, gross hematuria, and ecchymosis, in migraineurs. Migraineurs also have fewer platelet dense granules in their platelets than healthy control subjects (1.61+/-0.14 vs. 3.85+/-0.14), but there are no significant differences in platelet serotonin and adenine nucleotide content.
Advisors/Committee Members: Gunning, William.
Subjects: Biomedical research; Neurology
Keywords: platelet; serotonin; migraine; headache; bleeding; dense granule
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17.
Chen, Miao.
Moraxella catarrhalis-induced innate immune responses in human pulmonary epithelial cells and monocytes.
Degree: PhD, College of Medicine, 2009, University of Toledo Health Science Campus
► COPD is characterized by chronic inflammation in the lung, and its severe…
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▼ COPD is characterized by chronic inflammation in the lung, and its severe stageexacerbation is one of the major burdens of healthcare, causing diminished quality of life and highly increased mortality rate. Most common treatments for COPD could only improve the symptoms, but do not suppress the undergoing inflammations. Our study based on understanding the molecular basis of M. catarrhalis-induced innate immune responses on pulmonary epithelial cells and monocytes would help to identify new therapeutic target for COPD. M. catarrhalis has been proved to be the third leading cause of COPD. We found that M. catarrhalis could induce CCL20/MIP-3α expression mainly through TLR2-MyD88-TRAF-6-NF-κB/MAPK signaling pathway on pulmonary epithelial cells. Only TLR2, but not TLR4 signaling pathway can be activated on those cells during M. catarrhalis infection. Study on stable transfected HEK293 cells suggested that CD14 and MD2 are critical to activate TLR4 signaling; and sCD14 could enhance TLR2-mediated inflammatory responses. Thus our hypothesis is that lack of CD14 and MD2 might contribute to the silence of TLR4 signaling; and sCD14 released from monocytes could modulate TLR2- and TLR4-mediated inflammatory responses on airway epithelial cells. Both mCD14 and sCD14 expression levels are very low on naïve monocytic THP1 cells. 1α, 25-dihydroxy VD3 and specific ligands of TLR2 and TLR4 could increase mCD14 and sCD14 expression, which up-regulates both TLR2- and TLR4-mediated inflammatory responses on THP1 cells. Meanwhile, conditioned media from 1α,25-dihydroxy VD3 primed THP1 cells could activate TLR4 signaling pathway and enhance TLR2-mediated inflammatory responses on pulmonary epithelial cells under M. catarrhalis infection. Such amplified inflammatory responses could be abolished by CD14 antibody. All these data suggest that sCD14 could mediate a crosstalk between monocytes and pulmonary epithelial cells to mount an amplification loop through TLR2 and TLR4 during bacteria-induced inflammatory responses. Thus CD14 could be a potential therapeutic target to inhibit the abnormal inflammation during the pathogenesis of COPD or development of exacerbation.
Advisors/Committee Members: Pan, Z. Kevin.
Subjects: Biology; Biomedical research; Health; Immunology; Microbiology; Molecular biology
Keywords: COPD; CD14; Moraxella catarrhalis; pulmonary epithelial cells; inflammation; innate immunity
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18.
Chen, Yi Liang.
The N-terminus of a1 Subunit and Na/K-ATPase-Mediated Signal Transduction.
Degree: PhD, College of Medicine, 2009, University of Toledo Health Science Campus
► Recent studies have ascribed many non-pumping functions to theNa/K-ATPase and it has…
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▼ Recent studies have ascribed many non-pumping functions to theNa/K-ATPase and it has been demonstrated to interact with a variety of proteins via its cytosolic N-terminus domain (NT), which plays an essential role for its non-pumping functions. One of the proteins that interact with NT is caveolin-1 that is implicated in cellular cholesterol trafficking and homeostasis. Since we previously showed that the Na/K-ATPase regulated membrane trafficking of caveolin-1, in this study we have further revealed that the Na/K-ATPase is able to regulate cellular cholesterol distribution via its interaction with cavelin-1. Graded knockdown of the Na/K-ATPase leads to redistribution of cholesterol from membranes to the cytosol and this effect is independent of its pumping function. Moreover, this regulation is confirmed in α1+/- mouse liver. Functionally, the knockdown-induced redistribution appears to affect the cholesterol sensing in the endoplasmic reticulum because it activates the sterol regulatory element binding protein pathway in vivo. Interestingly, our subsequent study has demonstrated that plasma membrane cholesterol also regulates the cell surface Na/K-ATPase α1 subunit by modulating membrane trafficking of α1. Depletion of plasma membrane cholesterol leads to endocytosis of α1 and accumulation of α1 in the late endosome/lysosomes. Mechanistically, the cholesterol-regulated α1 trafficking appears to be related to cholesterol-α1 interaction at the cholesterol recognition/interaction amino acid consensus site (CRAC) within NT. Disruption of the cholesterol-α1 interaction by mutating the key amino acid in CRAC blunts the regulation of the Na/K-ATPase trafficking by cholesterol. Thus, our studies have revealed a reciprocal regulation between the plasma membrane Na/K-ATPase and cholesterol and the Na/K-ATPase may represent a potential plasma membrane cholesterol sensor for the regulation of cellular cholesterol. Furthermore, since the Na/K-ATPase is a signaling molecule that controls kinase activities, the reciprocal regulation between the Na/K-ATPase and cholesterol may serve as an important link between kinase cascades and lipid homeostasis. To study the physiological role of the receptor function of the Na/K-ATPase, we first revealed that Cav-1 KO mice developed salt-sensitive hypertension, which implicated the signaling Na/K-ATPase in blood pressure regulation. Furthermore, we generated NT-YFP-expressing transgenic mice and showed that renal NT-YFP expression results in salt-sensitive hypertension. Mechanistically, development of salt-sensitive hypertension is related to renal membrane α1 and sodium hydrogen exchanger 3 trafficking and urine excretion of the endogenous Na/K-ATPase ligand, marinobufagenin, which may influence renal sodium reabsorption and excretion. Taken together, this study has demonstrated that the Na/K-ATPase α1 subunit plays important roles in cellular cholesterol homeostasis and is also regulated by plasma membrane cholesterol content. Moreover, in vivo expression of NT-YFP affects α1 trafficking and leads to salt-sensitive hypertension, which warrants further examination of the physiological role of the Na/K-ATPase α1 in the renal and cardiovascular systems.
Advisors/Committee Members: Xie, Zi-Jian.
Subjects: Molecular biology
Keywords: cholesterol; Na/K-ATPase; metabolism; caveolin; hypertension
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19.
Chung, Yutein.
Identification of signaling pathways important for Borrelia burgdorferi-elicited IL-10 production by macrophages and their effects on suppressing antigen presenting cell immune responses.
Degree: PhD, College of Medicine, 2011, University of Toledo Health Science Campus
► Borrelia burgdorferi (Bb) is a tick-borne bacterium from the family Spirochaetes that…
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▼ Borrelia burgdorferi (Bb) is a tick-borne bacterium from the family Spirochaetes that is the causative agent for Lyme disease. These bacteria are notable for their ability to evade host defenses and persist extra-cellularly, even though infection elicits potent innate and adaptive immune responses. We previously demonstrated that host interleukin 10 (IL-10), an anti-inflammatory cytokine important for controlling excess inflammation, plays an important role in suppressing the immune-clearance of Bb. We hypothesize antigen-presenting cells (APCs) such as bone-marrow macrophages (BMM) and dendritic cells (BMDC) produce high-levels of IL-10 immediately upon recognition of Bb and this dysregulated IL-10 level subsequently suppresses the elicitation of pro-inflammatory mediators by the APCs against Bb. We also hypothesize that the production of IL-10 by APCs such as BMMs utilizes signaling pathways that are distinct from Bb-elicited pro-inflammatory mediators. Our results demonstrated that both cultured BMM and BMDCs rapidly produce IL-10 upon Bb-stimulation and this IL-10 suppressed the production of pro-inflammatory cytokines (e.g. IL-12), chemokines, reactive oxygen species, phagocytosis, and surface marker upregulation. Our data also indicate that IL-10 production by BMMs in response to Bb is dependent on surfaceToll-like receptor 2 (TLR2) yet independent of Bb phagocytosis/internalization. While most Bb-elicited pro-inflammatory mediators are also TLR2-dependent, they require that Bb be internalized. Bb-elicited IL-10 production by BMMs is dependent on signaling pathways involving both phosphotidylinositol-3 kinase (PI3-kinase) and mitogen-activating protein kinase (MAP kinase). On the other hand, the elicitation of most pro-inflammatory responses from BMMs by Bb is independent of both PI3-kinase and MAP kinase. Overall, our findings indicate that Bb stimulates APCs to produce dysregulated IL-10 through unique signaling pathways from those that produce inflammatory mediators and that the amount of IL-10 that are produced are sufficient to suppress many APC immune mechanisms that are critical for controlling bacterial infections. The delineation of these IL-10 specific signaling mechanisms should identify pathways that could be targeted to better control the development of Lyme disease.
Advisors/Committee Members: Wooten, R. Mark.
Subjects: Immunology; Microbiology
Keywords: Borrelia burgdorferi; macrophages; dendritic cells; interleukin 10; IL-10; toll-like receptor 2 TLR2; phagocytosis; OspA, PI3-kinase; MAP kinase p38; ERK 1/2
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20.
Dangeti, Venkata Srinivas Mohan Nimai.
Processing of Cisplatin Interstrand crosslinks (ICLs) by DNA repair proteins.
Degree: PhD, College of Medicine, 2012, University of Toledo Health Science Campus
► Interstrand crosslinks (ICLs) formed by cisplatin are unique lesions formed by the…
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▼ Interstrand crosslinks (ICLs) formed by cisplatin are unique lesions formed by the drug. They covalently crosslink both strands of DNA. Cisplatin induced cell death primarily occurs through the formation of various lesions on DNA. Hence, the persistence of the adducts is crucial for its cytotoxicity. DNA repair systems which ensure the integrity of the genome are mainly responsible for the development of resistance to the drug, due to their role in removing the damage on the DNA created by the drug. Repair of ICLs is thought to involve multiple repair pathways. Emerging evidence suggests that there is a replication dependent pathway which depends on stalling of replication forks, and a replication independent mechanism. The exact mechanistic details of cisplatin ICL repair remain poorly understood. Information from studies addressing the repair of ICLs formed by other agents cannot be directly applied to understand cisplatin ICL repair. Each lesion produces unique distortions, and as a result activates different DNA repair pathways. Using cell extracts and purified proteins, we propose a specific pathway for the processing of flanking DNA adjacent to cisplatin ICLs. This mechanism involves the action of two pathways, Base excision repair (BER) and Mismatch repair (MMR) which is activated due to conversion of cytosine adjacent to the cisplatin ICL. We demonstrate that proteins involved in BER –Uracil DNA glycosylase, Apurinic endonuclease, and DNA Polymerase β directly process this uracil. Finally, we showed that action of these BER proteins leads to the activation of MMR downstream of BER. We propose a novel mechanism in which this common mechanistic pathway acts adjacent to cisplatin ICL, but does not influence repair of damage caused by other crosslinking agents. It influences the overall rate of repair by interfering with the repair processes that act to resolve the crosslink.
Advisors/Committee Members: Patrick, Stephan.
Subjects: Biochemistry
Keywords: cisplatin; DNA repair; Interstrand crosslinks; Base Excision repair; Mismatch repair
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21.
DeAngelis, Anthony Michael.
CEACAM1: A Link Between Insulin and Lipid Metabolism.
Degree: PhD, College of Medicine, 2009, University of Toledo Health Science Campus
► The pathogenesis of T2DM is complex and is preceeded by the development…
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▼ The pathogenesis of T2DM is complex and is preceeded by the development of insulinresistance. Previous studies have shown CEACAM1 to play a major role in mediating insulin clearance in the liver and that altered CEACAM1 function can cause impaired insulin clearance, hyperinsulinemia, altered fat metabolism, and the development of insulin restance. In this work, three unique strategies were employed in order to gain a better understanding of the physiologic role of CEACAM1 in vivo. First, the metabolic phenotype of Cc1–/– mice which are homozygous for a null mutation of the Ceacam1 gene was characterized. As expected, these mice exhibited an impairment of insulin clearance, hyperinsulinemia, and altered fat metabolism. Moreover, hyperinsulinemiceuglycemic clamp studies revealed that the inbred Cc1–/– mice developed insulin resistance primarily in liver. Finally, despite substantial expression of CEACAM1 in pancreatic β-cells, insulin secretion in response to glucose in vivo and isolated islets was normal in Cc1–/– mice. This undermines the notion CEACAM1 is involved in regulating insulin secretion in the pancreas and suggests the principal role of CEACAM1 in insulin action is to mediate insulin clearance in liver. Next, an alternative approach at investigating the function of CEACAM1 in vivo was taken by generating a transgenic mouse model (Tg(ApoA1-Cc1)7Smn) overexpressing CEACAM1 specifically in the liver. Analysis of Tg(ApoA1-Cc1)7Smn transgenic mice confirmed expression of the CEACAM1 transgene in a liver specific manner. These mice are currently being utilized to perform a gain of function study in Cc1-/- mice. This will allow us to investigate whether or not restitution of CEACAM1 expression in the liver of Cc1-/- mice is sufficient to ameliorate impaired insulin clearance, insulin resistance, and subsequent metabolic abnormalities. Finally, human expression of CEACAM1 mirrors the combined expression of murine CEACAM1 and CEACAM2. Therefore, we attempted to generate a Cc1–/– / Cc2–/– double knockout mouse line in order to more accurately model CEACAM1 abnormality in humans and provide more accurate insights into the role of CEACAM1 in humans. Despite the unsuccessful first attempt at generating this mouse line, alternative strategies are being employed to simulate the concurrent absence of these two proteins in a mouse model. Taken together, this data highlights the pivotal role CEACAM1 plays in regulating circulating insulin levels through mediating insulin clearance in the liver.
Advisors/Committee Members: Najjar, Sonia.
Subjects: Genetics; Molecular biology; Physiological psychology
Keywords: CEACAM1; Lipid Metabolism; Insulin Resistance; Insulin Clearance; Diabetes
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22.
Detwiler, Jordyn A.
Dosimetric Evaluation of Three Partial Breast Irradiation Devices and the Dosimetric Effect of Tissue Thickness Surrounding a Multi-Lumen Partial Breast Applicator.
Degree: MS, College of Medicine, 2010, University of Toledo Health Science Campus
► Many High Dose Rate treatment planning systems that are in use fail…
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▼ Many High Dose Rate treatment planning systems that are in use fail to correct for heterogeneities. If the treatment planning system does not correct for heterogeneities, it would assume that the patient is receiving full scatter when in reality, the patient will possibly be underdosed. A 1cm diameter planning target volume for a lumpectomy cavity could extend beyond the skin or chest wall for the patient and could be a great problem when it comes to treatment with the MammoSite® single lumen breast applicator. A previous Monte Carlo study tested 3 MammoSite® balloon sizes at various depths beyond the planning target volume to see how much tissue would be needed to achieve full scatter. The results showed that on average, if there was no tissue beyond the prescription line of 1cm there would be a 10% dose reduction for the breast – skin interface. The purpose of this study is to use the Strut Adjusted Volume Implant (SAVI) multi-lumen breast applicator to re-create the measurements done with the MammoSite® balloon and expand these measurements to include tissue thicknesses less than the PTV. Previous simulations with the MammoSite® were done using Monte Carlo, with tissue thicknesses beyond the planning target volume of 0 – 10cm. This study will re-create these measurements using Metal Oxide Semiconductor Field Effect Transistors (MOSFETs) and also take measurements below the prescription line of 1cm due to the ability of the SAVI applicator to adjust dose to the skin.
Advisors/Committee Members: David, Pearson.
Subjects: Radiation
Keywords: Breast Brachytherapy; SAVI
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23.
Dickerson, Edward.
Quantitative Analysis of the Head Scatter and Jaw Transmission Correction Factor for Commissioning of Enhanced Dynamic Wedge Fields Using a MapCHECK 2 Diode Array.
Degree: MS, College of Medicine, 2012, University of Toledo Health Science Campus
► Quality assurance in radiation oncology treatment planning requires independent verification of dose…
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▼ Quality assurance in radiation oncology treatment planning requires independent verification of dose to be delivered to a patient through “second check†calculations for simple plans as well as planar dose fluence measurements for more complex treatments, such as intensity modulated radiation treatments (IMRT). Discrepancies between treatment planning system (TPS) and second check calculations created a need for treatment plan verification using a two dimensional diode array for Enhanced Dynamic Wedge (EDW) fields. While these measurements met clinical standards for treatment, they revealed room for improvement in the EDW model. The purpose of this study is to analyze the head scatter and jaw transmission effects of the moving jaw in EDW fields by measuring dose profiles with a two dimensional diode array in order to minimize differences between the manufacturer provided fluence table (Golden Segmented Treatment Table) and actual machine output. The jaw transmission effect reduces the dose gradient in the wedge direction due to transmission photons adding dose to the heel region of the field. The head scatter effect also reduces the gradient in the dose profile due to decreased accelerator output at increasingly smaller field sizes caused by the moving jaw. The field size continuously decreases with jaw motion, and thus the toe region of the wedge receives less dose than anticipated due to less head scatter contribution for small field sizes. The Golden Segmented Treatment Table (GSTT) does not take these factors into account since they are specific to each individual machine. Thus, these factors need to be accounted for in the TPS to accurately model the gradient of the wedge. The TPS used in this clinic uses one correction factor (transmission factor) to account for both effects since both factors reduce the dose gradient of the wedge. Dose profile measurements were made for 5x5 cm2, 10x10 cm2, and 20x20 cm2 field sizes with open fields and 10°, 15°, 20°, 25°, 30°, 45°, and 60° wedges for 6 MV and 18 MV beams and compared with TPS generated profiles. The transmission factor was adjusted for the 18 MV beam to obtain a better correlation between planned and measured dose gradient by reducing the gradient of the wedge in the TPS. This correction resulted in an average and maximum pass rate improvement for patient plans at a distance to agreement of 3% 3mm of 1.07% and 3.9% respectively. The off axis ratio data in the second check calculation software was also adjusted to bring the dose agreement between the initial TPS calculation and second check calculation within clinical standards. This study demonstrated the ability to adjust the EDW gradient in a treatment planning system to improve the differences in machine output specific to each machine and the manufacturer provided GSTT.
Advisors/Committee Members: Shvydka, Diana.
Keywords: head scatter; Enhanced Dynamic Wedge; EDW
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24.
Dong, Shuai.
The Na/K-ATPase/Caveolin-1 Interaction Regulates Cell Growth.
Degree: MS, College of Medicine, 2011, University of Toledo Health Science Campus
► The Na/K-ATPase α1 subunit contains a highly conserved caveolin-1 binding motif (CBM).…
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▼ The Na/K-ATPase α1 subunit contains a highly conserved caveolin-1 binding motif (CBM). We have previously shown that the interaction between the α1 CBM and caveolin-1 regulates the formation of caveolae, and intracellular trafficking and distribution of cholesterol. Here we demonstrate an important role of this interaction in control of cell growth. First, we find that knockdown of either the Na/K-ATPase or caveolin-1 inhibits cell proliferation and that this inhibition is greatly enhanced by modest depletion of cellular cholesterol. Second, the expression of wild-type α1, but not the CBM mutant, is capable of restoring normal growth in LLC-PK1 cells. Finally, we find that the expression of N-terminus of the α1 subunit inhibits the interaction between the Na/K-ATPase and caveolin-1. Consequently, it alters normal trafficking of caveolin-1 and cholesterol, and inhibits cell growth. Taken together, our new findings demonstrate that the highly conserved caveolin binding motif of the Na/K-ATPase plays an important role in regulation of cell growth by affecting the trafficking and distribution of caveolin-1 and cholesterol.
Advisors/Committee Members: Xie, Zijian.
Subjects: Biomedical Research; Pharmacology; Physiology
Keywords: Na/K-ATPase; Caveolin-1; Cholesterol; Cell growth
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25.
Dwyer, Trisha A.
Brain Hypometabolism and Seizures: The Dynamics of Hypoxia and Hypoglycemia in Brain Energy Homeostasis.
Degree: MS, College of Medicine, 2011, University of Toledo Health Science Campus
► Brain ischemia induces a metabolic insult that disrupts normal neuronal transmission and…
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▼ Brain ischemia induces a metabolic insult that disrupts normal neuronal transmission and leads to a prolonged hyperexcitable state. As a result of increased hyperexcitability, the brain may fire hypersychronous electrical discharges known as seizures. Seizures which become self-sustaining for 30 min or more, called status epilepticus (SE) are highly indicative of a poor prognosis. Thus, identifying how low oxygen and/or low glucose contribute to the generation of seizures may unveil mechanisms related to epileptogenesis, as well as provide a therapeutic target. Often, an imbalance in excitatory and inhibitory neurotransmission is associated with seizure development, which primarily involves glutamate and γ-aminobutyric acid (GABA) neurotransmission, respectively. GABARs are known for mediating the majority of fast inhibitory neurotransmission in the brain and play an integral role in the development of seizures. While GABARs are well-known for mediating anticonvulsant effects, their dysfunction during the development of seizures remains unclear. The lack of oxygen and/or low glucose may alter inhibition in the brain leading to an increased hyperexcitable state. In this thesis, we created a model of mild-to-moderate hypoxia/hypoglycemia that would allow investigation into the differential effects of hypoxia, hypoglycemia, or the combination on GABARs in the in vivo setting. We found that seizures were readily induced in rats when moderate doses of insulin were combined with or without oxygen. Rats treated with insulin alone (5U/kg) exhibited lethargy and developed myoclonic jerks, barrel rotations, and tonic-clonic seizures. No seizure activity was observed in rats treated with hypoxia (10% FiO2). Pulse oximetry data showed that rats treated with insulin had a decreased heart rate, while hypoxia and hypoxia/hypoglycemia treated rats displayed a decrease in peripheral oxygen saturation (SPO2). Future studies will be directed at assessing the differential effects of low oxygen and/or low glucose on GABARs, with a particular focus on the glycolysis-dependent modulation of GABAAR α1 subunit. A low energy state may disrupt the phosphorylation status of the α1 subunit, which could lead GABAR dysfunction and subsequent hyperexcitability. Although this mechanism bridges glycolysis with neuronal inhibition, whether glycolysis or particular glycolytic enzymes are absolutely necessary in maintaining normal neuronal excitability remains to be established.
Advisors/Committee Members: Greenfield, L. John.
Subjects: Biomedical Research; Neurosciences
Keywords: Ischemia; Hypoxia; Hypoglycemia; Seizures; Glyceraldehyde 3-phosphate dehydrogenase; GABA receptor; Glycolysis; Ketogenic diet; 2-deoxyglucose; Pentose phosphate pathway
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26.
Earl, Damien E.
Regulation of Neuronal L-type Voltage-Gated Calcium Channels by Flurazepam and Other Positive Allosteric GABAA Receptor Modulators.
Degree: PhD, College of Medicine, 2011, University of Toledo Health Science Campus
► Benzodiazpines (BZs) are clinically useful anxiolytics, sedatives, and anticonvulsants. Their mechanism of…
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▼ Benzodiazpines (BZs) are clinically useful anxiolytics, sedatives, and anticonvulsants. Their mechanism of action is positive allosteric modulation of γ-aminobutyric acid type A (GABAA) receptors, the main inhibitory neurotransmitter receptors in the mammalian central nervous system. Long-term administration of BZs and other positive allosteric GABAA receptor modulators, neurosteroids, barbiturates, and ethanol can lead to physical dependence manifested by a characteristic withdrawal syndrome. A common mechanism proposed to contribute to this withdrawal syndrome is functional up-regulation of L-type voltage-gated calcium channels (L-VGCCs). Our lab models BZ dependence using a 1-week oral treatment of rats with flurazepam (FZP) followed by 1 or 2 days of withdrawal. This treatment paradigm resulted in a near doubling of voltage-gated Ca2+ currents in hippocampal CA1 neurons. Enhanced L-VGCC-mediated Ca2+ influx may activate Ca2+/calmodulin-dependent protein kinase II (CaMKII), which potentiated excitatory synaptic function in CA1 neurons correlating with expression of FZP withdrawal anxiety. The current studies tested three hypotheses: 1) GABAA receptor modulators directly inhibit recombinantly expressed L-VGCCs containing neuronal α1 subunits, Cav1.2 or Cav1.3; 2) L-VGCC subunit expression is increased in the rat hippocampal CA1 region; and 3) CaMKII enhances CA1 excitatory synaptic function via activation and autophosphorylation at Thr286 and/or enhanced localization to the postsynaptic density (PSD). The findings suggested that while the barbiturate pentobarbital and ethanol directly inhibit L-VGCCs at clinically relevant concentrations, the concentrations of BZs and neurosteroids required to inhibit recombinant L-VGCCs were likely too high to be clinically relevant. Interestingly, Cav1.2 channels were more sensitive to inhibition by pentobarbital and FZP and were less sensitive to inhibition by the L-VGCC benzothiazepine (BTZ) antagonist, diltiazem, than Cav1.3 channels. Selective inhibition could independently block Ca2+ signaling cascades mediated by Cav1.2 and Cav1.3 L-VGCCs. Mutation studies revealed that the pentobarbital L-VGCC binding site may overlap that of dihydropyridines, and despite structural similarities amongst BTZs and BZs, the BZ L-VGCC binding site is distinct from that of BTZs. No alteration in L-VGCC subunit expression was observed in PSD-enriched CA1 homogenates or immunostained hippocampal slices as a function of FZP withdrawal. Taken together, the data suggested that mechanisms other than direct inhibition of L-VGCCs and increased L-VGCC subunit expression mediate the enhanced Ca2+ influx observed following long-term FZP treatment. Post-translational modifications and/or enhanced trafficking of L-VGCCs to the membrane due to persistent BZ enhancement of GABAA receptors are alternate possibilities. Additionally, after 2 days of FZP withdrawal, total CaMKIIα expression was decreased in CA1 PSDs with no alteration in the absolute amount of the autonomously active Thr286 autophosphorylated form of CaMKII. Alternate mechanisms of CaMKII activation by L-VGCC-mediated Ca2+ influx and for the loss of CaMKII from PSDs during FZP withdrawal are proposed.
Advisors/Committee Members: Tietz, Elizabeth.
Subjects: Neurosciences
Keywords: CNS depressants; benzodiazepines; GABA receptor; recombinant; calcium channels; CaMKII; electrophysiology; electron microscopy; neuronal plasticity; Western blot; Cav1.2; Cav1.3; hippocampus
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27.
Geng, Shuo.
Discovery of a New Dendritic Cell Subset Derived from Immature Granulocytes.
Degree: PhD, College of Medicine, 2011, University of Toledo Health Science Campus
► Compared to other leukocytes, dendritic cells (DCs) are extremely heterogeneous in terms…
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▼ Compared to other leukocytes, dendritic cells (DCs) are extremely heterogeneous in terms of developmental pathways and immunological properties. More than 10 DC subsets have been identified in mouse, which are distinguishable from each other by surface phenotypes, functions, tissue distributions, and developmental origins. Here we describe a novel subset of DCs that are derived from immature granulocytes, known as band cells. Large numbers of band cells are rapidly recruited to inflammatory sites, where some of them differentiate into this DC subset termed “grDCs.” In addition to showing a typical DC-like morphology, grDCs express many DC markers (MHC class II, CD11c, and CD205) and efficiently present peptide antigens to both CD8 and CD4 T cells. Importantly, grDCs retain the surface expression of Ly6G, which is not detectable on any of the currently known DC subsets, as well as several unique features of granulocytes. GeneChip analyses have revealed a cluster of genes that are selectively expressed by Ly6G+ grDCs, but not Ly6G- conventional DCs - this cluster includes cathelicidin (CRAMP), MMP9 and CD62L. CRAMP-deficient grDC exhibited a diminished bacterial killing activity, indicating a functional contribution of grDC-associated CRAMP. In a peritoneal E. coli infection model, grDC were found to internalize bacteria efficiently and present bacterial antigens to CD4 T cells. Not only have our data unveiled a previously iv unrecognized DC subset and its functions, they also suggest a new concept that granulocytes can directly participate in the induction of adaptive immunity via differentiation into grDCs.
Advisors/Committee Members: Takashima, Akira.
Subjects: Immunology
Keywords: leukocytes; grDCs; dendritic cells
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28.
Ghosh, Sumona.
A Novel Role for CEACAM1 in Hepatic Stellate Cell Activation in the Progression of Non-Alcoholic Steatohepatitis.
Degree: PhD, College of Medicine, 2012, University of Toledo Health Science Campus
► Hyperinsulinemia, a characteristic of insulin resistance, is a key factor in the…
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▼ Hyperinsulinemia, a characteristic of insulin resistance, is a key factor in the pathogenesis of a number of metabolic disorders such as obesity, fatty liver disease and type 2 diabetes mellitus. Regulation of both insulin secretion, from the beta cell, and insulin clearance, mainly by the liver, is vital to maintain insulin sensitivity. Insulin clearance is mainly mediated by Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1). Inactivation of CEACAM1 in the liver, or global null mutation of CEACAM1, decreases hepatic insulin clearance resulting in hyperinsulinemia and subsequent insulin resistance. High fat feeding for 30 days results in a >50% decrease in hepatic CEACAM1 expression and induces insulin resistance in C57BL/6 mice. Specific induction of CEACAM1 in the liver, driven by Apolipoprotein A1 promoter, protects against diet-induced insulin resistance by maintaining normal insulin clearance, even after 4 months of high fat feeding. This data promotes CEACAM1-dependent insulin clearance pathways as a molecular underpinning of metabolic diseases such as diet-induced obesity and non-alcoholic steatohepatitis (NASH). Long term high fat feeding induces features of NASH in Ceacam1 null mice: steatosis, inflammatory infiltration, oxidative stress and progressive fibrosis, thus implicating a role for CEACAM1 in the pathogenesis of NASH. Basal fibrosis observed in Ceacam1 null mice suggests a role for CEACAM1 in the initiation of fibrogenesis. Stable knockdown of CEACAM1 in cultured LX2 human stellate cells causes a state of activation including loss of lipid content, increased proliferation and expression of extracellular matrix components, generally observed in diseased liver. Together, the data provide an in vivo and in vitro demonstration of the critical role of CEACAM1 in the pathogenesis of metabolic diseases, in particular, obesity and NASH.
Advisors/Committee Members: Najjar, Sonia.
Subjects: Biomedical Research
Keywords: CEACAM1; insulin resistance; non-alcoholic steatohepatitis; fibrosis; hepatic stellate cell
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29.
Gonit, Mesfin.
The Androgen Receptor as a Transcriptional Co-activator: Implications in the Growth and Progression of Prostate Cancer.
Degree: PhD, College of Medicine, 2011, University of Toledo Health Science Campus
► Prostate cancer depends on the androgen receptor (AR) for growth and survival…
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▼ Prostate cancer depends on the androgen receptor (AR) for growth and survival even in the absence of androgen. In the classical models of gene activation by AR, ligand activated AR signals through binding to the androgen response elements (AREs) in the target gene promoter/enhancer. In the present study the role of AREs in the androgen-independent transcriptional signaling was investigated using LP50 cells, derived from parental LNCaP cells through extended passage in vitro. LP50 cells reflected the signature gene overexpression profile of advanced clinical prostate tumors. The growth of LP50 cells was profoundly dependent on nuclear localized AR but was independent of androgen. Nevertheless, in these cells AR was unable to bind to AREs in the absence of androgen. Gene expression profiling of LP50 cells showed that AR regulates two largely distinct gene expression programs, androgen-dependent and apo-AR dependent. Furthermore, a DNA binding domain mutant of AR which is unable to bind to ARE rescued androgen depletion insensitive proliferation and gene activation in LP50 cells depleted of endogenous wild type AR. Furthermore, ChIP-chip promoter tiling arrays revealed enrichment for AR in chromatin sites that are functional but lack ARE. To identify candidate transcription factors that tether AR to target genes in the absence of androgen, cis-elements of transcription factors in the AR interactome data set and AR chip peaks were used. We found direct interaction between AR and Elk-1 in both the C81 and C4-2 LNCaP variants of androgen depletion insensitive experimental model systems representing clinical advanced prostate cancer. AR dependent promoter activity of an Elk-1 driven promoter reporter construct and physical association between AR and Elk-1 by coimmunoprecipitation suggested that AR acts as a co-activator of Elk-1. Elk-1 was shown to be necessary for the proliferation of C81 and C4-2 cells. Expression profile studies further showed AR-dependent activation of gene clusters enriched for cell division function by Elk-1. This AR dependent gene regulation by Elk-1 was insensitive to androgen antagonist. The present study suggests that in advanced prostate cancer AR can support the progression of the tumor through ARE independent mechanisms by acting as a transcriptional co-activator for transcription factors such as Elk-1. This mechanism of action of AR is insensitive to hormone as well as antiandrogen. Hence, therapeutic strategies selectively targeting the interactions between AR and critical tethering proteins could be a novel approach for the management of advanced prostate cancer.
Advisors/Committee Members: Ratnam, Manohar.
Subjects: Biomedical Research
Keywords: Androgen receptor; Elk-1; prostate cancer; co-activator
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30.
Gordon, Ian R.
Feasibility Study of Intensity Modulation for Low Dose Rate External Beam Radiation Therapy.
Degree: MS, College of Medicine, 2010, University of Toledo Health Science Campus
► Previous studies have shown that low dose rate radiation therapy has radiobiological…
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▼ Previous studies have shown that low dose rate radiation therapy has radiobiological advantages for normal tissue. External irradiation techniques have been developed which use static blocked fields to irradiate a target which give a uniform dose rate over the area of the field but are unable to preferentially reduce dose to critical structures near the target. Intensity modulated radiation therapy techniques allow for the reduction in dose to critical structures but the standard technique of IMRT using a multi-leaf collimator has the disadvantage of not delivering a continuous dose rate across the area of the field being irradiated. Using compensator based IMRT compounds the benefits of IMRT with the benefits of low dose rate delivery and gives the patient a better treatment option than just one or the other individually. Because of the greater demands of IMRT on the linear accelerator a study of performance in the low dose rate ranges was carried out to determine if the accelerator was accurate enough to deliver this treatment with a non-standard range of machine parameters. The study entailed using a variety of dosimetry equipment to measure the basic beam parameter accuracy of the low dose rate techniques as compared to normal dose rate techniques and to ensure an accurate delivery of a low dose rate IMRT treatment. The outcome of this study showed that the machine does produce an accurate and dependable beam which allowed the measurement of a compensator based IMRT plan to be delivered as accurately as normal dose rate techniques.
Advisors/Committee Members: Parsai, E. Ishmael.
Subjects: Radiation
Keywords: LDREBRT
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