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Title: Neural differentiation of mesenchymal stem cells is dependent on the neuron restrictive silencer factor
Creator: Thompson, Ryan D.
Date: 2018
Collection: Electronic Theses & Dissertations
Description: Mesenchymal stem cells (MSCs) exist as an adult stem cell in major reservoirs primarily in the bone marrow and adipose tissue. Under normal physiologic conditions, MSCs serve mainly as the progenitor cell for adipocytes, chondrocytes, and osteocytes. The plasticity of MSCs has led researchers to investigate differentiation beyond their canonical lineages and since, in vitro studies have shown that MSCs can be induced to differentiate into renal cells, beta/islet cells, hepatocytes,...
Show moreMesenchymal stem cells (MSCs) exist as an adult stem cell in major reservoirs primarily in the bone marrow and adipose tissue. Under normal physiologic conditions, MSCs serve mainly as the progenitor cell for adipocytes, chondrocytes, and osteocytes. The plasticity of MSCs has led researchers to investigate differentiation beyond their canonical lineages and since, in vitro studies have shown that MSCs can be induced to differentiate into renal cells, beta/islet cells, hepatocytes, cardiomyocytes, and even neurons. Differentiated MSCs exhibit changes in gene marker expression, morphology, and even gain functional characteristics. Previously, our lab has shown that neural-like characteristics can be induced in MSCs by exposure to the cyclic adenosine monophosphate (cAMP) elevating compounds, forskolin and isobutylmethylxanthine (IBMX). In addition to short-term neural-like morphology changes, MSCs gain expression of neural markers as well as sensitivity to dopamine. However, a molecular mechanism to explain why cAMP elevating compounds would have a proneural effect in MSCs is lacking. Differentiation of stem cells into a mature phenotype is strongly driven by transcription factors within a cell. Some transcription factors control regulation of so many genes required for the mature differentiated cell type that they are termed master transcriptional regulators. For example, during osteogenesis, the master transcriptional regulator Runx2 is essential for differentiation of MSCs to osteocytes. Yang et al. demonstrated that silencing the master transcriptional regulator, NRSF, in MSCs could induce several neural characteristics. Therefore, I hypothesized and went on to show that forskolin and IBMX could be driving neural-like differentiation of MSCs by regulating NRSF. Neural differentiation of MSCs has also been studied from a tissue engineering perspective. In particular, it has been demonstrated in several types of stem cells that culture on very soft substrates can promote neural differentiation. This phenomenon shows that stem cell differentiation can also be influenced by physical characteristics in its environment. However, the molecular mechanisms explaining how cells can sense and respond to soft surfaces to affect differentiation are still vaguely characterized. We hypothesized that since soft surfaces induce neural-like differentiation in stem cells that maybe soft surfaces were somehow affecting NRSF. We go on to show that soft PDMS somehow affects NRSF within MSCs and that this is the main driver of neural-like differentiation from soft surfaces. The aims of both projects show that neural differentiation in MSCs can be induced by both small molecules and the physical environment. Seemingly disparate stimuli are connected due to their ability to downregulate expression of NRSF. These studies highlight the role of transcription factors in determining stem cell fate and show that their modulation can even transdifferentiate cells across their germ line barriers.
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Title: AUTOMATED PET/CT REGISTRATION FOR ACCURATE RECONSTRUCTION OF PET IMAGES
Creator: Khurshid, Khawar
Date: 2018
Collection: Electronic Theses & Dissertations
Description: The use of a CT attenuation correction (CTAC) map for the reconstruction of PET image can introduce attenuation artifacts due to the potential misregistration between the PET and CT data. This misregistration is mainly caused by patient motion and physiological movement of organs during the acquisition of the PET and CT scans. In cardiac exams, the motion of the patient may not be significant but the diaphragm movement during the respiratory cycle can displace the heart by up to 2 cm along...
Show moreThe use of a CT attenuation correction (CTAC) map for the reconstruction of PET image can introduce attenuation artifacts due to the potential misregistration between the PET and CT data. This misregistration is mainly caused by patient motion and physiological movement of organs during the acquisition of the PET and CT scans. In cardiac exams, the motion of the patient may not be significant but the diaphragm movement during the respiratory cycle can displace the heart by up to 2 cm along the long axis of the body. This shift can project the PET heart onto the lungs in the CT image, thereby producing an underestimated value for the attenuation. In brain studies, patients undergoing a PET scan are often not able to follow instructions to keep their head in a still position, resulting in misregistered PET and CT image datasets. The head movement is quite significant in many cases despite the use of head restraints. This misaligns the PET and CT data, thus creating an erroneous CT attenuation correction map. In such cases, bone or air attenuation coefficients may be projected onto the brain which causes an overestimation or an underestimation of the resulting CTAC values. To avoid misregistration artifacts and potential diagnostic misinterpretation, automated software for PET/CT registration has been developed that works for both cardiac and brain datasets. This software segments the PET and CT data, extracts the brain or the heart surface information from both datasets, and compensates for the translational and rotational misalignment between the two scans. The PET data are reconstructed using the aligned CTAC, and the results are analyzed and compared with the original dataset. This procedure has been evaluated on 100 cardiac and brain PET/CT data sets, and the results show that the artifacts due to the misregistration between the two modalities are eliminated after the PET and CT images are aligned.
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Title: Novel role of intracellular triacylglycerol in lifespan regulation in Saccharomyces cerevisiae
Creator: Handee, Witawas
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Triacylglycerol (TAG) is one of the fundamental molecules of life that exists in all living organisms. TAG is a non-polar lipid which has a role in controlling cellular energy stockpiles, lipid homeostasis, and cellular signaling. Conventional thought holds that cells accumulate TAG only when they consume excess energy, but a growing body of research reveals that TAG accumulation also takes place when cells are exposed to stresses. This suggests a potential life-preserving role for TAG. Our...
Show moreTriacylglycerol (TAG) is one of the fundamental molecules of life that exists in all living organisms. TAG is a non-polar lipid which has a role in controlling cellular energy stockpiles, lipid homeostasis, and cellular signaling. Conventional thought holds that cells accumulate TAG only when they consume excess energy, but a growing body of research reveals that TAG accumulation also takes place when cells are exposed to stresses. This suggests a potential life-preserving role for TAG. Our studies have discovered that high accumulation of intracellular TAG in budding yeast, S. cerevisiae, correlates with extended chronological lifespan. In general, wild yeast exhibit larger volumes of TAG and significantly longer lifespans than laboratory yeast. Additionally, increasing intracellular TAG levels in laboratory strains by either abolishing TAG lipases or enhancing TAG biosynthesis results in lifespan extension, whereas blocking TAG production leads to premature aging.The TAG-associated lifespan mechanism is unlikely dependent on several well- characterized longevity pathways such as the Target-of-Rapamysin (TOR) and the RAS/ PKA pathways. While TAG accumulation does not protect cells from stresses such as UV light, osmolarity, and acidity, our data suggest that high-TAG yeast may benefit fromdecreased reactive oxygen species (ROS) or increased resistance to oxidative damages. In order to quantitatively measure ROS level in yeast cells, we have developed a normalization technique that is applicable to aging studies via using the fluorescent probe, 2’,7' -dichlorofluorescein diacetate. The results demonstrate that yeast with high TAG abundance display lower intracellular ROS than that of wild-type yeast. On the other hand, yeast with low TAG exhibit significantly increased ROS and oxidative damages. These results support the mitochondrial free radical theory of aging which suggests that longevity can be extended by reducing cell injury from ROS. Subsequently, we offer the radical sink hypothesis proposing a TAG-mediated longevity mechanism by which TAG can intercept ROS before they impair other vital macromolecules and then safely transfer to lipid droplets.Because TAG metabolism and most lifespan regulations are highly conserved from yeast to human, it is possible that this lifespan regulation can universally apply to other organisms. It is important to note that these results are consistent with an observation in modern societies called obesity paradox, a phenomenon by which overweight individuals benefit from lower overall mortality rate than normal or underweight people. Our findings thus demonstrate an overlooked function of TAG that could potentially readjust future healthcare guidelines in order to increase greater a healthy life of the advanced age.
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Title: Understanding the protein-protein interactions of heme a synthase and their implications for cytochrome c oxidase assembly
Creator: Herwaldt, Emily
Date: 2014
Collection: Electronic Theses & Dissertations
Description: ABSTRACTTHE PROTEIN-PROTEIN INTERACTIONS OF HEME A SYNTHASEByEmily HerwaldtHeme a is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. The heme a molecule is synthesized from heme o within the mitochondria by a multi-spanning inner membrane protein, heme a synthase (Cox15 in yeast). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. In an effort to...
Show moreABSTRACTTHE PROTEIN-PROTEIN INTERACTIONS OF HEME A SYNTHASEByEmily HerwaldtHeme a is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. The heme a molecule is synthesized from heme o within the mitochondria by a multi-spanning inner membrane protein, heme a synthase (Cox15 in yeast). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. In an effort to increase our understanding of heme a insertion into cytochrome c oxidase, we investigated the protein-protein interactions that occur with Cox15 in Saccharomyces cerevisiae. Cox15 in S. cerevisiae exists in six protein complexes ranging in size from ~120 kDa - 1 MDa as observed via blue native PAGE (BN-PAGE). The two largest complexes at approximately 750 kDa and 1 MDa are reminiscent of the respiratory supercomplexes containing both complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). The large 750 kDa and 1 MDa Cox15 complexes were not observed in yeast strains in which the supercomplexes are unable to form, thus supporting the hypothesis that Cox15 is present in the respiratory supercomplexes. In addition, Cox15 was found to interact with one of the catalytic subunits of the cytochrome bc1 complex, Cyt1, and we propose that Cox15 and Cyt1 interact within the supercomplexes. No other proteins from the cytochrome bc1 complex or cytochrome c oxidase were found to interact with Cox15, although if Cox15 is present in the respiratory supercomplexes, by definition, it would seem that Cox15 must also interact (at least indirectly) with the other components of the respiratory supercomplexes. Of the lower four Cox15-containing complexes ranging from ~120 - 440 kDa, the complex at 120 kDa was the most prominent, indicating that the majority of the Cox15 observed by BN-PAGE is represented by this species. Although 120 kDa is ~1.5 times larger in molecular weight than monomeric C-terminal tagged Cox15, we were unable to identify other proteins that interact with Cox15 in this 120 kDa band. Because it is accepted that molecular weights of proteins are over-estimated via BN-PAGE due to the effect of detergent, we hypothesize that this lowest complex represents monomeric Cox15. Experiments to test the composition of the remaining Cox15-containing complexes revealed that approximately 30% of Cox15 interacts with itself in homo-oligomeric complexes. In addition, experiments to test if other proteins interacted with Cox15 revealed that cytochrome c oxidase assembly factors may exist with Cox15 in one of the Cox15-containing complexes. It does not appear, however, that assembly factors of cytochrome c oxidase represent predominant protein interactions with Cox15. Finally, Cox15 was shown to interact with the cytosolic heat shock proteins, Ssa1 and Hsc82. Deletions of Ssa1 and Hsc82, however, indicated that these proteins are not part of the Cox15-containing complexes observed via BN-PAGE. Based on previous studies implicating cytosolic heat shock proteins in mitochondrial protein uptake, we predict that Ssa1 and Hsc82 are involved in the import of Cox15 into the mitochondria.
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Title: Roles of the mitochondrinal single-stranded DNA-binding protein at the mitochondrial DNA replication fork
Creator: Oliveira, Marcos Túlio
Date: 2011
Collection: Electronic Theses & Dissertations
Description: The mitochondrion is one of the most important and versatile eukaryotic organelles, responsible for the bulk of energy production in the cell, and involved in apoptosis, signaling, cellular differentiation, and control of cell cycle and cell growth. It possesses its own genome, the mitochondrial DNA (mtDNA), which encodes polypeptides that are essential subunits of the complexes that form the energy-producing respiratory chain. mtDNA replication is thus an important process that maintains...
Show moreThe mitochondrion is one of the most important and versatile eukaryotic organelles, responsible for the bulk of energy production in the cell, and involved in apoptosis, signaling, cellular differentiation, and control of cell cycle and cell growth. It possesses its own genome, the mitochondrial DNA (mtDNA), which encodes polypeptides that are essential subunits of the complexes that form the energy-producing respiratory chain. mtDNA replication is thus an important process that maintains proper mitochondrial function, accomplished by the coordinated action of three main protein components that work directly at the mtDNA replication fork: DNA polymerase gamma (pol gamma), which catalyzes DNA synthesis per se; mtDNA helicase (also known as Twinkle), which unwinds double-stranded DNA to provide a single-stranded DNA (ssDNA) substrate for pol gamma; and mitochondrial single-stranded DNA-binding protein (mtSSB), which binds ssDNA to protect it against damage and to coordinate the functions of pol gamma and mtDNA helicase. The general aim of my thesis work was to examine the roles of mtSSB at the mtDNA replication fork by investigating the biochemical and physiological performance of a group of mtSSB variants bearing alanine substitutions or deletions of amino acid residues conserved across animal species. We purified 9 recombinant human mtSSB (HsmtSSB) proteins, which maintained their homotetrameric state and bound ssDNA with only slightly different affinities. However, they exhibited very distinct capacities to stimulate the DNA polymerase activity of human pol gamma (Hspol gamma) and the DNA unwinding activity of human mtDNA helicase (HsmtDNA helicase) in vitro. Whereas the variants HsmtSSBΔN (Δ1-9), ΔC (Δ126-132) and ΔNΔC (Δ1-9, Δ126-132) stimulated Hspol gamma ~2-fold higher than HsmtSSBwt (wild type), the variants HsmtSSBloop23 (Δ51-59), α1 (Y83A/Q84A) and loop45-1 (Y100A/G101A/E102A) exhibited an ~40% reduction as compared to HsmtSSBwt. We developed a molecular model of mtSSB-pol gamma interaction that explains these and other biochemical data published previously, and that suggests how a group of Hspol gamma mutations associated with various human diseases may disturb such interactions. Interestingly, the variants HsmtSSBloop12 (E33A/G34A/K35A) and loop45-2 (K106A/N107A/N108A), which did not exhibit altered capacity to stimulate Hspol gamma, were indeed defective in stimulating HsmtDNA helicase, presumably by failing to interact with its C-terminal tail. We also evaluated mtSSB in Drosophila S2 cells by knocking down the endogenous protein (DmmtSSB) and expressing variants of DmmtSSB equivalent to those of HsmtSSB. Endogenous DmmtSSB knockdown and overexpression of DmmtSSB variants caused reduction of mtDNA copy number under conditions of mitochondrial homeostasis, and impeded mtDNA repletion during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Preliminary analysis of mtDNA replication intermediates from cells overexpressing DmmtSSB variants indicated that the defects in mtDNA replication are associated with the binding sites of the transcription termination factor DmTTF. Our findings suggest that mtSSB uses a repertoire of structural elements to interact functionally with pol gamma and mtDNA helicase, to guarantee proper mtDNA replication in animal cells. Understanding the mechanisms of mtDNA replication via further biochemical, physiological and structural studies will provide valuable insights into the processes in which the mitochondrion is the key regulator in eukaryotic cells.
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Title: PERTURBATION OF ASTROCYTIC KEAP1-NRF2-ARE PATHWAY AND GLUTAMATE TRANSPORTER EXPRESSION IN SPINAL ASTROCYTIC DEGENERATION
Creator: Wiwatratana, Duanghathai
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Several cellular mechanisms are known to be involved in methylmercury (MeHg) induced central nervous system (CNS) toxicity, including the dysregulation of intracellular Ca2+, redox, and glutamate homeostasis. However, the factors that make particular neurons susceptible to MeHg toxicity, and the latency period of neurological signs and symptoms, have not yet been clearly delineated. For example, the spinal dorsal root ganglia (DRG) is the primary target of MeHg. Mercury (Hg) granules are...
Show moreSeveral cellular mechanisms are known to be involved in methylmercury (MeHg) induced central nervous system (CNS) toxicity, including the dysregulation of intracellular Ca2+, redox, and glutamate homeostasis. However, the factors that make particular neurons susceptible to MeHg toxicity, and the latency period of neurological signs and symptoms, have not yet been clearly delineated. For example, the spinal dorsal root ganglia (DRG) is the primary target of MeHg. Mercury (Hg) granules are first detected in spinal cord motor neurons (SMNs) in the non-symptomatic phase, whereas Hg granules are detected in glia later, following with neurological symptoms (Møller-Madsen, 1991). This finding suggested that the latent period (non-symptomatic phase) is associated with Hg accumulation in neurons, while the symptomatic phase occurs following Hg accumulation in glia, and the susceptibility is not associated with Hg granule accumulation in cells (Møller-Madsen, 1991). Astrocytes generally provide glutathione (GSH) for neurons to detoxify toxic insult. In the spinal cord, MeHg might perturb the antioxidant pathway, Keap1-Nrf2-ARE pathway in the spinal cord astrocytes (SCAs) consequently contribute to DRG or SMN susceptibility to MeHg toxicity. In this study, the comparative responses of different SCAs maturity to a non-toxic MeHg concentration (0.1 μM) suggested that the fully mature SCAs (Day in vitro 30; DIV30), were more susceptible to MeHg than SCAs on DIV14. The perturbation of the Keap1-Nrf2-ARE pathway in SCAs (DIV 30) during exposure to sub-toxic MeHg concentration (0.50 μM) caused a biphasic increase in antioxidant genes such as Keap1, Nrf2, Gclc, Abcc1 mRNAs expression. The concomitant increase of glutamate transporter Slc7a11 encoded for the system Xc-, and Slc1a3 encoded for EAAT1, and Slc1a2 encoded for EAAT2 expression during MeHg exposure might suggest the cooperative expression or function of these glutamate transporters. This concomitant expression was further demonstrated in studies using Nrf2-knockout (Nrf2-KO) derived SCAs. The increase of basal Slc7a11 mRNA, was concurrent to the increase of basal Slc1a3 and Slc1a2 mRNA expressions in Nrf2-KO derived SCA. The function of time of MeHg exposure indicated that Nrf2-KO derived SCAs were more susceptible to MeHg than the wild-type (WT)-derived SCAs. The pronounced susceptibility of Nrf2-KO derived SCAs was mainly due to the loss of GSH) metabolism and transport genes Gclc, GPx1, GPx4, and Abcc1 mRNAs in this genotype. MeHg significantly reduced these mRNA expressions in both genotypes. However, not all Nrf2-ARE regulated genes were affected by MeHg in similar ways in these genotypes. For example, MeHg induced the increase of Slc7a11 mRNA expression in WT-derived SCAs, but it appears to cause the reduction of this mRNA expression in Nrf2 KO-derived SCAs. Administration of antioxidant N-acetyl-L-cystine (NAC) in pretreatment (NP), co-treatment (CO), and post-treatment of MeHg (MP) prevented the reduction of SCAs metabolic functions for over 160h. The mechanism of NAC action in preventing MeHg induced SCAs degeneration is primarily due to its thiol antioxidant property.In conclusion, this study suggests that age and genetic predisposition contribute to SCAs susceptibility to MeHg toxicity. The dysregulation of the antioxidant pathways and glutamate homeostasis in SCAs potentially contributes to SMNs or DRG susceptible to MeHg.
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Title: The right size : the hormonal and transcriptional regulation of growth in Drosophila melanogaster
Creator: Gokhale, Rewatee Hemant
Date: 2017
Collection: Electronic Theses & Dissertations
Description: Regulation of final organ size is a complex developmental process involving the integration of systemic, organ-specific and environmentally regulated processes. Together these processes enable co-ordination of organ growth with body growth and the achievement of correct organ size. Dysregulation in these processes causes over- or under growth of organs resulting in compromised organ function. Understanding the mechanisms contributing to regulation of organ size is therefore key to...
Show moreRegulation of final organ size is a complex developmental process involving the integration of systemic, organ-specific and environmentally regulated processes. Together these processes enable co-ordination of organ growth with body growth and the achievement of correct organ size. Dysregulation in these processes causes over- or under growth of organs resulting in compromised organ function. Understanding the mechanisms contributing to regulation of organ size is therefore key to understanding organ function. My research has focused on understanding two distinct aspects of developmental growth control. Firstly, what role does systemic hormonal signaling play in regulating final organ size? Second, what is the role of transcriptional regulation of the Insulin Receptor (InR) gene in regulating final organ size? I have addressed these questions through a variety of genetic and biochemical tools in the model system of the common fruit fly Drosophila melanogaster. In order to understand the role of hormonal signaling, I used the model of the wing imaginal disc to show that growth retardation in one part of the imaginal disc results in coordinated reduction in growth rate in the unperturbed part. Further, I show that this is mediated through systemic signaling by the insect hormone 20-hydroxyecdysone. Lastly, I demonstrate that systemic ecdysone signaling interacts with organ-autonomous insulin signaling to mediate growth coordination across the imaginal disc. To understand the role of transcriptional regulation of InR, I generated transgenic flies with increased dosage of InR. Using this transgenic fly line, I identified the minimal cis-regulatory region of InR and the effects of increased dosage of InR on aspects of fly physiology. I further show that the InR cis-regulatory region consists of multiple enhancers, which are capable of driving tissue-specific reporter gene expression. Lastly, I outline a strategy to test the role of modified transcriptional feedback to InR by the transcription factor dFoxo. Together, my results would be of broad interest to developmental biologists and contribute to our understanding of the mechanisms organ and body size regulation.
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Title: Dissecting the role of mixed lineage kinase 3 (MLK3) in breast cancer invasion and metastasis
Creator: Rattanasinchai, Chotirat
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "MLK3 is a mitogen-activated protein kinase kinase kinase (MAP3K) protein which can activate multiple MAPK pathways. MLK3 has been recently shown to be critical for breast cancer metastasis. In this thesis, the mechanisms through which MLK3 signaling controls discrete steps of cancer invasion and metastasis have been investigated. MLK3 was found to regulate the expression of FOS-related antigen-1 (FRA-1), a member of activator protein-1 (AP1) transcription factor family. Overexpression of...
Show more"MLK3 is a mitogen-activated protein kinase kinase kinase (MAP3K) protein which can activate multiple MAPK pathways. MLK3 has been recently shown to be critical for breast cancer metastasis. In this thesis, the mechanisms through which MLK3 signaling controls discrete steps of cancer invasion and metastasis have been investigated. MLK3 was found to regulate the expression of FOS-related antigen-1 (FRA-1), a member of activator protein-1 (AP1) transcription factor family. Overexpression of catalytically active MLK3, but not catalytically inactive MLK3, induced expression of FRA-1, suggesting that MLK3 utilizes its kinase activity rather than scaffolding function to control FRA-1 levels. In addition, MLK3 overexpression was sufficient to drive cancer cell migration in non-invasive, estrogen receptor positive (ER+) breast cancer cells. Conversely, deletion of MLK3 in highly metastatic, triple-negative breast cancer (TNBC) cells decreased FRA-1 expression at both the transcript and protein levels. Similarly, FRA-1 level was reduced upon treatment of TNBC cells with CEP-1347 or URMC-099, two structurally distinct, adenosine triphosphate (ATP)-competitive, MLK inhibitors. These data, together with the findings in ER+ breast cancer cells, indicate that MLK3 kinase activity is required for FRA-1 expression. MLK3 is capable of activating several MAPK pathways. Experiments utilizing small molecule inhibitors targeting specific MAPK pathways identified JNK and ERK pathways as mediators for MLK3-induced FRA-1 expression. FRA-1 is an oncogenic transcription factor controlling a subset of invasion genes. The work described in this thesis demonstrates a role for MLK3 in regulating matrix metalloproteinase (MMP) levels in breast cancer cells. In ER+ breast cancer cells, MLK3 overexpression induced expression of MMP-1 and MMP-9, two well-characterized, FRA-1 target genes. Silencing of FRA-1 in MLK3-overexpressing ER+ breast cancer cells attenuated MMP-1 and MMP-9 expression indicating that FRA-1 is a required intermediary in MLK3- induced MMP regulation. Furthermore, MLK3 deletion or MLK inhibitor treatment was sufficient to decrease both MMP-1 and MMP-9 levels in TNBC cells. MMPs are known to facilitate cancer invasion. Overexpression of MLK3 in ER+ breast cancer cells, which induces both MMP-1 and MMP-9, enhanced the ability of these cells to invade through a thin layer of Matrigel and this invasion was attenuated upon FRA-1 silencing. Moreover, MLK3 deletion or MLK inhibitor treatment also blocked Matrigel invasion of highly invasive TNBC cells. Consistent with a role for MMP-1 in vascular intravasation, deletion of MLK3 which downregulates MMP-1 expression rendered TNBC cells defective in both endothelial permeability and transendothelial migration. Upregulation of FRA-1 and MMP-1 was observed in circulating tumor cells (CTCs) derived from TNBC-bearing mice when compared with parental TNBC cells, further supporting the role of MLK3/ MMP-1 axis in vascular intravasation. High levels of MMP-1 in breast cancer patients are strongly associated with poor prognosis, including increased distant metastases, shortened recurrence, and poorer overall survival. This investigation has deciphered key components of the MLK3 signaling pathways that control the transcription factor FRA-1 and MMP target genes during cancer invasion and intravasation. These studies support the idea that targeting MLK3 is a promising therapeutic strategy for combating TNBC metastasis."--Pages ii-iii.
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Title: Stability, activity and genome-wide association of the Drosophila Retinoblastoma tumor suppressor Rbf1
Creator: Acharya, Pankaj
Date: 2011
Collection: Electronic Theses & Dissertations
Description: The Retinoblastoma protein (RB) is a well known tumor suppressor that controls cell cycle and developmentally regulated gene expression. Germ-line mutation of RB is closely linked with retinoblastoma in early childhood and osteosarcoma in adolescence, and is mutated in about half of all human cancers. During normal growth, interactions between E2Fs and RB family proteins, including the Drosophila RB homolog, Rbf1 are regulated by phosphorylation by cyclins and cyclin-dependent kinases (CDKs)...
Show moreThe Retinoblastoma protein (RB) is a well known tumor suppressor that controls cell cycle and developmentally regulated gene expression. Germ-line mutation of RB is closely linked with retinoblastoma in early childhood and osteosarcoma in adolescence, and is mutated in about half of all human cancers. During normal growth, interactions between E2Fs and RB family proteins, including the Drosophila RB homolog, Rbf1 are regulated by phosphorylation by cyclins and cyclin-dependent kinases (CDKs) and proteolytic destruction by the proteasome. To better understand the mechanism for RB family protein instability, we characterized Rbf1 turnover in Drosophila, and the protein motifs required for its destabilization. We show that specific point mutations in a C-terminal instability element (IE) stabilized Rbf1, but sacrifice repression activity. Rbf1 is destabilized especially in actively proliferating tissues of the larva, indicating that controlled degradation of Rbf1 is linked to developmental signals. The positive linkage between Rbf1 activity and its destruction indicates that the instability and activity relation is similar to that observed in the case of transcriptional activators such as VP16 and Myc. Physical and functional targets of RB and its paralogs p107/p130 have been studied largely in cultured cells, but the full biological context of this family of proteins' activities will likely be revealed only in whole organismal studies. To identify direct targets of the major Drosophila RB counterpart in a developmental context, we carried out ChIP-Seq analysis of Rbf1 in the embryo. The association of the protein with promoters is developmentally controlled; early promoter access is globally inhibited, while later in development Rbf1 was found to associate with promoter-proximal regions of approximately 2,000 genes. In addition to the conserved cell cycle-related genes, a wholly unexpected finding was that Rbf1 targets many components of the insulin, Hippo, JAK/STAT, Notch and other conserved signaling pathways. Rbf1 may thus directly affect output of these essential growth-control and differentiation pathways by regulation of receptor, kinase and downstream effector expression. Rbf1 was also found to target multiple levels of its own regulatory hierarchy. Bioinformatic analysis indicates that different classes of bound genes exhibit distinct promoter motifs, suggesting that the context of Rbf1 recruitment involves diverse transcription factors, which may allow for independent regulation of Rbf1 bound genes. Many of these targeted genes are bound by Rbf1 homologs in human cells, indicating that a conserved role of retinoblastoma proteins may be to adjust the set point of interlinked signaling networks essential for growth and development.
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Title: Effects of methylmercury in the dopamine synthesizing pheochromocytoma PC12 cell line
Creator: Tiernan, Chelsea Tate
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Methylmercury (MeHg) is a potent bioaccumulative neurotoxicant that targets discrete neuronal populations, including the nigrostriatal dopamine (NSDA) neuronal system. Epidemiological evidence has implicated chronic exposure to MeHg as an environmental risk factor for Parkinson disease (PD), and experimental analyses using in vivo animal and in vitro cell culture models have demonstrated that acute exposure to MeHg alters DA homeostasis, including release, reuptake, and metabolism. The...
Show moreMethylmercury (MeHg) is a potent bioaccumulative neurotoxicant that targets discrete neuronal populations, including the nigrostriatal dopamine (NSDA) neuronal system. Epidemiological evidence has implicated chronic exposure to MeHg as an environmental risk factor for Parkinson disease (PD), and experimental analyses using in vivo animal and in vitro cell culture models have demonstrated that acute exposure to MeHg alters DA homeostasis, including release, reuptake, and metabolism. The purpose of the studies described in this dissertation is to characterize MeHg-induced changes in DA synthesis, release, reuptake, and metabolism, and to investigate mechanisms by which MeHg exerts its neurotoxic effects in the pheochromotocytoma (PC12) cell line. MeHg causes a concentration- and time-dependent increase in DA release. Higher concentrations (5μM) are associated with an increased incidence of cell death at later time points (60-120 min). However 2μM MeHg induces DA release by 60 min without altering cell viability. Thus this concentration and time-point was selected to examine other indices of DA homeostasis. MeHg-induced DA release is abolished by inhibition of vesicular exocytosis with reserpine, but not inhibition of membrane transport with desipramine. A role for synthesis in MeHg-induced DA release was indicated by an increase in the concentration of intracellular DA and the rate of decline of intracellular DA following acute treatment with the tyrosine hydroxylase (TH) inhibitor α-methyltyrosine (AMT). MeHg stimulates DA synthesis indicated by an increase in DOPA accumulation following treatment with the DOPA decarboxylase inhibitor NSD-1015. This is supported by the observation that MeHg elevates phosphorylation of TH at serine reside 40, without altering the total amount of TH. Moreover, MeHg-induced DA release is dependent upon DA synthesis because pre-treatment with AMT abolishes MeHg-induced DA release. MeHg induces aberrant DA metabolism. Intracellular concentrations of DOPAC are decreased, while intracellular concentrations of the intermediate metabolites DOPAL and DOPET are increased. This metabolomic profile suggests that MeHg inhibits the oxidation of DOPAL to DOPAC and thus inhibits aldehyde dehydrogenase (ALDH). MeHg does not directly impair ALDH activity. Instead, inhibition may be indirect because MeHg inhibits mitochondrial respiration and ATP synthesis, and decreases availability of the ALDH cofactor nicotinamide adenine dinucleotide (NAD). To assess the roles of extracellular and intracellular calcium (Ca2+) in altered DA release and metabolism, undifferentiated PC12 cells were exposed to MeHg in both the absence and presence of extracellular and/or intracellular Ca2+. Removal of intracellular but not extracellular Ca2+ attenuates MeHg-induced DA release. MeHg-impaired DA metabolism is not influenced by chelation of either Ca2+ source. The present findings are consistent with the follow conclusions: 1) MeHg-induced DA release is dependent upon DA synthesis and vesicular exocytosis, 2) MeHg impairs DA metabolism by indirectly inhibiting ALDH, 3) release of Ca2+ from intracellular stores triggers MeHg-induced DA release, and 4) aberrant DA metabolism is not affected by changes in intracellular Ca2+ homeostasis.
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Title: Pathology at the neuromuscular junction in mouse models of spinal bulbar muscular atrophy
Creator: Poort, Jessica Erin
Date: 2014
Collection: Electronic Theses & Dissertations
Description: Spinal bulbar muscular atrophy (SBMA) is a progressive, late onset neuromuscular disease that results in muscle weakness and atrophy, as well as motoneuron death in men. While pathology at the neuromuscular junction (NMJ) is noted in numerous neurodegenerative diseases, disease-related changes at the NMJ in SBMA have not been explored. Characterizing such changes is not only important for determining whether the NMJ has any role in the functional changes underlying motor dysfunction, but also...
Show moreSpinal bulbar muscular atrophy (SBMA) is a progressive, late onset neuromuscular disease that results in muscle weakness and atrophy, as well as motoneuron death in men. While pathology at the neuromuscular junction (NMJ) is noted in numerous neurodegenerative diseases, disease-related changes at the NMJ in SBMA have not been explored. Characterizing such changes is not only important for determining whether the NMJ has any role in the functional changes underlying motor dysfunction, but also in determining how such potential pathology at the NMJ develops as disease progresses. If for example, pathology emerges first at the NMJ followed by motoneuron death, then the NMJ offers future promise as a therapeutic target for preventing or reversing symptoms of SBMA before motoneurons are lost. We evaluated three different mouse models of SBMA, one overexpressing a wildtype androgen receptor (AR) exclusively in muscle fibers (so called "myogenic" model), a second which expressed the endogenous AR gene with the first exon of the human mutant AR gene "knocked in" (the so called "knock-in" model), and a final model that broadly expresses a full length human AR transgene harboring the SBMA mutation (the so called "97Q" model). Using both confocal microscopy and electron microscopy, I found that all three mouse models show a pathological fragmentation of the NMJ suggestive of functionally weakened synapses. Other changes at the neuromuscular synapse suggesting decreases in synaptic strength that were found in some but not all models include a decline in the number of docked vesicles ready for release in nerve terminals, a widening of synaptic clefts, simplified postsynaptic folds, and an abnormal accumulation of synaptic vesicle and neurofilament proteins. Retrograde axonal transport of endosomes was also characterized in the 97Q model using live imaging confocal microscopy. Despite previously published data, I found no evidence for a disease-related defect in retrograde transport in the 97Q model. The strikingly abnormal morphology of NMJs in all three models raises the possibility that synaptic function is impaired. Such synaptic dysfunction may contribute to or underlie the impairments in motor function associated with SBMA.
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