The first study examined the interaction between low doses of the multi-kinase inhibitor sorafenib and the histone deacetylase inhibitor vorinostat in colon cancer cells. Sorafenib and vorinostat synergized to kill HCT116 and SW480 cells. In SW480 cells, sorafenib+vorinostat toxicity correlated with CD95 activation and CD95-stimulated autophagy. Drug lethality in SW480 cells was blocked by knock down of CD95. In SW620 cells that are patient matched to SW480 cells, sorafenib+vorinostat toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and tumor cell killing, whereas knock down of LASS6 in SW480 cells suppressed CD95 activation. In HCT116 cells, sorafenib+vorinostat did not increase CD95 plasma membrane levels, weakly induced caspase 8 association with CD95, and knock down of CD95 enhanced drug lethality. In HCT116 cells sorafenib+vorinostat treatment caused CD95-dependent autophagy that was a protective signal. Thus, treatment of tumor cells with sorafenib+vorinostat activates CD95 that promotes viability via autophagy or degrades survival via extrinsic or intrinsic pathways. Drug-induced activation of the de novo ceramide synthesis pathway plays a key role in CD95 activation. The second project explores the mechanism by which the combination of 17AAG, an hsp90 inhibitor, and PD184352, a MEK1/2 inhibitor alters survival in colon cancer cells. 17AAG and PD184352 synergized to kill HCT116 and SW480 cells. In HCT116 cells drug-exposure increased CD95 plasma membrane levels In SW620 cells, 17AAG and PD184352 toxicity was significantly reduced that correlated with a lack of CD95 activation and lower expression LASS6. Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and tumor cell killing. In Mia Paca2 cells, a pancreatic cell line, inhibition of caspase 8 or overexpression of c-FLIP-s suppressed cell killing by PD184352 and 17AAG exposure. Drug lethality in Mia Paca2 cells was blocked by knock down of CD95. Additionally, overexpression of Bcl-xL or knockdown of caspase 9 decreased cell killing in 17AAG and PD184352 combination treatment. Thus, 17AAG+PD184352 exposure activates the extrinsic and intrinsic apoptotic pathways to kill Mia Paca2 cells. This document was created in Microsoft Word 2000.

In order for oligodendrocyte progenitor cells (OPCs) to differentiate into fully mature, myelinating oligodendrocytes, they must be specified at the correct times and undergo coordinated changes in both gene expression and morphology. As oligodendrocytes differentiate, they transition from a bipolar morphology into a morphology characterized by a complex network of multiple processes, which will eventually generate membranous structures necessary for myelination of axonal segments. As changes are observed in cellular morphology, oligodendrocytes also undergo changes in their gene expression profile and express genes necessary for both early and later stages of development such as olig1 and myelin basic protein (mbp), respectively. Data from our laboratory demonstrate that autotaxin (ATX), also referred to as phosphodiesterase Iα/autotaxin (PD-Iα/ATX), is involved in all of these processes as a multifunctional protein by regulating lysophospholipid signaling and cell-extracellular matrix interactions. Previously, our laboratory has identified ATX as an oligodendrocyte-secreted factor present in the extracellular environment that via a newly-identified functional domain, named the MORFO domain (modulator of oligodendrocyte remodeling and focal adhesion organization), can regulate adhesion of oligodendrocytes to naturally occurring extracellular matrix (ECM) proteins and ultimately the establishment of the oligodendrocyte’s complex process network. In vitro data presented in this dissertation suggest that lysophosphatidic acid (LPA), via production from ATX’s well characterized lysophospholipase D (lysoPLD) domain, can induce the expression of myelin basic protein (mbp) and the establishment of membranous structures by differentiating oligodendrocytes, both necessary for the initial stages of myelination. Interested in relating these functions to an in vivo model and due to the early embryonic lethality of atx-null mice, we utilized the zebrafish as an in vivo model. The in vivo data presented in this dissertation demonstrate that atx expression in the zebrafish is evolutionarily conserved within vertebrates. Interestingly, in both mouse and the zebrafish, atx was found expressed by cells of the cephalic floor plate in addition to differentiating oligodendrocytes. Functionally the in vivo data presented in this dissertation confirmed ATX’s role in stimulating mbp expression during later stages of oligodendrocyte development. In addition, a novel function for ATX was revealed by the studies undertaken as part of this dissertation that has never been described before. More specifically, based on the timing of atx expression and the phenotype seen upon atx knock-down, the data presented here suggest that ATX, released by the cephalic floor plate, regulates early oligodendrocyte development and/or specification. Taken together, these data identify ATX as a major regulator for early as well as late developmental stages of the oligodendrocyte lineage.

RNA degradation plays a fundamental role in maintaining cellular homeostasis, along with being a part of normal regulatory mechanisms, whether it occurs as a surveillance mechanism eliminating aberrant mRNAs or during RNA processing to generate mature transcripts. 3’-5’ exoribonucleases are essential mediators of RNA decay pathways, and one such evolutionarily conserved enzyme is polynucleotide phosphorylase (PNPase). The human homologue of this fascinating enzymatic protein (hPNPaseold-35) was cloned a decade ago in the context of terminal differentiation and senescence through a novel ‘overlapping pathway screening’ approach. Since then, significant insights have been garnered about this exoribonuclease and its repertoire of expanding functions. hPNPaseold-35 has progressed a long way from being just a 3’-5’ exoribonuclease to a functionally relevant molecule implicated in a multitude of diverse and important biological effects. hPNPaseold-35 plays central roles in diverse physiological processes including growth inhibition, senescence, mtRNA import, mitochondrial homeostasis, and RNA degradation, all while primarily being localized in the mitochondrial IMS (inter membrane space). hPNPaseold-35 also holds immense promise as a therapeutic agent due to its ability to degrade specific miRNA (miR-221) and mRNA (c-myc) species, and this property can be exploited in treating malignancies that are characterized by upregulation of harmful miRNA or mRNA molecules. But apart from these two targets, little is known about any other targets hPNPaseold-35 may degrade. Thus, the primary objective of this dissertation research was to identify targets other than c-myc or miR-221 that hPNPaseold-35 could directly degrade to discover newer and biologically relevant therapeutic targets for the treatment of hPNPaseold-35 –associated disease states. In order to do this we performed extensive microarray analyses following hPNPaseold-35 overexpression and depletion in mammalian cell lines, and were able to identify transcripts that could be potentially regulated by hPNPaseold-35 directly or indirectly. Apart from this we also analyzed the 3’UTR of c-myc in order to identify any specific sequence or secondary structural elements necessary for hPNPaseold-35 mediated degradation. Lastly, we identified certain residues in hPNPaseold-35 that have been under positive natural selection through evolution.

Nicotine is one of the most commonly used drugs among adolescent populations. Given the fact that adolescence is a unique developmental stage, during which nicotine has long-term effects on future drug-taking behavior, it is essential to understand how early exposure to nicotine during adolescence may affect the abuse liability of other drugs. We hypothesize that repeated exposure to low doses of nicotine in adolescence induce age-specific enhancement of the rewarding effects of several drugs of abuse in the conditioned place preference (CPP) test. Furthermore, we predict that these changes in behavioral responses are mediated by nicotine-induced brain region-specific increases in the expression of ΔFosB, a member of the Fos family of transcription factors, through activation of neuronal nicotinic receptors. We used mice as a model system to investigate the effects of adolescent nicotine exposure on responses to cocaine, amphetamine, and morphine in adulthood. We found that exposure to nicotine during the early phase of adolescence (postnatal day 28) enhanced cocaine CPP, acute locomotor activity, and locomotor sensitization in adulthood. Our data demonstrate that nicotine priming effects on cocaine are affected by the dose, duration, method of administration, age of exposure, and mouse strain. These data strongly suggest that nicotine intake during adolescence may cross-sensitize the brain to the rewarding effects of cocaine. A follow-up study was undertaken to determine if this enhancement applies to other drugs of abuse. The repeated exposure to 0.5 mg/kg nicotine (subcutaneous) during early adolescence resulted in significant enhancement of amphetamine and morphine preference in a CPP test, but had no effect on the somatic signs of morphine withdrawal. In addition, we investigated the possible neuronal mechanisms underlining enhancements to behavioral responses using both in vivo and in vitro techniques. Our results showed that nicotinic antagonists, with varying subtype selectivity, administered during adolescence prior to nicotine exposure diminished cocaine enhancement in CPP. This suggests that the enhancement of cocaine behavioral responses is mediated by neuronal nicotine receptors (mainly β2* and α7). Finally, studies of ∆FosB revealed significant effects of age and nicotine pre-treatment in nucleus accumbens (NAc), but not in the prefrontal cortex (PFC). Indeed, nicotine pre-treatment was able to significantly increase ∆FosB levels in NAc of early adolescent mice compared to adult mice. This accumulation of ∆FosB persisted for several weeks. Further studies are needed to fully examine the mechanisms of action underlying the observed changes in cocaine rewards.

Nicotine is one of the first and most commonly abused drugs in adolescence. According to The Center for Disease Control, every day more than 6000 adolescents try their first cigarette and over 3000 of them become daily smokers. Smoking among adolescents is a strong predictor of future drug abuse and dependence in adulthood. A number of studies has suggests that adolescents pre-exposed to nicotine may suffer permanent disruption of the brain’s reward systems through changes in dopamine receptor function. We hypothesize that nicotine exposure during adolescence causes long lasting neurobiological alterations that increase the likelihood of cocaine use in adulthood. Furthermore, it activates a neurobiological mechanism that is shared by many drugs of abuse, which will increase susceptibility to their rewarding effects. The work in this thesis contributes to the further understanding of this critical developmental period. Conditioned-place-preference, acute locomotor and locomotor sensitization pardigms were used to examine changes in cocaine sensitivity in adulthood. Testing was performed on adult ICR mice that were exposed to nicotine (0.1 or 0.5 mg/kg, S.C., b.i.d.) or saline during adolescence (postnatal days 28 or 46) or adult (postnatal day 70). Data showed that a 7-day exposure to the higher dose of nicotine (0.5 mg/kg) altered cocaine-induced responses. In contrast, neither 1 day exposure nor a low dose of nicotine (0.1 mg/kg) elicited this effect. A follow-up study was undertaken to determine if this enhancement generally applies to other drugs of abuse. Pre-exposure to 0.5mg/kg nicotine during early adolescence demonstrated significant enhancement to morphine reward, but it failed to increase d-amphetamine preference in a CPP model. Further research will be required in order to more fully examine the mechanisms of action for the observed changes in cocaine rewards. In summary, these findings suggest that early adolescent nicotine exposure leads to changes in cocaine reward and sensitivity during adulthood in both dose and duration matters. Indeed, the adolescent brain is uniquely vulnerable to the effects of nicotine on subsequent drug reward.

Acute behavioral responses to ethanol have predictive value for determining an individual's risk of long-term drinking behavior. Although the neurobiology of alcohol abuse is complex, prior studies from our laboratory demonstrated differential myelin-associated gene expression (MAGE) in medial prefrontal cortex (PFC) as one potential mechanism influencing acute ethanol behaviors between C57BL/6J (B6) and DBA/2J (D2) mice. Our laboratory and others have also shown MAGE is reduced in PFC of alcoholics. Herein, I have extended these findings through expression profiling of PFC into chronic models of ethanol self-administration from non-human primates and mice. Together, these results suggest that regulation of MAGE may be relevant to behavioral phenotypes witnessed in alcoholism. The pathogenesis of alcoholism progresses through multiple stages of drug exposure and withdrawal, however, genetic predisposition is also a major contributing factor for this disease. Therefore, I tested the hypothesis that not only does ethanol have direct effects on MAGE, but also variation in basal MAGE within the PFC is a molecular endophenotype underlying ethanol behavioral sensitivity. Bioinformatics of basal MAGE across the BXD recombinant inbred panel (n=29), derived from B6 and D2 mice, revealed a densely correlated myelin gene network associated with several ethanol behavioral phenotypes. Literature association tools identified Fyn kinase as potential regulator of MAGE. Fyn knockout mice are known to be more sensitive to the sedative-hypnotic properties of ethanol in the loss of righting reflex (LORR) paradigm. Microarray analysis of Fyn knockout mice revealed a significant decrease in MAGE, suggesting MAGE may be an underlying factor for LORR. In support of this premise, microarray analysis of genetic variance in LORR across Inbred Long Sleep and Inbred Short Sleep mice, as well as congenics for the Lore5 quantitative trait locus, also demonstrated an inverse relationship between MAGE and duration of LORR. The hypothesis was further investigated using cuprizone to model demyelination in B6 mice and test them in a battery of acute ethanol behaviors. Cuprizone-treated mice had a significantly greater duration in LORR (p < 0.01), demonstrating that myelin is an important contributor to the genetic variance in LORR. Thus, through genetic, genomic, and pharmacological tools I have ‘molecularly triangulated’ a myelin gene network as a contributing factor influencing acute ethanol behavioral sensitivity. The ability of myelin to alter acute ethanol sensitivity may warrant a prospective study of myelin in humans as a predictive molecular phenotype for an individual’s risk of developing alcohol dependence. Additionally, further genomic dissection of MAGE architecture and associated networks may aid in developing novel pharmacotherapies for an alcohol use disorder. Supported by NIAAA grants F31 AA018615 to SPF

Abstract: Folylpoly-γ-glutamate synthetase (FPGS) catalyzes the formation of polyglutamate forms of the reduced folates and antifolates such as methotrexate (MTX) and pemetrexed; this allows the retention of folates and antifolate cancer drugs inside the cell. The enzyme activity of FPGS is essential for cell proliferation and survival. The mouse fpgs gene contains two promoters spaced 10 kb apart which are activated in a tissue-specific manner. The upstream promoter (P1) and exons A1a and A1b are used in some differentiated tissues, mainly liver and kidney, whereas the downstream promoter (P2) and exon 1 are used in rapidly dividing cells. In contrast, the human fpgs gene expresses virtually all transcripts from the downstream promoter. In order to more faithfully mimic human folate metabolism in the mouse, we have deleted the upstream promoter and the associated two small exons of fpgs in the mouse genome by homologous recombination. Homozygous deletion mice survive embryonic development, grow to adulthood, and reproduce through several generations, they appear to be normal. The results of Q-RT-PCR analysis on RNA from adult mouse liver of three different genotypes (A1aA1b +/+, +/-, -/-) indicated that deletion of P1 results in the release of promoter interference of P2, and activation of the downstream P2 promoter is increased by 3-5 fold. Interestingly, the total FPGS mRNA expression in KO mouse liver is 20-100 fold lower than in liver from wild-type mice. However when the FPGS activity was measured using an FPGS enzyme assay, the liver of knockout mice appeared to have only 2 fold lower enzyme activity than liver from wild-type mice. In conclusion, we have successfully generated a mouse which reflects human folate metabolism much closer than seen in wild type mice. The FPGS- humanized mouse liver model would be an appropriate in vivo tool for the study of the antifolate drug toxicity and inhibition.

The Hsp90 chaperone complex associates with the telomerase enzyme, facilitating the assembly of the ribonucleoprotein complex. While previous data from our laboratory indicate that Hsp90 and p23 remain stably associated with (functionally active) telomerase, more recent experiments suggest that these chaperones associate with telomeres independent of telomerase, presumably through a specific interaction with telomere binding proteins. The current study examines the novel interactions between TRF2, TRF1, TIN2 and TPP1 and molecular chaperones (Hsp90, Hsp70, p23). In vitro and in cell experiments have shown an interaction between TRF1 and TRF2 and the molecular chaperones Hsp90 and Hsp70. Inhibition of Hsp90 using drugs that specifically block ATPase activity results in an increased association of TRF1 and TRF2 with Hsp90 to presumably stabilize the telomere associated proteins to the telomere. A definitive explanation as to the mechanisms underlying the chaperone/telomere associated protein interaction has yet to be determined and further studies examining chaperones’ contribution to telomere structure and function are underway. A better understanding of the telomeric proteins and Hsp90 and their roles in nuclear events is important, as both have extremely important functions in the cell. Our current working hypothesis is that chaperone proteins associate with TRF2, TRF1, TIN2 and TPP1 to facilitate telomeric protein-protein interactions and protein-telomere binding in both cancer and normal cells. The interaction between chaperones and telomere binding proteins may eventually provide a better understanding of telomeric structure and function. Defining the mechanisms of telomeric protein regulation is important in the development of new therapeutic approaches for targeting telomeres to induce dysfunction. Clinical trials are underway employing drugs targeting Hsp90 in cancer cells and given the results here, these Hsp90 compounds likely cause telomere alterations.

Evidence for the timing and frequency of monitoring chronic conditions in primary care is often of low quality or absent. Recently, a methodology based upon repeated measures of monitoring tests has been developed to evaluate monitoring strategies which differ by frequency and the length of interval. In studies using this approach, it has been shown that there is potential for substantial over-detection and over-treatment through routine monitoring. The aim of this thesis was to extend the existing methodology to outcomes with highly skewed distributions and tests that are interpreted as dichotomous or categorical states. These methods were applied to find evidence for the intervals of monitoring for microalbuminuria in people with type 1 diabetes and screening for diabetic retinopathy. Statistical models were used to describe the progression of albuminuria and retinopathy and estimate the rates of false positive and false negative tests over time. Simulation was then used to create hypothetical cohorts of people being monitored and screening at different frequencies and for different baseline risk. The impact of changing the timing of tests within the programme was measured and evaluated. The basic framework of the methodology can be usefully extended to tests that have categorical outcome or highly skewed distributions. More frequent monitoring of microalbuminuria in people with type 1 diabetes, and more frequent screening of diabetic retinopathy leads to disproportionate numbers of false positive results and potential over-treatment or referral to specialist services. As with any other clinical intervention, monitoring and screening intervals should be based upon solid evidence. Monitoring for microalbuminuria and screening for diabetic retinopathy less frequently would reduce the rates of false positives tests but the implications of less surveillance need further investigation.

<strong>Background</strong>. Pathological processes in Parkinson’s disease (PD) start long before the first symptoms appear and by the time the disease is clinically established the results of neurodegeneration may be irreversible. Efforts to prevent or stem disease progression need to start in early disease and good characterization and new markers of early PD are urgently needed. <strong>Objectives</strong>. This thesis aims to characterize early disease stages in three projects. Firstly, clinical features of PD within 3 years of diagnosis will be explored in an incident cohort of patients and controls, using a range of tools to cover the whole breadth of clinical presentation of PD. Secondly, functional imaging studies in PD published so far will be examined through a meta-analysis to identify the most robust functional imaging markers. Thirdly, a functional MRI resting-state study in early PD will be performed to identify reproducible differences between patients and matched control subjects. <strong>Results</strong>. The cohort analysis found that age was a strong predictor of disease severity, independent of disease duration, while gender was seen to affect disease severity depending on the body region. A meta-analysis of all published functional imaging studies across all disease stages showed abnormal activations in the Basal Ganglia but also in a wide range of motor and non-motor brain areas. Dopamine supplementation normalized activations in the Basal Ganglia and some other areas, while other circuits remained resistant to medication suggesting non-dopaminergic abnormality. In the resting-state study, the Basal Ganglia Network showed greatly reduced connectivity in early PD compared to controls, which normalized on administration of dopaminergic medication. Reduced BGN connectivity was also validated on a separate group of PD subjects achieving very good separation of patients from controls. <strong>Conclusions</strong>. The effect of gender and age on early presentation of PD has potential significance for early diagnosis and choice of outcome measures for clinical trials. Within the realm of imaging, traditional task-based fMRI studies fail to show a clear and reproducible pattern of activations making this method unfeasible for early diagnostic testing. In contrast, resting-state fMRI connectivity in the Basal Ganglia Network appears to be a promising and reliable method even in the early stages of PD. Clinical profiling and resting imaging changes offer avenues for developing future biomarkers in early PD.