Neurofibromatosis type 1 results from mutations in NF1, a gene that encodes Neurofibromin. This common genetic condition is associated with tibial pseudarthrosis (PA), whose etiology is unknown but thought to involve defective bone-repairing osteoprogenitors. The main objective of my thesis was to delineate the causal determinants of the poor osteogenic potential of Nf1-/- osteoprogenitors. I showed that increased Epiregulin and TGFb1 expression does not contribute to the reduced osteogenic differentiation of Nf1-/- osteoprogenitors, and contrary to all expectations, that this phenotype is likely independent from MAPK/ERK constitutive signaling. Using a RNA-Seq approach, I identified changes in pro-inflammatory and extracellular matrix gene signatures as putative determinants of the impaired differentiation of Nf1-/- osteoprogenitors. Finally, I obtained preliminary data pointing to inhibition of RUNX2 activity upon loss of Nf1 function. These results suggest unexpected interactions between Neurofibromin and proximal cell signaling/adhesion components that impact not one but multiple downstream signaling pathways.

L-DOPA Induced Dyskinesia (LID) is the motor complication involving an overproduction of movement that arises from L-DOPA treatment of Parkinson's disease (PD). Despite the high incidence of the development of LID, the etiology of this side effect and variability in its speed of onset is still poorly understood. Previous work in the lab discovered that subjects with PD who had developed LID had less striatal mitochondrial DNA (mtDNA), relative to non-dyskinetic PD and healthy subjects. The focus of this research was thus to identify mechanisms by which reduced mtDNA might affect the function of the striatal neurons typically affected by LID. Through treating primary murine striatal cultures with low doses of ethidium bromide (EtBr) to selectively inhibit mtDNA replication and transcription, it was determined that mtDNA reduction reduces mitochondrial respiration in neurons and astrocytes, but selectively increases glycolytic activity in astrocytes. Further, decreased mtDNA selectively reduces the expression of mitochondrial creatine kinase in neurons, which is accompanied by a reduction in phosphocreatine quantity. Creatine and phosphocreatine act as crucial energy distributing molecules to facilitate the transfer of mitochondrially-synthesized ATP-phosphates to other sites of high energy demand in the cytosol. Consequently, reduced phosphocreatine production, arising from reduced mtDNA, may impair efficient energy transfer in the striatum. This may contribute to the development of maladaptive signaling pathways associated with LID.

Cardiac arrest is a leading cause of death. Even with the best cardiopulmonary resuscitation (CPR), many patients still die or suffer severe organ damage. The reintroduction of blood flow at the start of CPR after systemic ischemia causes additional damage to organs, such as the heart, beyond that caused by the ischemia itself. Ischemia/reperfusion (I/R) injury is a complex pathological event involving processes that can lead to disruptions in the cell membrane and cellular dysfunction. Loss of membrane integrity may allow an influx of calcium (Ca2+) into cardiomyocytes, leading to hypercontracture and cell death. Methods to improve the endogenous membrane resealing capacity of cells that are overwhelmed due to pathological disruptions, such as I/R injury, are needed to prevent cardiomyocyte death, because the ability of the myocardium to regenerate is limited. Using an in-vitro cardiomyocyte model exposed to simulated I/R (hypoxia/reoxygenation, H/R), the tri-block copolymer Poloxamer 188 (P188), with its unique hydrophobic/hydrophilic chemical properties, was administered during reoxygenation and assessed for its ability to provide membrane repair and cellular protection. P188 protected cardiomyocytes from H/R injury by repairing cell membranes, reducing LDH release, and decreasing Ca2+ influx. Additionally, it was shown that the majority of Ca2+ influx during H/R was through tears in the cell membrane, which were targeted by P188, rather than through Ca2+ channels and exchangers not targeted by P188. Determining the mechanism of action of a compound administered during CPR on the cellular dysfunction caused during I/R injury could aid to improve CPR practices in the future.

Modulation of neurotransmitter exocytosis by activated Gi/o coupled G-protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is the Gβγ and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) interaction. There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR and the specificity to effectors in vivo are not known. In this dissertation, a quantitative MRM analysis of neuronal Gβ and Gγ subunits was developed to detect neuronal Gβ and Gγ subunits interaction with receptors and effectors and understand the specificity to α2a adrenergic receptors (α2aARs) and SNARE. The specificity of Gβ and Gγ subunits to α2aARs in both adrenergic (auto α2aARs) and non-adrenergic neurons (hetero α2aARs), and SNARE in presence of epinephrine were identified. Moreover, the microarchitecture of the Gβγ and SNARE interaction was examined by understanding the molecular interaction of Gβγ and SNAP25 and identifying 47 synaptic proteins that may be in complex with Gβγ and SNARE. These studies form the basis for further investigations to determine the effect of G protein specificity on its downstream signaling, identify potential pathophysiological states in which the Gβγ-SNARE interaction may be dysregulated, and yield additional insights into Gi/o-coupled GPCR-mediated regulation of exocytosis.

Diabetes mellitus affects millions of people. Although diabetics can lead relatively normal lives, all treatments for the disease are symptomatic and not curative. The purpose of this investigation was to determine whether the sensitivity to opiates and other selected centrally-acting drugs in animals is altered by streptozotocin (STZ)-induced diabetes. A second objective was to determine which aspect of the diabetic syndrome primarily was responsible for the altered sensitivity. Other experiments were performed in an attempt to elucidate the mechanism whereby this altered sensitivity occurred. STZ-induced diabetes or dextrose-induced transient hyperglycemia did not have a significant effect on the duration of hexobarbital-induced anesthesia. Similarly, following 5 days treatment with phenobarbital, the duration of hexobarbital-induced anesthesia was reduced equally in both control and STZ-induced diabetic mice. STZ-induced diabetes did not alter the acute oral LD50 of nicotine. The antinociceptive potency of morphine as determined by the tail-flick test was significantly decreased (p < 0.05) in STZ-induced diabetic mice and mice pretreated with equimolar doses of hypertonic dextrose or fructose. STZ-induced diabetic rats and spontaneously diabetic mice were also significantly less sensitive to the antinociceptive effects of morphine as quantitated by the tail-flick test. The ability of morphine to inhibit phenquuinone-induced writhing was attenuated in STZ-induced diabetic mice. Hypoglycemic mice were significantly more sensitive to morphine in the tail-flick test. Insulin reversal of dextrose-induced and STZ-induced diabetic hyperglycemia returned sensitivity to morphine-induced antinociception in the tail-flick test to control values. Pretreatment with the non-metabolizable sugar 3-0-methylglucose at a dose equimolar to the doses of dextrose and fructose had no effect on morphine potency. The antinociceptive potencies of phenazocine and levorphanol were altered similarly to that of morphine, but the potencies of methadone, propoxyphene and meperidine were not altered by changes in blood glucose levels. The LD50 of morphine but not methadone was significantly decreased in STZ-induced diabetic mice. These results confirm the selectivity of the STZ-induced diabetes to alter the sensitivity of morphine and not methadone, and are provocative since they show that the lethal effect of morphine is altered in the opposite direction from the antinociceptive potency. Levels of morphine in the brains of STZ-induced diabetic and insulin-treated STZ-induced diabetic mice were not significantly different from control mice. The durations of action of morphine in STZ-induced diabetic and control mice were similar, although the level of antinociception in the diabetic mice was lower at all time points. STZ-induced diabetes in mice did not alter serum osmolarity and brain water content. Mice receiving various pretreatments (STZ-induced diabetes, STZ-induced diabetes plus insulin, dextrose, fasting or fasting plus insulin) were subjected to analyses of their serum glucose levels, serum insulin levels, and brain glucose levels. From these data only blood glucose levels correlated (inversely) with the antinociceptive potency of morphine. The results of these experiments led to the hypothesis that the hyperglycemia was the aspect of diabetes principally responsible for selectively affecting the potency of certain opiate-like pharmacologic agents.

One-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a host of metabolites critical to tumor propagation. Although drug-targeting of cytosolic 1CM remains a clinically-relevant mainstay, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in lung, colon, breast, glioma, and liver cancer and, overall, is the fifth-most differentially expressed metabolic enzyme in cancer cell versus normal tissue. Despite the unequivocal oncogenic importance and therapeutic potential of SHMT2, there are no clinically relevant (i.e. active in vivo) inhibitors of this enzyme. In this dissertation work, we sought to design, synthesize, and characterize pharmacodynamics of our 5-substituted pyrrolo[3,2-d]pyrimidine antifolates synergistically dual-targeting mitochondrial SHMT2 and cytosolic 1CM, the latter specifically at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase). By depleting SHMT2-derived formate, these compounds potentiated their own inhibition of the downstream formate-dependent GARFTase and AICARFTase. We generated these compounds (AGF291, AGF320, and AGF347) by melding structures of SHMT2 cofactor 5,10-methylene tetrahydrofolate with our previously reported purine inhibitors and confirmed enzyme targets with in vitro targeted metabolomics in H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma) human tumor cell lines as well as in vitro cell-free assays. Transport assays revealed significant uptake by both the proton-coupled folate transporter (narrow physiological niche, but commonly expressed in many solid tumors) and the reduced folate carrier (major tissue folate transporter). Subcellular fractionation of MIA PaCa-2 and GlyB Chinese hamster ovary cells revealed AGF347 to be heavily (>98%) polyglutamylated in both cytosol and mitochondria with mitochondrial uptake partially mediated by the mitochondrial folate transporter. Intracellular glycine depletion secondary to SHMT2 inhibition by all compounds also depleted cellular ROS scavenging capacity as reflected in decreased GSH/GSSG ratio. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice with tumor growth delay (T-C) of 61 days and one out of five treated mice tumor-free 120+ days after treatment. In vivo metabolomics on these xenografts confirmed inhibition of purine biosynthesis. Collectively, the work in this dissertation establishes the exceptional therapeutic potential of dual-targeting mitochondrial and cytosolic 1CM.

Abstract not available

3,4-Methylenedioxymethamphetamine (MDMA) is a ring-substituted amphetamine with a potential for abuse. Although originally developed by Merck, MDMA is an illegal drug that is popular recreationally, and is more recently being touted as a therapeutic agent. Unlike some other drugs in the amphetamine class, the mechanism(s) by which MDMA produces its subjective effects are not well understood. MDMA is a selective serotonin (5-HT) neurotoxin. Exposure to MDMA can lead to lasting reductions in brain 5-HT and 5-HT axonal markers. Somewhat paradoxically, its acute pharmacological effects involve a dramatic acute increase in serotonin (and other monoamine) levels in the brain and the periphery. MDMA is also a direct agonist at several different monoaminergic receptors. Although these pharmacological properties of MDMA are known, they don't appear to fully explain the subjective of effects of MDMA, which include feelings of well-being and euphoria. One unfortunate notion held by many MDMA users is that the drug is safe, or at least safer than many other illegal drugs. This is a notion that is strengthened by MDMA's current and past use as a psychotherapeutic agent, although definitive safety/efficacy reports have yet to appear in the literature. In recent years, there has been a renewed push to acknowledge the potential utility of MDMA in the treatment of conditions such as post-traumatic stress disorder. MDMA has been reported to damage a number of organ systems in addition to its properties as a selective 5-HT neurotoxin in the brain. Furthermore, recreational MDMA users develop tolerance, which results in a need to increase the dose to achieve the same subjective effects, thereby also increasing the risk for dose-related adverse effects. A number of research laboratories have demonstrated that abstinent MDMA users develop both a loss of brain 5-HT markers, in addition to potential functional consequences of 5-HT neurotoxicity, including deficits in cognitive function, endocrine modulation, and sleep regulation. Although these effects have been well-described, the mechanism by which MDMA leads to neurotoxicity remains unclear, and multiple theories have been suggested. There are many unanswered questions when it comes to MDMA. Without knowing more about how MDMA acts in the body and how it produces toxicities, use of the drug constitutes a significant risk. Not only are the acute, systemic and potentially fatal effects of MDMA problematic, but longer term functional consequences secondary to serotonin depletion may pose significant problems for abstinent MDMA users as they age. In light of the drug's popularity, the need for answers and increased public awareness has never been more pressing. Although MDMA is classified by the Drug Enforcement Agency (DEA) as schedule I, popular musicians have begun to positively reference MDMA in their lyrics, which has likely contributed to the observed rise in MDMA-related hospital visits and fatalities. Communities, parents, and healthcare professionals must make a more concerted effort to raise public awareness of the potential dangers of MDMA use.

Includes bibliographical references.

Although it is more than 100 years since Robert Koch discovered the tubercle bacillus, and more than 40 years since effective chemotherapy became available, tuberculosis remains a major cause of morbidity and mortality in the world to-day. A major contributor to the morbidity is anaemia. This anaemia falls under the classification of anaemia of chronic disorders, the pathogenesis of which has not been fully elucidated. With the advent of recombinant human erythropoietin (Epo ), it has become evident that a blunted Epo response to the anaemia plays a major role. The mechanism (s) involved still have to be elucidated. The aims of, this study were to evaluate serum Epo levels and iron parameters in anaemic patients with active pulmonary tuberculosis (PTB) and investigate the effect of tumour necrosis factor alpha (TNFa) produced by activated macrophages in PTB patients on Epo production in vitro. Furthermore, the mechanism involved in Epo gene expression was investigated. Haematological and biochemical parameters (including serum iron and Epo) were I studied prospectively in four groups each of 1 O subjects. Group I comprised newly diagnosed non-pregnant individuals with pulmonary tuberculosis (PTB), haemoglobin below 110 g/L, and having no apparent dissemination or other I associated systemic illnesses. Group II were age and sex matched PTB patients with haemoglobin levels greater than 130 g/L. Group III ·consisted of otherwise healthy ' people with demonstrated absolute iron deficiency anaemia with haemoglobin corresponding to those in Group I. Group IV consisted of 10 healthy non-anaemic volunteers. For matching degrees of anaemia, the serum Epo was significantly lower in Group I than in Group III patients. In PTB, therefore, the Epo response to anaemia is attenuated. With anti-tuberculous therapy there was a significant increase in the Hb and serum iron levels in the Group I patients which correlated with a fall in the levels of the inflammatory marker C-reactive protein (CRP). This argues for the degree of inflammation being casually related to the anaemia. To further investigate the effect of inflammation on Epo production, blood samples were collected. from individuals in Groups I, II and III and the peripheral blood mononuclear cells (I>BMC) were assayed for the cytokine TNFa. The incubation of supernatant fractions (SNF) of the anaemic PTB group with HepG2 cells resulted in a marked inhibition of Epo production by these cells. Dose response studies showed that increasing concentration of SNF resulted in a progressive reduction in Epo production, which could be reversed by the presence of anti-TNFa antibodies in the: medium. Thus TNFa is capable of inhibiting Epo production and may play a role in the blunted Epo response to anaemia seen in patients with PTB. In order to characterize the mechanisms involved in oxygen sensing, the murine Epo gene was studied to define the sequences within the enhancer involved in oxygen sensing and Epo gene expression. To this end, transfection experiments of deleted, mutated and re-iterated enhancer sequences located 3' to the poly (A) signal sequence were carried out in HepG2 cells and in the non-Epo producing lung fibroblastoid cell line a23. Transcription factor binding to the enhancer was investigated by DNA I footprint analysis and revealed that at least three sites within a 96 nucleotide sequence 0f the Epo enhancer were critical. Oxygen regulated operation was dependent on sites ' ' within the first 25 nucleotides. In both HepG2 and a23 cell lines the same two critical sites in the 5' region of the enhancer were necessary for function. Sequences located 3' to this region modulated enhancer function but did not themselves convey oxygen regulated operation. This study has contributed to the understanding of the pathophysiology of the I anaemia of tuberculosis in that in this disease, TNFa released from activated macrophages was capable of inhibiting Epo production in vitro. This may explain the attenuated Epo response to anaemia in PTB patients. Furthermore, three critical sites on the Epo enhancer were shown to be essential for oxygen sensing and Epo gene expression. It can be postulated, therefore, that TNFa may disrupt the interaction of transcription factors with critical sites of oxygen sensing on the enhancer and prevent the increased production of Epo. Further work is required to clarify this postulate.