Addiction is a chronic disease that characterize as habitual or compulsive involvement in an activity despite it¡¦s bring negative consequences. Some of psystimulants such as cocaine or amphetamine cause a strong reinforcing effects even after prolonged abstinence periods. Such illegal drugs not only hurt on the adult health but also result in fetal physiological damage. For example, that babies born to mothers who abuse with cocaine bring prematurely delivered, low birth weights, smaller head circumferences and increased heart disease in adult offspring. Mesolimbic dopamine system include nucleus accumbens (NAc) and ventral tegmental area (VTA) are critical regions for the neural adaptations that contribute to addiction. VTA that receives inputs from a large number of brain regions. For example, it receives glutamatergic inputs from prefrontal cortex, or GABAergic inputs from NAc. It has been known that VTA play a major role in the acquisition and expression of learned addictive behaviors. Results from many neuropharmacological studies in animal models indicate that exposure to cocaine or some other drugs of abuse seems to induce long-term potentiation (LTP) ¢w like changes of synaptic plasticity among neurons in VTA region. LTP was first described in hippocampus, a region that associated with memory formation, and were found widespread events in many mammalian brain sites. In the present time, theories and investigation indicated that memory and addiction might shared the similar neural circuitry and signal pathways. In general, LTP can be separate into two main phases : induction and maintenance phases. Many of molecules participate in induction phase such as calcium/calmodulin-dependent protein kinase II (CaMKII), cyclic AMP (cAMP), phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC). However, until now there was only one molecule has been found associated with LTP maintenance¡Xprotein kinase M£a (PKM£a). PKM£a is a brain specific, constitutively active form of PKC that does not need Ca2+ or diacylglycerol (DAG) for its activation. Molecular evidences showed that PKM£a is translated uniquely by PKM£a mRNA which is generated under the control of an internal promoter in the PKC£a gene. Recently, investigators introduced a PKM£a selective inhibitor¡XZIP, to hippocampus or insular cortex both successful to eliminate long-term spatial memory or conditioned taste aversion (CTA) behavior, respectively, on rat. Therefore, exclude PKM£a by specific inhibitors and then result in abolish long-term synaptic potentiation which had already established seem to be a leading candidate for cure addiction. Here we showed that blocked of PKM£a activity in VTA dopaminergic neuron eliminated mEPSCs or AMPAR/NMDAR ratio increment elicited by cocaine. Otherwise, our results also presented that myristoylatedinhibitory peptide¢wZIP had no effect on spike timing-dependent long-term potentiation in rats previously injected with saline but remarkably restored spike timing-dependent long-term potentiation in VTA dopamine neurons in slices prepared from rats that received single or multiple cocaine exposure. Furthermore, our western blot analyses showed that both single and five consecutive cocaine injections induced a significant increase in PKM£a level in VTA or NAc. Moreover, our ex vivo cocaine incubation results indicated that multiple kinases activation or de novo protein synthesis was required for PKM£a increment. The most important, our data provided the first physiological evidence between PKM£a and drug addiction when intracranial administered specific PKM£a inhibitors to VTA reversed cocaine-induced conditioned-place preference (CPP) behavior. Finally, we investigated the behavioral effect of cocaine-induced locomotor sensitization in an open field apparatus. Our data showed that peri-adolescent (P21) rats exhibited prominently increased in either acute or repeated cocaine-induced locomotor activity than mid-adolescent (P28) and post-adolescent (P41). Interestingly, applied to high dosage cocaine (30 mg/kg) rescued the acute locomotor response in P28 rats but not behavioral sensitization. We further examined the locomotion on rats that were exposed to cocaine in utero after single or multiple cocaine injection. However, cocaine-induced increase in locomotor activity was lower in P21 rats which exposed to cocaine during pregnancy but no significantly difference in P28 rats. Surprisingly, single high dose cocaine treatment caused a marked reduction in locomotor activity on P21 rats prenatally exposed to cocaine. Otherwise, we also provided the first evidences that repeated cocaine injection in pregnant rats induced a significant decreased to KCC2 level in PFC regions prepared from P20 rat. In conclusion, results from our current studies demonstrate for the first time that persistently active PKM£a is necessary in (1) mEPSC facilitation induced by single cocaine exposure; (2) cocaine-induced enhancement in AMPAR/NMDAR ratio; (3) single or repeated cocaine-induced LTP but not in LTP induced by spike-timing stimulation; and (4) cocaine conditioned place preference in the VTA. In addition, our results also present evidence that the expression of PKM£a is increased by either single or repeated cocaine exposure. Furthermore, our behavioral or Western blotting consequence of cocaine treatment in utero was reflected by the diminishion in the sensitivity of locomotor activity in postnatal rats to cocaine and KCC2 level in PFC regions.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 78-93).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 102-114).

Thesis (Ph. D.)--Medical College of Ohio, 2003. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Howard Rosenberg. Document formatted into pages: iv, 144 p. Title from title page of PDF document. Includes bibliographical references (p. 104-132).

Protein kinase CK2 phosphorylates wheat eIF2, eIF3, eIF4B, eIF5 and three 60S ribosomal proteins. The substrate specificity of CK2[alpha] toward various plant initiation factor substrates was altered in vitro through holoenzyme formation in the presence of regulatory [beta]-subunits. This presents a potential mechanism through which the differential expression and sub-cellular distribution of CK2 [beta]-subunits could regulate phosphorylation of various CK2 substrates in plants. Our analysis of initiation factor phosphopeptides produced by in vitro phosphorylation identified 20 CK2 phosphorylation sites in eIF2[alpha], eIF2[beta], eIF3c, eIF4B, and eIF5. Native wheat eIF5 was prepared in the presence of phosphatase inhibitors and analyzed by mass spectrometry. Native wheat eIF5 was determined to be a phosphoprotein containing at least 3 phosphorylation sites. The C-terminal CK2 site (S451) of native eIF5 was completely phosphorylated, and tryptic fragments containing the other in vitro CK2 two sites (S209, T240) also appear to be partially phosphorylated. Many of the CK2 phosphorylation sites identified are in conserved binding domains of the yeast multifactor complex (eIF1/eIF3/eIF5/eIF2/GTP/Met-tRNAi[superscript Met). This observation lead to the hypothesis that CK2 phosphorylation may regulate the formation of plant multifactor complexes. The results presented here suggest that plant initiation factors are capable of forming complexes similar to those previously reported in yeast. The in vitro interaction of initiation factors within these complexes appears to be enhanced by phosphorylation of eIF2, eIF3c, and eIF5 by CK2. Site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites not only prevents the CK2 mediated increase in interaction with eIF1, but also resulted in reduced stimulation of translation initiation in vitro. / text

The specific interactions of macromolecules along with the activity of enzymes are central to all aspects of biology. It is well recognized that when the relative concentration or activity of macromolecules is perturbed, it can lead to human diseases. Thus, the development of simple methods for the detection of macromolecules and the activity of enzymes in complex environments is important for understanding biology. Moreover, the development of methods for measuring interactions allows for the testing of inhibitors that can be used as tools or drugs for improving human health. Towards this goal, a promising new method has been developed, which is the focus of this thesis, called split-protein reassembly or protein fragment complementation. In this method, a protein reporter, such as the green fluorescent protein or firefly luciferase, is dissected into two fragments, which are attached to designed adaptor proteins. The designed split-protein systems only produce a measurable signal, either fluorescence or luminescence, when a specific macromolecular interaction or activity is present. In this thesis, I have extended previous research on the direct detection of DNA using split-protein sensors utilizing a red fluorescent protein, dsRED from Discosoma that allows for multiplexed DNA detection. I have designed a new split-luciferase based sensor for detection of poly (ADP-ribose) or PAR, which plays a key role in the response to DNA damage and have applied it for monitoring the activity of poly (ADP-ribose) glycohydrolase that controls PAR levels in the cell. Furthermore, I have significantly expanded upon a three-hybrid split-luciferase system for identifying protein kinase inhibitors. I have designed and tested two orthogonal peptide based chemical inducers of dimerization based on BAD and p53mt conjugates. I have studied these chemically induced dimerization systems in detail in order to begin to provide a theoretical basis for the observed experimental results. Finally, in a less related area, I have developed methods for producing water soluble semiconductor nanoparticles called Quantum Dots (QDs), with potential application in biological imaging. I have developed methods for functionalizing the QDs with orthogonal peptides, which can be potentially used for the assembly of high affinity non-covalent QD targeted proteins.

The RAS/RAF/MEK/ERK pathway is the prototypical cellular signal transduction cascade and has been the focus of intense scrutiny over the last two decades. As a mitogenic pathway, its activation is a potent driver of cellular growth and survival, and its deregulation underlies many cancers. While RAS family GTPases have long been recognized as prolific human oncogenes, a landmark study in 2002 also established the RAF family kinase as a bona fide oncogene (Davies et al., 2002). Indeed, aberrant RAS-RAF signaling underlies nearly one-third of all human cancers (Wellbrock et al., 2004). Notably, mutations in RAF are found with astounding frequency in certain cancers (e.g. 70% of malignant melanomas) (Dhomen and Marais, 2007). These findings have identified intercepting aberrant RAF function as an ideal therapeutic target. RAF is a Ser/Thr protein kinase and its activity is strictly regulated by a core complex of at least three proteins, namely, KSR, CNK and HYP (Claperon and Therrien, 2007). The mechanism by which the KSR/CNK/HYP complex regulates RAF function remains enigmatic. In particular, the function of KSR in regulating RAF activity is highly controversial. The work described in this thesis was conducted with the aim of: i) understanding the interactions that underlie formation of the KSR/CNK/HYP complex, and ii) elucidating the mechanism by which the complex regulates RAF function. I have attempted to accomplish these aims using a combination of structural biology, biochemistry and cell biology approaches. I begin by presenting the structure of the SAM domain mediated interaction between CNK and HYP. I describe a model for how the CNK/HYP interaction in turn serves to recruit KSR to form the higher-order KSR/CNK/HYP complex. Subsequently, I describe the allosteric mechanism by which KSR controls RAF activation via the formation of specific side-to-side kinase domain heterodimers of KSR and RAF. Lastly, I describe a potential mechanism by which RAS directly mediates the attainment of the side-to-side dimer configuration of RAF through its own ability to form dimers. The acquisition of the side-to-side dimer configuration is essential for aberrant RAF signaling in cancers, suggesting future RAF inhibition strategies could be aimed at preventing dimer formation.

We hypothesized that alterations in cardiac myofilaments are associated with hereditary canine DCM. DCM myofilaments demonstrated a reduction in EC50 and a modest decrease in maximum activity compared to non-failing dog samples. Treatment of myofilaments with the calcium sensitizer, bepridil, showed a reduction in EC50. Desmin and tropomyosin phosphorylation was increased in DCM. Desmin protein levels were increased in DCM. Total troponin I phosphorylation was unchanged, but S23/S24 phosphorylation was reduced in DCM. Myofilament-associated PKC-δ and -ζ were elevated in DCM, PKC- ε was modestly reduced, and PKC-α showed no change. These data are the first investigation of cardiac myofilaments in naturally occurring canine DCM, and support the hypothesis that alterations in cardiac myofilaments are associated with DCM. / OVC Pet Trust (operating funds)

Establishing the cellular and molecular basis for cardiovascular disease and the application of tools to manipulate the cardiovascular system genetically provide potential for new forms of treatment against cardiovascular disease, including: atherosclerosis, myocardial ischemia, cardiac hypertrophy and heart failure. Heme oxygenase-1 (HO-1) is an enzyme that has potential for the treatment of cardiovascular diseases (CVD). Atherosclerotic plaques express high levels of HO-1. Advanced plaques are stabilized in part through the separation of plaque constituents from the blood by the fibrous cap made up of smooth muscle cells. Protection of smooth muscle cells from apoptosis in the fibrous cap may be a means of promoting plaque stability in patients. Here we show that expression of HO-1 in human vascular smooth muscle cells renders them resistant to apoptosis mediated by oxidative stress. The cytoprotective mechanism mediated by HO-1 is mediated in part through protein kinase B (Akt). Plaque rupture may lead to myocardial infarction. Tissue recovery after mycocardial infarction requires neovascularization for improved tissue perfusion. A novel cell type recently discovered in the circulation has been characterized as an endothelial progenitor cell (EPC) and appears capable of promoting neovascularization of post-infarct tissue, thereby enhancing tissue recovery and perfusion. Most EPCs transplanted into the infarct environment do not survive or are not retained to function in neovascularization. Here we show that expression of HO-1 and its cytoprotective partner Akt protect EPCs in an infarct environment and promote EPC function in an infarct environment. Oxidative stress can result in maladaptive cardiomyocyte hypertrophy. In a model of oxidative stress-induced myocyte hyperterophy we demonstrate the expression of HO-1 prevents cellular hypertrophy through antioxidant mechanisms and regulation of the transcription nuclear factor kappa B (NF-κB). Atherosclerotic plaque vulnerability is determined by the composition of the lesion. We demonstrate that HO-1 deficient mice have more calcified and fibrotic lesions. This may have implications in the management of late stage atherosclerosis. Collectively, this work demonstrates new insights into the molecular mechanisms of cardiovascular cells under stress that may have implications for strategies aimed at treating CVD using HO-1. / Thesis (Ph.D, Physiology) -- Queen's University, 2009-04-21 15:31:14.05

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors. The beta-arrestins, adaptor proteins involved in GPCR desensitization, may also act as scaffolds for signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. The MAPK family, which includes the extracellular-signal regulated kinases (ERK) 1 and 2, promotes cellular differentiation and proliferation. Herein, the activation of ERK1/2 upon stimulation of the GPCR bradykinin type 2 receptor (B2R) with bradykinin was examined. Various B2R mutants with modified C-termini were employed to examine the temporal kinetics of ERK1/2. One of these receptor mutants displayed a loss of beta-arrestin binding as well as greatly enhanced ERK1/2 activation, compared to the wild-type receptor, when a cluster of serine/threonine residues important for B2R internalization was mutated. The other receptor mutants exhibited a correlation between their affinity for beta-arrestin and the intensity of ERK1/2 activation. Data from a mouse embryonic fibroblast cell line null for beta-arrestin suggested that beta-arrestin is involved in late-phase ERK1/2 activation by the B2R. These data point to the involvement of beta-arrestin in the activation of the ERK1/2 MAPKs through the B2R.