Cancer is one of the leading cause of death in modern world, so there is an emerging demand for better diagnostic tools and more specific less toxique therapeutics. Nanoparticles offers characteristics that could fullfill such perspectives. They can easily target tumor by ehanced permeation and retention effect (EPR). Nanoparticles can combine more than one imaging properties, so we can say that they are multimodal, some of them could combine diagnostic and therapeutic molecules in one nanoparticle, which is now highly popular topic of nanoparticles for theranostics . The aim of this thesis was to characterize new multimodal glycogen-based nanoparticle. Glycogen is an ideal structure for nanoparticle design. Glycogen is part of natural dendrimers group which are easily to modify. Glycogen's size is suitable for EPR effect. We have evaluated biological characteristics of five different types of modified glycogen. The in vitro experiments were carried on HepG2 cells. We have set time curve of cellular uptake of this glycogen probes, evaluated cytoplasmatic localization and for the first time we have carried MTT assay. Biodistribution studies on CD1-Nude mice were performed by using non-invasive method for measuring in vivo fluorescence. In conlusion we've provided some of the biological characteristics of new...

The aim of this study has been to explore the site specific phosphorylation pattern of rat heart glycogen synthase paying particular attention to phosphorylations that are important to the in vivo control of enzyme activity. This problem has been approached using techniques of immuneprecipitation of 32P labeled synthase from hormonally responsive, freshly isolated adult rat cardiomyocytes. Identification of the active subunit of rat heart glycogen synthase was accomplished by immuneprecipitating synthase from 32P-labeled cardiomyocytes and performing Western blot analysis on DEAE-cellulose fractions containing synthase activity. Using these methods, glycogen synthase activity has been localized to a protein of 88,000 daltons. Reverse phase HPLC analysis of synthase tryptic peptides from either hormone responsive cardiomyocytes or synthase treated in vitro with cAMP-dependent protein kinase and protein phosphatase-1 (PP-1) resulted in finding six reproducible peaks of phosphopeptides. The incorporation of radioactivity into peaks 1 and 2 was associated with both the treatment of cardiomyocytes with epinephrine and the in vitro phosphorylation of rat heart synthase with cAMP-dependent protein kinase. These same two peaks are selectively dephosphorylated when cAMP-dependent kinase treated synthase is incubated with protein phosphatase-1. This dephosphorylation of peaks 1 and 2 are coincident with the conversion of synthase from the D to the I form. Peak 3 is dephosphorylated upon treatment of cardiomyocytes with insulin and hyperphosphorylated in cardiomyocytes derived from alloxan diabetic animals. Taken together these results demonstrates the direct relationship between the phosphopeptides in peaks 1 and 2 and the inhibition of synthase activity in response to epinephrine treatment in the cell. This inhibition can be explained by the activity of cAMP-dependent protein kinase which can duplicate the intracellular, epinephrine-stimulated synthase phosphopeptide pattern. This inhibition can be relieved in vitro by protein phosphatase-1 which dephosphorylates peaks 1 and 2. The effect of insulin and alloxan diabetes is localized to peak 3 whose phosphorylation is unaffected in vitro by either cAMP-dependent protein kinase or protein phosphatase-1.

Indiana University-Purdue University Indianapolis (IUPUI) / The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Glycogen synthase (GS) is the rate-limiting enzyme for glycogen synthesis. Recent evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these diseases. Herein, we describe the discovery, characterization, and development of small molecule inhibitors of GS through a multicomponent study including biochemical, biophysical, and cellular assays. Adopting an affinity-based fluorescence polarization assay, we identified a substituted imidazole molecule (H23), as a first-in-class inhibitor of yeast glycogen synthase 2 (yGsy2) from the 50,000 ChemBridge DIVERSet library. Structural data derived from X-ray crystallography at 2.85 Å, and enzyme kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2. Medicinal chemistry efforts examining over 500 H23 analogs produced structure-activity relationship (SAR) profiles that led to the identification of potent pyrazole and isoflavone compounds with low micromolar potency against human glycogen synthase 1 (hGYS1). Notably, several of the isoflavones demonstrated cellular efficacy toward suppressing glycogen accumulation. In an alternative effort to screen inhibitors directly against human GS, an activity-based assay was designed using a two-step colorimetric approach. This assay led to the identification of compounds with submicromolar potency to hGYS1 from a chemical library comprised of 10,000 compounds. One of the hit molecules, hexachlorophene, was crystallized bound to the active site of yGsy2. The structure was determined to 3.15 Å. Additional kinetic, mutagenic, and SAR studies validated the binding of hexachlorophene in the catalytic pocket and its non-competitive mode of inhibition. In summary, these two novel assays provided feasible biochemical platforms for large-scale screening of small molecule modulators of GS. The newly-developed, potent analogs possess diverse promising scaffolds for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation. / 2021-07-01

No description available.

Type 1 diabetes (T1D) is the result of the autoimmune-mediated destruction of the pancreatic beta-cells leading to the inability to produce insulin sufficiently and, in turn, regulate blood glucose levels. Abnormal levels of blood glucose, specifically hyperglycemia, have been linked to many diabetic complications, with Brownlee proposing decreased GAPDH activity and the resultant increase in four main pathways as the mechanism(s) leading to these complications. Though skeletal muscles play a major role in glucose uptake, they are believed to be relatively protected against these complications as they are able to regulate their glucose uptake. However, evidence is accumulating that skeletal muscles are adversely affected in T1D, particularly with respect to their mitochondrial function. This led us to consider that the skeletal muscles of those with T1D would experience substrate overload (high intracellular lipids and recurrent, high levels of intracellular glucose), which would initiate a negative spiral whereby substrate excess would damage mitochondria - leading to an impaired ability to utilize these substrates - further worsening the substrate overload. Therefore, the objective of this study was to investigate glycogen and intramyocellular lipid (IMCL) content in the muscles of mice and humans with T1D, as well as the potential downstream effects in the form of post-translational modifications (PTMs), mitochondrial content, and lipofuscin accumulation. The Akita T1D mouse model was used to assess substrate overload in uncontrolled diabetes, whereas human participants were used to investigate substrate overload in the presence of insulin therapy. Assessment of glycogen and IMCL content revealed no difference between controls and diabetic cohorts in both the rodent and human study, indicating the lack of substrate overload. Post-translational modifications did not significantly change between Akita and wild-type mice; however, there was a main effect of diabetes on acetylation levels within Akita mice. Lastly, most mitochondrial properties, except for subsarcolemmal pixel density, did not differ either between diabetic and non-diabetic subjects in the human study. Thus, despite mitochondrial complex impairments in diabetic subjects, its extent was not significant enough to cause alterations to the mitochondria as a whole and result in mitochondrial degradation and lipofuscin formation. This study has provided novel insight into the metabolic properties of skeletal muscle during diabetes. Although there was no indication of substrate overload, diabetes still resulted in some changes to PTM levels and mitochondrial pixel density. However, the effects of these changes did not significantly alter the muscle and resulted in pathway impairments of those that were studied. This could be due to an adaptive mechanism in mice, although future studies are needed to confirm this hypothesis. In the human study, healthy, well-controlled individuals could explain why there was hardly any difference seen, suggesting that controlling glycemic levels was imperative in preventing diabetic complications in muscle. / Thesis / Master of Health Sciences (MSc)

Masters of Science / The infants of women who smoke during pregnancy have a lower birth mass than those born of women who abstain. Animal studies reveal that reduced growth due to maternal nicotine exposure during gestation is accompanied by lung hypoplasia. Biochemical analysis suggests that these lungs contain more cells which implies that lung damage occurs. In this study we examined the in vivo effects of maternal nicotine exposure (lmg/Kg/day), the equivalent of 32 cigarettes per day, on the following parameters of fetal and neonatal Wistar rat lung:(i) the content and distribution of glycogen in fetal and neonatal lung (ii) the status of connective tissue in neonatal lung (iii) the cell composition of the alveoli in neonatal lung. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Transmission electron microscope (TEM) techniques were employed to investigate the characteristics and composition of the alveolus The results show clearly that maternal nicotine exposure elevates pulmonary alveolar macrophage numbers'(PAM's) and lung glycogen levels. The quantity of elastic fibres in 1 day old neonates was significantly reduced but no changes in the quantity of reticulin and collagen fibres was observed. As a result of this change in connective tissue status, emphysema-like lesions and alveolar collapse was evident in the lungs of nicotine-exposed pups. TEM investigations revealed that changes to the composition of alveoli occurred. These included increased numbers of type II pneumocytes with high numbers of lamellar bodies with degenerative changes. Thickening of the blood-air barrier was also observed. The effect of maternal nicotine exposure has been documented in this study. However, it has not been possible to pinpoint the mechanisms involved but explanations have been proposed. Further research is required to elucidate the mechanisms by which nicotine produces these effects. Information thus obtained could help prevent the harmful effects to the fetus and neonate caused by smoking during pregnancy.

<p>  </p> <p>Breast cancer is a significant public health concern being the second leading cause of cancer-related death in women, with a projected 43,250 deaths in the US in 2022. However, cancer progression to metastatic sites is the primary cause of death in breast cancer patients. A hallmark of cancer is the dysregulation of cellular metabolism. Cancer cells have the ability to hijack their metabolism and drive cellular processes supporting cancer progression. As cancer cells continue through the metastatic cascade, they are challenged with various bioenergetic processes that can be supported by the influx of glucose. Thus, altering glycogen accumulation, where glucose is stored in cells, may be beneficial in supporting cancer progression. In this study, we aim to determine what drives glycogen accumulation in metastatic cells and if it is utilized to support cancer progression. We employed the non-metastatic MCF10A-<em>ras</em> and the metastatic MCF10CA1a cells for these studies. Our results demonstrate that metastatic MCF10CA1a have 20-fold accumulation of glycogen compared to the MCF10A-<em>ras</em> cells. Utilizing 13C6-glucose flux analysis, surprisingly, most of the glucose incorporated into glycogen of the MCF10CA1a cells was in the M+5 glucose labeling pattern instead of the expected M+6 pattern which occurs when glucose is directly converted to glycogen. We showed that glycogen was accumulated due to increased gluconeogenesis through cataplerosis (PEPCK) utilizing inhibitors of the enzyme. Additionally, in a pulse-chase experiment using 13C6-glucose flux analysis, there was an approximate 50% reduction in labeled glucose in glycogen, 3 hours after removing the label, suggesting that the MCF10CA1a cells also have a rapid turnover of glycogen. Glucose can be released through two mechanisms, glycogenolysis or glycophagy. Utilizing siRNAs to a rate limiting steps in each pathway, results suggest both glycogenolysis (PYGL) and glycophagy (GAA) are necessary to support cell migration, a critical step in metastasis of the MCF10CA1a cells. Thus, glycogen metabolism is dysregulated in the MCF10CA1a breast cancer cells such that they have increased glycogen accumulation and that glycogen is required to support cell migration. Further understanding the mechanism by which glucose is accumulated and released in a specific cancer and in specific steps or stressors in cancer progression may contribute to potential therapeutic targets to help mitigate metastasis, and potentially breast cancer mortality.</p>

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No description available.

Cocaine is a highly abused psychostimulant with repeated use potential culminating in addiction, a disease associated with compulsive drug seeking, use and high rates of relapse despite adverse consequences. It is well established that cocaine acts by binding to and blocking monoamine transporters therefore increasing synaptic extracellular monoamine concentrations. Cocaine also increases extracellular levels of the excitatory amino acid glutamate within the neural circuitry comprising the ascending dopamine system. Cocaine induces a number of behavioral and neurochemical manifestations following acute and repeated administration. As such, elucidating the molecular mechanisms involved in the behavioral and neuromodulatory effects of cocaine are critical to the development of effective pharmacotherapies for cocaine addiction. The overall aim of this research was to identify a novel kinase that may be involved in the behavioral effects of cocaine. Thus, we chose to investigate glycogen synthase kinase-3 (GSK3), which has recently gained attention as being critical in dopaminergic and glutamatergic signal transduction. GSK3 is a critical mediator of many intracellular signaling systems. The activity of GSK3 is regulated by several kinases including Akt, with inactivation occurring via phosphorylation of the inhibitory serine-21(α-isoform) and serine-9 (β-isoform) residues. It is well established that acute cocaine administration causes hyper-locomotion in animal models and that repeated cocaine administration elicits a sensitized or increased response to the locomotor-stimulating properties of the drug. The studies outlined herein sought to determine whether non-selective and selective inhibition of GSK3 would regulate acute cocaine-induced hyper-locomotion. Further, we investigated the role of GSK3 in the development of cocaine-induced locomotor sensitization. Results of the research outlined herein demonstrate that pharmacological inhibition of GSK3 reduced both the acute behavioral responses to cocaine and the long-term neuroadaptations produced by repeated cocaine, therefore suggesting a role for GSK3 in the behavioral manifestations associated with cocaine exposure. Previous studies have assessed the role of the dopamine D1 receptor in locomotor behaviors. As cocaine indirectly activates dopamine D1 receptors, we investigated whether activation of GSK3 was necessary for the expression of dopamine D1 receptor-mediated behaviors. To assess the role of GSK3 in dopamine D1 receptor-induced hyperactivity, GSK3 was inhibited prior to administration of the selective dopamine D1 receptor agonist SKF-82958. Selective inhibition of GSK3 reduced ambulatory and stereotypic activity produced by SKF-82958. These data implicate a role for GSK3 in the behavioral manifestations associated with dopamine D1 receptor activation. To further assess the importance of GSK3 in cocaine-induced behaviors we investigated the role of GSK3 in various facets of cocaine-conditioned reward. We show that selective inhibition of GSK3 prevented the development of cocaine-conditioned reward using a conditioned place preference paradigm, indicating a reduction in the rewarding properties of cocaine. Relapse to drug-seeking can be precipitated by certain stimuli including the drug itself, drug-paired contextual cues and stress. Memory of drug-paired cues is highly resistant to extinction and the molecular mechanisms underlying relapse have not been clearly defined. Our results demonstrate that inhibition of GSK3 interfered with the reconsolidation of cocaine-associated contextual memories by preventing the retrieval of cocaine conditioned place preference. Inhibition of GSK3 in a neutral environment 24 hours prior to the test for reinstatement, however, did not prevent reinstatement of cocaine place preference following a cocaine priming injection. Thus, our results indicate that GSK3 serves an important role in cocaine-conditioned reward and is a critical intracellular signaling protein for the development of cocaine place preference. GSK3 is also essential to the reconsolidation and subsequent retrieval of cocaine-associated contextual cues. In addition to studying the role of GSK3 in cocaine-induced behaviors, we assessed the neuromodulatory effects of cocaine on GSK3 activity. As stated previously, the activity of GSK3 is regulated by a number of kinases including Akt (protein kinase B). Recent evidence suggests that psychostimulants regulate the activity of Akt and subsequently GSK3 in various brain regions. Here, the ability of cocaine to regulate the activity of Akt and GSK3 was investigated. Enzymatic activity was assessed by determining protein phosphorylation in the brain. Mice administered acute injections of cocaine showed a significant decrease in phosphorylated Akt (Thr. 308) and GSK3β; in the caudate putamen as determined by Western blot analysis. Cocaine did not alter pAkt (Thr. 308) or pGSK3β; in the nucleus accumbens or frontal cortex. The role of dopaminergic and glutamatergic receptors on cocaine-induced attenuation of pAkt (Thr. 308) and pGSK3β; was also assessed. Blockade of the dopamine D1, D2 or glutamatergic NMDA receptor prevented cocaine-induced attenuation of pGSK3β; in the caudate putamen. Only blockade of the dopamine D2 receptor prevented the effect of cocaine on pAkt (Thr. 308) levels in the caudate putamen. The results of the present study indicate that the activity of Akt and GSK3 is selectively regulated in the brain following acute cocaine, an effect that is contingent upon both dopaminergic and glutamatergic receptor regulation. In summary, the experiments described in this dissertation tested the initial hypothesis that GSK3 mediates acute cocaine-induced hyperactivity and locomotor sensitization. Acute cocaine administration increased the phosphorylation of GSK3 in the caudate putamen, therefore enhancing kinase activity. Further, the increase in GSK3 activity following cocaine administration is contingent upon activation of the dopamine D1 and D2 receptors and the glutamatergic NMDA receptor. Results presented herein also demonstrate a role for GSK3 in cocaine-conditioned reward. Selective inhibition of GSK3 prevented the development of cocaine conditioned place preference. Inhibition of GSK3 also prevented the retrieval of cocaine contextual memories, therefore playing an important role in reconsolidation. Thus, the results presented in this dissertation indicate that GSK3 is a neuromodulator of cocaine-induced behaviors and may be an important factor underlying cocaine addiction. / Pharmacology