Cardiac ischemia, stroke and some neurodegenerative disorders are all characterized by cell damage and death due to low oxygen levels. Comparative studies show that anoxia tolerant model systems present a unique opportunity to study "survival" instead of death in the complete absence of oxygen. The freshwater turtle (Trachemys scripta elegans) is unique in its ability to survive total oxygen deprivation for hours to days, as well as reoxygenation insult after anoxia. The broad objective of this study is to understand the modulation of key molecular mechanisms involving stress proteins and VEGF that offer neuroprotection and enhance cell survival in the freshwater turtle through anoxia and reoxygenation. In vivo analyses have shown that anoxia induced stress proteins (Hsp72, Hsp60, Grp94, Hsp60, Hsp27, HO-1); modest changes in the Bcl2/Bax ratio and no change in cleaved caspase-3 expression suggesting resistance to neuronal damage. These results were corroborated with immunohistochemical evidence indicating no damage in turtle brain when subjected to the stress of anoxia and A/R. To understand the functional role of Hsp72, siRNA against Hsp72 was utilized to knockdown Hsp72 in vitro (neuronally enriched primary cell cultures established from the turtle). Knockdown cultures were characterized by increased cell death associated with elevated ROS levels. Silencing of Hsp72 knocks down the expression of Bcl2 and increases the expression of Bax, thereby decreasing the Bcl2/Bax ratio. However, there was no increase in cytosolic Cytochrome c or the expression levels of cleaved Caspase-3. Significant increase in AIF was observed in the knockdown cultures that increase through anoxia and reoxygenation, suggesting a caspase independent pathway of cell death. / Expression of the master regulator of hypoxia, HIF1 alpha and its target gene, VEGF, were analyzed at the mRNA and protein levels. The results showed no significant increase in HIF-1 alpha levels but anoxia VE GF The levels of stress proteins and VEGF returned to control levels during reoxygenation suggesting robust ROS protection mechanisms through reoxygenation. The present study thereby emphasizes Trachemys scripta as an advantageous model to examine anoxia and reoxygenation survival without major damage to the brain due to it's modulation of molecular mechanisms. / by Shailaja Kesaraju. / Thesis (Ph.D.)--Florida Atlantic University, 2008 / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

The hypothalamus is the central regulator of systemic energy homeostasis, and its dysfunction can result in extreme body weight alterations. This small (3-4 mm in thickness in human) neuro-endocrine brain region, located just above the median eminence, is comprised of cell types that subserve specific metabolic and behavioral aspects of the control of body weight, as well as hepatic glucose production, body temperature, autonomic physiology, neuroendocrine axes, serum osmolarity and circadian rhythms. Insights into the complex cellular physiology of this region are critical to the understanding of obesity pathogenesis and its prevention and treatment; however, human hypothalamic cells are largely inaccessible for direct study. My thesis research focused on establishing an in vitro model for understanding the molecular neurophysiology of obesity using, as "proof-of-principle", neurons derived from human pluripotent stem cells (hPSCs) derived from individuals with monogenic forms of obesity. Three related projects are described in details: I. Differentiation of hypothalamic-like neurons from human pluripotent stem cells (Chapter 2) This project was designed to establish an in vitro model for studying hypothalamic cell-molecular physiology in neurons derived from hPSCs. After screening several morphogens and other molecules affecting neuronal differentiation, we developed a protocol that combined early activation of sonic hedgehog signaling followed by timed NOTCH inhibition resulting in the generation of hypothalamic arcuate nucleus (ARC)-like neurons. Neuronal cells expressing pro-opiomelanocortin (POMC), neuropeptide-Y/agouti-related protein (NPY/AgRP) were generated from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) obtained from patients with monogenic forms of obesity. These hypothalamic-like neurons accounted for over 90% of differentiated cells and exhibited transcriptional profiles characteristic of specific hypothalamic neurons (and explicitly lacking pituitary markers). Importantly, these cells displayed hypothalamic neuronal characteristics, including production and secretion of neuropeptides and responsiveness to metabolic hormones such as insulin and leptin. Nkx2.1 progenitor cells at 12 days of differentiation from iPSC integrated into the hypothalamus following injection into the lateral ventricle of NSG mice. Single cell transcriptome analysis of day 27 hESC-derived hypothalamic neurons enabled us to identify specific hypothalamic cell types (e.g. POMC, NPY, MC4R) based on transcript signatures. These findings, in the aggregate, supported the utility of these cells for elucidation of aspects of the cellular/molecular neurophysiology of body weight regulation. II. Using stem cell-derived hypothalamic neurons to investigate the neurophysiology of obesity caused by prohormone convertase 1/3 deficiency (Chapter 3). My second project investigated the use the hPSC-differentiated hypothalamic neurons to assess the cellular physiology of hESC-derived hypothalamic neurons with induced knockdown or mutations of proprotein convertase subtilisin/kexin type 1 (PCSK1, encodes prohormone covertase 1/3 (PC1/3)). Congenital hypomorphism for this gene causes a rare autosomal disorder that impairs the processing of specific proproteins to their more bioactive derivatives, affecting, for example, the processing of POMC, proinsulin and proglucagon. The consequences of inactivating mutations of PCSK1 include obesity, possibly due to impaired function of anorexigenic POMC arcuate neurons. To understand the molecular neurophysiology of the obesity in PC1/3-deficient subjects, we generated PCSK1 deficient hESC lines with CRISPR or by knocking down PCSK1 with shRNA, and assessed the POMC processing in the hypothalamic ARC-like neurons made from these lines. The ratios of adrenocorticotropic hormone (ACTH)/POMC, αMSH/POMC and β endorphin (BEP)/POMC proteins were significantly decreased, while total quantities of POMC peptides were greatly increased in PCSK1-deficient hESC-derived neurons, indicating impaired POMC processing caused by reduced PC1/3 protein. These results are consistent with the elevated plasma POMC and ACTH intermediates levels of in humans segregating for hypomorphic mutations of PCSK1, and the impaired pituitary POMC processing in the PC1/3 mutant mice. Interestingly, in day 28 PC1/3-deficient neurons, in addition to upregulation of POMC gene expression and protein, we found increases in some of the "downstream" proteolytic enzymes for POMC processing and the "upstream" transcription factor that regulates PCSK1 expression. The molecular mechanisms underlying the invocation of these possibly compensatory processes are under study. These findings provide confidence that the hypothalamic neurons generated by the techniques described in Chapter 2 display molecular phenotypes consistent with a mutation in one of the important neuropeptide processing pathways. III. Using iPSC-derived neurons to investigate the molecular pathogenesis of obesity in Bardet-Biedl Syndrome (Chapter 4). To further investigate the use of iPSC-derived neurons in the study of the neurobiology of obesity, I analyzed structural and molecular physiologic phenotypes cells derived from patients with Bardet-Biedl Syndrome (BBS). BBS is a rare autosomal recessive disease characterized by multiorgan dysfunction, including polydactyly, hyperphagic obesity, retinal degeneration, renal cysts and cognitive impairments. Eighteen discrete genes have been implicated in specific instances of BBS, and all cognate proteins that have been identified encode constituents of the basal body of the primary cilium. The primary cilium has also been implicated in other clinical obesities, including the Alstrom syndrome, and the effects of a highly prevalent FTO allele on body weight. We found that ciliogenesis and neurite outgrowth were affected in both BBS1 and BBS10 mutant iPSC-derived neurons as reflected by longer primary cilia, shorter neurite length, and fewer processes. Furthermore, insulin-induced AKT phosphorylation at Thr308 was greatly reduced in both BBS1 and BBS10 mutant neurons compared to controls. Overexpression of BBS10 fully restored insulin signaling in BBS10 mutant neurons by rescue of the tyrosine phosphorylation of insulin receptor. Co-immunoprecipitation assays indicated that both BBS1 and BBS10 interacted physically with the insulin receptor. Leptin signaling was also investigated in BBS mutant fibroblasts and neurons. Both BBS mutations impaired leptin-mediated pSTAT3 activation in both cell lines by affecting either the trafficking or the quantities of leptin receptor in these cells. These data demonstrate that BBS proteins are essential for insulin and leptin signaling in neurons and fibroblasts, in a cellular context independent of the effects of obesity. These studies further confirm the ability of iPSC-derived neurons to reflect aspects of the molecular pathophysiology of the patients from whom they are obtained, and to enable studies of these phenotypes in circumstances isolated from the secondary effects of adiposity per se.

A new era of pain management could be on the horizon. How Terry Snutch turned snail venom into a multi-million dollar painkiller.

Thesis (M.S.)--University of Alabama at Birmingham, 2009. / Title from PDF t.p. (viewed June 30, 2010). Additional advisors: Lori McMahon, Stephen Watts. Includes bibliographical references (p. 31-35).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 130-169).

Background: Light-adaptation is a multifaceted process in the retina that helps adjust the visual system to changing illumination levels. Many studies are focused on the photochemical mechanism of light-adaptation. Neural network adaptation mechanisms at the photoreceptor synapse are largely unknown. We find that large, spontaneous Excitatory Amino Acid Transporter (EAATs) activity in cone terminals may contribute to cone synaptic adaptation, specifically with respect to how these signals change in differing conditions of light. EAATs in neurons quickly transport glutamate from the synaptic cleft, and also elicit large thermodynamically uncoupled Cl- currents when activated. We recorded synaptic EAAT currents from cones to study glutamate-uptake events elicited by glutamate release from the local cone, and from adjacent photoreceptors. We find that cones are synaptically connected via EAATs in dark ; this synaptic connection is diminished in light-adapted cones. Methods: Whole-cell patch-clamp was performed on dark- and transiently light-adapted tiger salamander cones. Endogenous EAAT currents were recorded in cones with a short depolarization to -10mV/2ms, while spontaneous transporter currents from network cones were observed while a local cone holding at -70mV constantly. DHKA, a specific transporter inhibitor, was used to identify EAAT2 currents in the cone terminals, while TBOA identified other EAAT subtypes. GABAergic and glycinergic network inputs were always blocked with picrotoxin and strychnine. Results: Spontaneous EAAT currents were observed in cones held constantly at -70mV in dark, indicating that the cones received glutamate inputs from adjacent photoreceptors. These spontaneous EAAT currents disappeared in presence of a strong light, possibly because the light suppressed glutamate releases from the adjacent photoreceptors. The spontaneous EAAT currents were blocked with TBOA, but not DHKA, an inhibitor for EAAT2 subtype, suggesting that a / non-EAAT 2 subtype may reside in a basal or perisynaptic area of cones, with a specialized ability to bind exocytosed glutamate from adjacent cones in dark. Furthermore, these results could be artificially replicated by dual-electrode recordings from two adjacent cones. When glutamate release was elicited from one cone, the TBOA-sensitive EAAT currents were observed from the other cone. Conclusions: Cones appear to act like a meshwork, synaptically connected via glutamate transporters. Light attenuates glutamate release and diminishes the cone-cone synaptic connections. This process may act as an important network mechanism for cone light adaptation. / by Matthew JM Rowan. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Since the first successful derivation of human embryonic stem cells (hESC), rapid progress has been attained in the development of strategies in differentiation of these cells into various neural lineages, with the fundamental objective of using these cells for replacement and repair of damaged neuronal circuits in the central nervous system (CNS). Of particular interest are midbrain dopaminergic (mDA) neurons, which play a central role in regulation of voluntary movement. Degeneration or loss of function of mDA neurons in the nigrostriatal pathway is associated with Parkinson disease (PD). Stromal-Derived Inducing Activity (SDIA) is recognized as one of the most efficient methods in restricting ESC differentiation to a dopaminergic lineage, and refers to the property of mouse stromal cell lines such as PA6 or MS5 to cause ESC to differentiate to DA neurons. Although this strategy has been extensively used to generate mDA neurons from hESC, the biochemical nature of SDIA is yet unknown.  In the present study mDA neurons were generated from the BG01V2 hESC line by SDIA. To examine whether SDIA exerts its effect directly on hESC and is responsible for early dopaminergic induction, neural progenitor cells (NPC) were enyzmatically isolated from the co-cultures and allowed to differentiate in feeder-free conditions. The isolated cells were committed to a mesencephalic neural lineage, and were capable of maintaining their phenotype and developing into postmitotic mDA neurons in feeder-free conditions. The mDA neurons showed neuronal excitability and dopamine transporter function. The in vitro proliferation and differentiation of the NPC was also investigated by a BrDU incorporation assay. Next, the maintenance of cellular memory and capacity for proliferation of the mesencephalic NPC was assessed. The NPC could be expanded in vitro by five-fold as neurospheres for up to two weeks while retaining their DA differentiation potential, but did not retain a stable phenotype over extended periods of time. Preliminary transplantation experiments of neurospheres in striatal lesioned animals indicated, however, that these cells could survive and conserve their phenotype in vivo. To gain additional insight into the biochemical role of SDIA in early dopaminergic induction of hESC, the separate contributions of cell surface activity and secreted factors were examined. The data revealed that the PA6 cell surface activity promoted cell survival and was mainly responsible for enhanced neurogenesis of hESC, whereas secreted factors provided DA lineage-specific instructions. In order to identify the soluble factors responsible for the DA phenotype-inducing component of SDIA, the gene expression profile of PA6 cells was compared to that of cell lines lacking the DA-inducing property. A number of soluble factors known to be associated with CNS development that were highly expressed in PA6 cells were identified as potential DA differentiation-inducing candidates. These differentially-expressed genes included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). When these factors, termed SPIE, were applied to the hESC, they induced dopaminergic neuronal differentiation of hESC line, BG01V2 and other karyotypically normal hESC lines in vitro. Thus, it appears that SPIE comprises the DA phenotype-inducing property of SDIA. This may provide a simple and direct means of differentiating hESC to form DA neurons in a single step, without a requirement for co-culture, animal cell lines, or animal products. / QC 20100916

La presencia de glucógeno en las neuronas ha sido motivo de controversia durante las pasadas décadas. Sin embargo, está aceptado que las neuronas expresan la maquinaria necesaria para sintetizar glucógeno, pero no para degradarlo. La presencia de la glucógeno sintasa (GS) en las neuronas es un misterio y no existe ningún estudio que analice cuál es su función fisiológica en este tipo neuronal. Recientemente se ha establecido un paralelismo entre la GS neuronal y el caballo de Troya, ya que múltiples estudios han demostrado que una hiper-activación de la GS y la consecuente acumulación de glucógeno desencadenan la entrada en apoptosis. A pesar de ello, la neurona gasta energía para su transcripción y traducción, lo que hace pensar que su presencia en la neurona es importante para su correcto funcionamiento. Los objetivos de esta tesis doctoral son los siguientes: 1. Caracterización del metabolismo de glucógeno y de su presencia en neuronas. 2. Estudio de la maquinaria de degradación de glucógeno en neuronas y su regulación en hipoxia 3. Análisis de la síntesis de glucógeno en neuronas expuestas a hipoxia. 4. Evaluación de la función biológica de la GS en neuronas bajo condiciones de hipoxia. Los resultados de esta tesis revelan que las neuronas tienen una síntesis de glucógeno activa, y, además, lo acumulan en condiciones basales. Además, poseen la maquinaria necesaria para la degradación de glucógeno y degradan sus propias reservas en condiciones de hipoxia. La capacidad neuronal de degradación de glucógeno está presente en una situación in vivo, ya que las neuronas del modelo Drosophila melanogaster movilizan sus reservas en condiciones de hipoxia, y las neuronas de Purkinje en el ratón lo hacen tras una anoxia post-mortem prolongada. La maquinaria de degradación del polisacárido, desconocida hasta el momento, está mediada por la expresión de la glucógeno fosforilasa (GP). Las neuronas expresan la isoforma cerebral del enzima, pero no la muscular, como en el caso de los astrocitos. Esta isoforma está presente tanto en neuronas en cultivo como en neuronas procedentes de cortes de cerebro de ratón adulto. La hipoxia causa la defosforilación y activación de la GS. La GS sintetiza glucógeno activamente, aunque los niveles netos del polisacárido disminuyen en hipoxia. Por tanto, está teniendo lugar un ciclo aparentemente fútil en donde la síntesis y degradación se encuentran activas. Finalmente se ha demostrado que el metabolismo del glucógeno forma parte de la maquinaria de protección que activa la neurona para resistir a la hipoxia. En consecuencia, las neuronas que carecen la GS tienen una mortalidad más elevada que aquellas que sí que expresan el enzima. En el modelo de la mosca, el metabolismo del glucógeno neuronal también juega un papel en la tolerancia a la hipoxia y moscas con niveles reducidos de GS específicamente en la neurona muestran un empeoramiento en la respuesta tras un período de bajo oxígeno. En conclusión, esta tesis presenta evidencias de que las neuronas poseen un metabolismo activo de glucógeno que, además, juega un papel clave en la tolerancia de estas células a condiciones de hipoxia. / The presence of glycogen in neurons has been a matter of debate for the past decades. However, it is accepted that neurons express the necessary machinery to synthesize glycogen, but not for degrading it. The presence of Glycogen Synthase (GS) in neurons is a mystery and there is no study which approaches its physiological function in this cellular type. Recently a parallelism has been drawn between GS and the Trojan horse, since several studies have shown that an over-activation of GS and an accumulation of glycogen cause apoptosis in the neurons. Nevertheless, neurons waste energy for transcribing and synthesizing the protein, which suggests that its presence might be important for the normal functioning of the neurons. The aims of the thesis are the follows 1) Characterization of glycogen metabolism and its presence in neurons 2) Study of the glycogen degradation machinery in neurons and its regulation in hypoxia 3) Analysis of the synthesis of glycogen in neurons exposed to hypoxia 4) Evaluation of the biological function of GS in neurons under hypoxia conditions The results of this thesis reveal that there is an active glycogen synthesis under normal conditions. In addition, they express the necessary machinery to degrade the polysaccharide and degrade it under hypoxia conditions. The neuronal capacity of glycogen degradation is present in the in vivo situation, both in neurons from Drosophila melanogaster and from mice. The glycogen degradation machinery is mediated through the expression of glycogen phosphorylase (GP). Neurons express the brain isoform of the enzyme, but not the muscle, as astrocytes do. This isoform is present in neuronal cultures, as well as in neurons from adult mice brain slices. Hypoxia causes the dephosphorylation and activation of GS. GS actively synthesizes glycogen, although the global glycogen levels diminished in hypoxia. Indeed, an apparent futile cycle is taking place under hypoxia, where both synthesis and degradation are activated. Finally, we have demonstrated that glycogen metabolism is part of the protection machinery the neuron activates for tolerating the hypoxia conditions. Consequently, neurons without GS have a higher mortality rate that these who actively express the enzyme. In Drosophila melanogaster, flies with reduced GS levels specifically in neurons have an impaired phenotype in their reponse to hypoxic conditions. In conclusion, this thesis presents evidences that show neurons have an active glycogen metabolism which plays a key role in the neuronal response to hypoxia.

A series of studies is described here which explore the functional organisation of face- and object-processing neurones in the adult vervet monkey brain. This fundamental issue in high-level vision is addressed by the use of a novel molecular mapping technique that was developed for this purpose. / In the first study, the temporal dynamics of c-fos and zif268 expression were delineated in detail in the rat visual cortex. Knowing the precise temporal parameters of up-regulation (after onset of sensory stimulation) and down-regulation (after offset of sensory stimulation) of these genes was integral to optimising the temporal aspects of the stimuli to be used for subsequent mapping experiments. This study provided the critical information for devising stimuli with corresponding temporal parameters to those of c-fos or zif268 so that one could take advantage of the disparity between the expression of their mRNA and protein products in order to visualise activated neurones. / In the second study, the newly developed molecular mapping technique was validated in the rat auditory, visual and multisensory systems. First, bimodal audiovisual stimuli were designed using the data obtained from the first study. Then, through the combined histological detection of the mRNA and protein products of zif268, discrete populations of neurones responsive to either component of the bimodal stimulus were visualised. It was also observed that a third population of neurones was found that responded to the stimulation through both sensory modalities. The combined results from these two studies set the stage for addressing the issue of the organisation of face- and object-selective neurones of the inferior temporal cortex in the vervet monkey brain. / In the third study, the functional organisation of face- and object-selective neurones was examined using the molecular mapping technique. Based on the data gathered from the first two studies, suitable stimuli containing two distinct object classes (conspecific faces and non-face familiar objects) were designed with appropriate temporal parameters. / Finally, the last study provided an opportunity to address the issue of hemispheric asymmetry of function in the context of face processing in the non-human primate brain. Results support the notion that there may indeed be phylogenetic explanations for the hemispheric asymmetry observed in the human brain.

No description available.