Browsing by Author "Tjalkens, Ronald B., committee member"
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Item Open Access Amino acid transmitters and the neural control of feeding and energy homeostasis(Colorado State University. Libraries, 2016) Dicken, Matthew S., author; Hentges, Shane T., advisor; Tamkun, Michael M., committee member; Amberg, Gregory C., committee member; Tjalkens, Ronald B., committee memberConsuming the correct number of calories to maintain a healthy bodyweight is a delicate balancing act between intake and energy expenditure, and humans in modern society seem to have a keen knack for throwing the balance off-center. In the U.S. alone, more than 1/3 of adults are obese based on the body mass index scale, and $147 billion is the estimated annual medical cost for obesity in the United States. On the other end of the feeding spectrum, anorexia in the U.S. has been steadily rising since the 1960s, and has the highest mortality rate of any mental illness. While great strides have been made in understanding the neuronal regulation of energy balance, there is a need to more fully understand the homeostatic systems within the hypothalamus that are so powerful that they are able to drive individuals to poor health or death, often even in the face of consciously fighting their urges. Two groups of functionally opposed neurons contained within the arcuate nucleus of the hypothalamus, Neuropeptide Y / Agouti-related peptide (NPY/AgRP) and proopiomelanocortin (POMC) cells (the so-called first order feeding neurons), have been extensively studied for their roles in energy homeostasis—mostly through research into the peptides they are named after. There is clear evidence that peptides play an essential role for the function of NPY/AgRP and POMC cells, but what about simple amino acid transmitters? While it is known that GABA is packaged and released by NPY/AgRP cells and that this release is relevant to feeding behavior, there is still a dearth of information about this aspect of the circuitry, very much an area waiting to be mined. This study focuses on better understanding the functional release and relevance of amino acid transmitters packaged in both NPY/AgRP and POMC cell populations. Evidence is presented here for the conclusive release of both GABA and glutamate from POMC cells within intact circuitry. For NPY/AgRP neurons, evidence is presented for a shift in functional release of GABA from these neurons onto POMC cells depending on feeding state, corroborated by concurrent in situ hybridization experiments. Using a combination of electrophysiology and in situ hybridization approaches, evidence is also provided that mRNA levels of glutamate decarboxylase can act as a general proxy for functional GABA release. Altogether, these results indicate that amino acid transmitters play a significant role in first order feeding neuron physiology. Not only does this warrant further study on the significance of each transmitter alone and their purpose in comparison with the peptides released, but also the interplay between POMC cell and NPY/AgRP cell amino acid transmitters and their many shared downstream targets. Imbalances in proper glutamatergic and GABAergic signaling may significantly contribute to obesity, and advancing this area of study could lead to correcting those imbalances to restore healthy energy homeostasis.Item Open Access An investigation of the molecular complexities that regulate molting in decapod crustaceans(Colorado State University. Libraries, 2015) Pitts, Natalie Lynn, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Tjalkens, Ronald B., committee member; Tsunoda, Susan, committee memberMolting in decapod crustaceans is regulated by the interaction of two hormones, molt inhibiting hormone (MIH) and ecdysteroids. Ecdysteroids are steroid hormones secreted from the molting gland or Y-organ (YO) and fluctuations in hemolymph ecdysteroid titers regulate progression through the molt cycle. Secretion of ecdysteroids is controlled by the peptide hormone MIH, which is synthesized and released from the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). The field of crustacean endocrinology has mainly focused on understanding the molecular underpinnings of MIH’s action on ecdysteroid production in the YO. The goal of this dissertation was to examine how MIH synthesis and secretion from the XO/SG complex contributes to molt cycle progression. Blackback land crabs, Gecarcinus lateralis, were induced to molt via autotomy of five or more walking legs (multiple limb autotomy or MLA). ESG were collected from intermolt, premolt, and post-molt animals and changes in expression of Gl-MIH and mTOR signaling pathway components were investigated. There was a significant effect of molt stage on Gl-MIH and mTOR signaling pathway gene expression in the ESG of G. lateralis. Continuous elevation of MIH transcript abundance during pre and post molt indicates that MIH titers in the hemolymph are not regulated by changes in transcript abundance. Molting also significantly increased expression of Gl-Akt, Gl-mTOR, Gl-Rheb, and Gl-S6K in one or more molt stages. Akt inhibits the tuberous sclerosis complex allowing for the activation of Rheb. Rheb is a GTPase that binds and activates the mechanistic target of rapamycin (mTOR). mTOR activates S6 kinase (S6K), increasing protein synthesis. ESG of naturally molting green crabs, Carcinus maenas, were also collected from intermolt, early premolt, and post molt animals. Molting had little effect on gene expression in C. maenas, confirming previous findings that molt progression is regulated post transcriptionally. This dissertation identifies a novel nitric oxide (NO) binding protein in the SG of C. maenas. The hypothesis is that NO negatively regulates MIH secretion from the SG thereby controlling molt progression. This unidentified endogenous binding protein allows NO to be present in the SG for a prolonged period and can therefore continually regulate neuropeptide release. Localization of the enzyme that produces NO (nitric oxide synthase; NOS) and MIH in the SG of C. maenas, G. lateralis, and Metacarcinus magister is consistent with the hypothesis that NO is a regulator of neuropeptide release in the crustacean SG. The second goal of this dissertation was to explore why some crustacean populations or individuals within a population are refractory to molt induction. The Bodega Bay population of C. maenas is refractory to molt induction techniques and a similar phenomenon is observed in G. lateralis. Some G. lateralis induced to molt via MLA did not enter premolt 90 days post induction and were classified as "blocked." These animals underwent a second molt induction technique, eyestalk ablation (ESA), and YO, brain (Br), and thoracic ganglia (TG) were collected at 1, 3, and 7 days post ESA. Gene expression of MIH and mTOR signaling pathway genes was examined in all three tissues (see Figure 3 for MIH signaling pathway components and there interactions). Results from this experiment suggested that a similar mechanism of molt resistance exists between C. maenas and G. lateralis. ESA did not increase hemolymph ecdysteroid titers of blocked animals, whereas ESA significantly increased ecdysteroid titers in control and intermolt animals. Gl-MIH expression in the ESG and expression of many Gl-MIH signaling components in the YO were upregulated in blocked animals, suggesting that the blocked animals were in a "hyper-repressed" state, and therefore resistant to molt induction by ESA and MLA. In both species, MIH is expressed in the Br and TG. The hypothesis is that MIH secretion from these other central nervous system (CNS) tissues contributes to a resistance to molt induction techniques. Expression of MIH signaling pathway genes is unchanged in the Br and TG in response to ESA. These data suggest that MIH does not activate a signaling pathways in CNS tissues but like the ESG, MIH is synthesized and secreted from these tissues. This experiment also supports the growing body of literature that mTOR inhibition activates downstream transcription factors which are important in maintaining energy homeostasis in times of environmental stress.Item Open Access Characterization of selected gene expression patterns as potential markers for oocyte quality in young versus old mares(Colorado State University. Libraries, 2010) Rodrigues, Bernardo de Lima, author; Clay, Colin M., advisor; Carnevale, Elaine M., advisor; Tjalkens, Ronald B., committee member; Bouma, Gerrit J., committee member; Bruemmer, Jason E., committee memberAs a female ages a series of alterations in normal physiology take place, during this process fecundity decreases. The reproductive system starts to shut down as a consequence of hormonal, histological and anatomical changes, and in the middle of this process, playing a critical role, is the oocyte. As in women, fertility decreases with aging in mares. Recently the mare has been suggested as a promising model to study age-related infertility in women due to similarities in the reproductive cycle and similar age-associated reproductive changes. During the last decades the use of assisted reproductive techniques (ART) in human and veterinary medicine to treat infertility has increased. Unfortunately, ART can only partially compensate for declining fertility- particularly the age related decline in fertility. Therefore, we need to understand the mechanisms involved in age-associated infertility and improve both the diagnostic tools and the techniques currently used in ART. In that regard the identification of reliable oocyte quality markers is of great interest, specifically of extrinsic markers in follicular cells and follicular fluid (FF). Follistatin (FS1) and anti-müllerian hormone (AMH) have been suggested as potential oocyte quality markers. In addition, the rate of apoptosis in follicular cells has also been suggested to be a good indicator of oocyte quality In this study, our goal was to use the young and old mare model to obtain competent and incompetent oocytes, respectively, to try and elucidate the involvement of apoptosis of follicular cells and/or of the oocyte in the determination of oocyte quality. Oocytes, follicular fluid, granulosa and cumulus cells were collected through transvaginal follicular aspirations from young ( 4-10 years) and old (>20 years) mares. Preovulatory follicles were aspirated 30-36 h post induction of follicular maturation, which was performed by administration (i.v) of a combination of deslorelin and hCG when the biggest follicle reached 35 mm. We used real time PCR to examine expression of pro-apoptotic ( CASP2, CASP 3) and pro-survival (XIAP) genes, as well as of FST and AMH expression in these cell types. In addition we measured androgens and estrogens in FF and calculated the androgens to estrogens ratio to assess follicular atresia. We also sought to determine FF concentrations of FST and AMH, and relate it to oocyte quality. There was no difference in CASP3 expression levels in granulosa and cumulus cells between the two age groups. In addition, there was no difference in CASP2 and CASP3 mRNA expression in oocytes from young and old mares. XIAP mRNA levels were expressed 3.3 fold higher in oocytes from young when compared to old mares, and there was a tendency for XIAP to be more highly expressed in granulosa cells of young mares. In contrast, the levels of XIAP mRNA in cumulus cells were 1.46 fold higher in old when compared to young mares. There was no difference in the expression levels of AMH in granulosa cells between young and old mares, but in cumulus cells there was a tendency for AMH to be higher expressed in cells from old vs. young mares. Unfortunately we were not able to analyze AMH FF concentrations. FST mRNA levels in oocytes were similar between the age groups, but FST concentrations in FF of preovulatory follicles from young mares (197 ± 16.7 ng/mL) were higher (p=0.02) than in FF from old (153.3 ± 22.7 ng/mL) mares. In both age groups FST FF concentrations in preovulatory follicles significantly decreased when compared to mid-estrus and post-deviation follicles. In conclusion, we believe that our data suggest that FST follicular fluid levels can be a non-invasive marker to assess oocyte quality in the horse, and that FST levels decrease in preovulatory follicles of the horse. In addition, expression levels of caspase-3 in follicular cells, and caspases 3 and 2 in the oocyte, does not seem to be involved in the mechanism of fertility loss in the old mare. Finally, XIAP mRNA levels may be important for oocyte quality in the horse.Item Open Access Cognitive effects of exposure to manganese in drinking water in California school children(Colorado State University. Libraries, 2010) Murphy, Eryn, author; Reif, John S., advisor; Tjalkens, Ronald B., committee member; Chen, Peter, committee memberThe hypothesis tested in this study was that low level exposure to manganese in children through drinking water is associated with impaired cognitive performance on standardized tests of intellectual function. The study was based on the pathological and toxicological effects of exposure to manganese in laboratory animals and recent epidemiologic evidence showing an association between exposures to manganese in drinking water and decreased intellectual function (IQ) as well as hyperactive behaviors in children. A clear analogy with lead exists. California has a statewide monitoring system for drinking water manganese which was used to identify school districts for water sampling and analysis. Target school districts were identified for sampling from the California Department of Public Health Drinking Water Program. 100 schools within those cities were identified with manganese concentrations ranging from 20 µg /L to over 900 µg /L. Water samples were collected (n=81), analyzed for manganese concentration (ppb), and grouped into three exposure categories High (>30µg/L), Low (2 -29µg/L), and ND (<2µg/L). Cognitive assessment was determined from standardized test score data for 3rd, 4th, and 5th grade children from the California Standardized Testing and Reporting Program (STAR) for each school. Analysis of Variance, Analysis of Covariance, and Mixed Effect General Linear Regression analyses were used to analyze the data and adjusted for covariates including age, gender, ethnicity, parental education and economic status. The results of this study did not suggest mean test scores to be significantly different between high, low, and non-detect manganese exposure groups when adjusted for confounders. Mean test scores were not highest in the non-detect exposure group and lowest in the high exposure group as predicted.Item Open Access Compensatory responses to oxidant stresses in vitro and in vivo(Colorado State University. Libraries, 2013) Khademi, Shadi, author; Hamilton, Karyn L., advisor; Miller, Benjamin F., advisor; Hickey, Matthew S., committee member; Tjalkens, Ronald B., committee memberEmerging evidence shows that reactive oxygen species (ROS) are not merely damaging agents causing random destruction to cell structure and function, but that they act as modulators of physiological processes (such as cell adaptation to physical exercise) by regulating gene transcription and protein synthesis. The exact redox signaling pathways involved in cell adaptations to oxidative stress are unknown. Since various stimuli can induce oxidative stress under different conditions in vivo and in vitro, different models are warranted to study the cell signaling pathways involved in compensatory responses to oxidative stress. The following investigation comprises a series of experiments with the overall aim of elucidating the role of redox sensitive pathways in inducing cellular responses to oxidative stress in vitro and in vivo. The experiments tested the general hypothesis that changes in the redox state of the cell, through hypoxia, contractile activity or direct application of hydrogen peroxide (H2O2), would cause antioxidant compensatory responses and cell adaptations. The specific aims of the experimental series were: 1) to determine whether pulmonary edema, evoked by cerebral hypoxia in the presence of systemic normoxia, will be accompanied by sympathetic activation, increased oxidative stress, and upregulation of endogenous antioxidant pathways, 2) to determine whether electrical stimulation (Es) induced contractile activity of cultured murine myotubes would induce energetic stress, redox sensitive signaling, and mitochondrial biogenesis, and 3) to determine whether treatment with H2O2 would result in a greater rate of mitochondrial biogenesis compared to control, and whether the increase would be maintained during co-treatment with either an exogenous antioxidant (vitamin C) or a nuclear erythroid 2 -related factor 2 (Nrf2) activator that increases transcription of endogenous antioxidants. Studies in aim 1 demonstrated that under pathologic conditions such as isolated cerebral hypoxia with systemic normoxia, tissue specific patterns of compensatory responses to the hypoxic stressor exist. Further, this study showed that the differences in lung and brain redox signaling pathways during hypoxia can have different systemic outcomes through modulation of the sympathetic nervous system (SNS). Studies in aim 2 demonstrated a unique model of contractile activity in vitro, which was successful in simulating the cellular adaptations to a single bout of endurance exercise such as greater rate of cytosolic protein synthesis, upregulation of antioxidants and mitochondrial protein markers as well as AMP activated protein kinase (AMPK). Studies in aim 3 demonstrated that H2O2 did not increase mitochondrial biogenesis. Further, increasing Nrf2 activation maintained the rate of mitochondrial protein synthesis during H2O2 treatment, while treatment with the exogenous antioxidant failed to restore the H2O2 induced decreases in mitochondrial biogenesis during H2O2 treatment. Collectively, we have used different models of oxidative stress in vitro and in vivo to evaluate some of the mechanisms involved in cell adaptations responses. Findings from these experiments provide insight into understanding the role of redox signaling in pathologic and non-pathologic circumstances and can help future therapeutic recommendations for battling the consequences of oxidative stress on health.Item Embargo Infectious disease, age, and environmental contaminants as neurotoxicants that modulate glia and contribute to neurodegenerative pathology(Colorado State University. Libraries, 2024) Latham, Amanda Shellee, author; Moreno, Julie A., advisor; Basaraba, Randall J., advisor; Tjalkens, Ronald B., committee member; Santangelo, Kelly S., committee member; Elf, Jessica, committee memberNeurodegenerative disease cases are expected to double over the next twenty years. These diseases, which include Alzheimer's Disease (AD) and Parkinson's Disease (PD), are incurable with a largely unknown etiology. It is acknowledged within the field that age is the greatest risk factor for neurodegenerative disease, and that genetics and environmental factors, such as neurotoxicants and infectious agents, likely play a role. Despite this knowledge, it is not entirely understood why select individuals are pushed into a state of disease, while others progress into a state of normal brain aging. This is further complicated by the shared neuropathology between brain aging and neurodegenerative disease, which includes blood-brain barrier (BBB) modulation, gliosis, misfolded protein accumulation, and loss of function or degradation of neurons. To address these gaps in our understanding, the studies herein provide valuable insight as to how infectious disease, specifically through infection with Mycobacterium tuberculosis, contributes to the progression of neuropathology, evaluates an alternative model of brain aging that better recapitulates human disease, and provides mechanistic understanding of the neuroprotective and neurotoxic roles of glia in disease. Altogether, these data elucidate the etiology and mechanisms that drive neurodegenerative disease, as well as possible therapeutic avenues that may bring us one step closer to a cure.Item Open Access Performance-computation tradeoffs in detection and estimation(Colorado State University. Libraries, 2023) Damale, Pranav U., author; Chong, Edwin K. P., advisor; Pezeshki, Ali, committee member; Tjalkens, Ronald B., committee member; Cavalieri, Renzo, committee memberDetection and estimation problems involve challenging tasks that often demand real-time, accurate results. Algorithms able to produce highly accurate results are often computationally expensive or inefficient. Naturally, we need to tailor algorithms to the specific needs of problems to optimally trade off between computation and accuracy. To explore this ever-present tradeoff, this dissertation describes three distinct problems in detection and estimation and our contribution to the decision-making process for choosing the best algorithms for solving these problems. First, we look at tradeoffs involved in designing a low-cost, camera-based autonomous gait acquisition and analysis system for inspecting gait impairments in mice. Specifically, we give a detailed description of our detection and classification algorithms for gait-event detection and gait-parameter extraction. Using the videos acquired in a live-animal study, we validate the performance of our system for assessing recovery in a mouse model of Parkinson's disease. Next, we analyze the tradeoffs involved in designing a modified data association algorithm for tracking multiple objects using measurements of uncertain origins, such as radar detection with false alarms and missed detection. Specifically, we explore the performance of the distance-weighting probabilistic data association approach in conjunction with the loopy-sum product algorithm and, using simulation data, we analyze its performance in terms of tracking accuracy and computation against other state-of-the-art data association methods for tracking multiple targets in clutter. Finally, to address the ill-conditioning of linear minimum mean square error estimation, we develop four approximate Wiener filter formulas that do not directly involve the inverse of the observation covariance matrix. Using real data, we evaluate the performance-complexity tradeoff for our approximated filters. The common underlying theme that connects our solutions to these distinct problems is that our decisions for selecting various parameters in each solution are based on the performance-computation tradeoff. Throughout this dissertation, we employ various methods to handle this tradeoff, such as receiver operating characteristics analysis and line-search procedure. Our analysis is beneficial for choosing the best algorithm to optimally trade off between performance and computation.Item Open Access Relationships between plasma cytokines, leukocyte telomere length, serum lipid profile, and nutrient intake in healthy adults following a 4-week dietary intervention study(Colorado State University. Libraries, 2016) Harbison, Gregory James, author; Ryan, Elizabeth P., advisor; Bailey, Susan M., committee member; Tjalkens, Ronald B., committee member; Weir, Tiffany L., committee memberColorectal cancer is the third most commonly diagnosed cancer worldwide and the fourth leading cause of cancer-related death. The etiology of colorectal cancer is predominately attributed to modifiable lifestyle factors that promote chronic inflammation, and only 20% of colorectal cases are credited to hereditary syndromes. Specifically, recent nutritional studies have suggested that diet modification is a promising lifestyle intervention for reducing systemic inflammation and promoting colorectal cancer prevention and remission. In particular, rice and navy beans have been identified as two foods with anti-inflammatory and anti-neoplastic properties that warrant evaluation for chemoprevention through dietary supplementation in humans. In this study, plasma cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, TNF, and VEGF) and leukocyte telomere length were measured at baseline, two weeks, and four weeks in individuals with and without a history of colorectal cancer who consumed a diet supplemented with rice bran, navy beans, or a placebo-control for 28 days. Serum lipid profile and nutrient intake were also measured. At baseline, the three diet intervention groups had no significant differences in cytokine concentration, telomere length, or lipid profile. At the end of the study, individuals with a history of colorectal cancer who consumed the navy bean supplemented diet had significantly higher plasma TNF and VEGF concentrations than individuals consuming the control diet. Otherwise, at the end of the study, no significant differences in cytokine concentration or telomere length between groups existed. Additionally, compared to males, females with a history of colorectal cancer had significantly longer telomeres at baseline but not at four weeks. Females with a history of colorectal cancer also had significantly lower IL-4, IL-6, and IL-10 at baseline, but no significant difference was found at four weeks. Linear correlation analysis on repeated measures that adjusted for sex, age, and total energy intake showed significant correlations between several study variables. Telomere length was inversely correlated with age, serum triglyceride level, carbohydrate intake, and saturated fat intake. IL-2 and IL-4 concentrations were inversely correlated with α-Tocopherol intake. IL-8 was inversely correlated with vitamin B3 intake. VEGF was positively correlated with vitamin B9 intake. Total serum cholesterol was positively correlated with saturated fat intake and inversely correlated with β-Carotene intake. Serum LDL was inversely correlated with β-Carotene intake, and serum HDL was positively correlated with intake of saturated fat and linolenic acid. Triglyceride level was inversely correlated with intake of β-Carotene and fiber and was positively correlated with selenium intake. Finally, comparison of two experimental methods for telomere length measurement showed positive but inconclusive correlations.Item Open Access Role of vascular hyperpolarization in muscle blood flow regulation in healthy humans(Colorado State University. Libraries, 2013) Crecelius, Anne Renee, author; Dinenno, Frank A., advisor; Chicco, Adam J., committee member; Tjalkens, Ronald B., committee member; Earley, Scott, committee memberThe following investigation composes a series of experiments with the overall aim of determining the role for vascular hyperpolarization via activation of inwardly-rectifying potassium (KIR) channels and Na+/K+-ATPase in the regulation of vascular tone in response to muscle contractions and ischaemia in young, healthy humans. We tested the general hypothesis that activation of KIR channels and Na+/K+-ATPase contributes in large part to the vasodilatory, hyperaemic, and sympatholytic responses observed in these conditions and this contribution is greater than that of other vasodilators, specifically nitric oxide (NO) and prostaglandins (PGs). The specific aims of each experiment were: 1) to determine whether K+-stimulated vascular hyperpolarization via activation of KIR channels and Na+/K+-ATPase mediates contraction-induced rapid vasodilatation in the human forearm; 2) to determine whether vascular hyperpolarization via activation of KIR channels and Na+/K+-ATPase contributes to the hyperaemic response at the onset of repeated muscle contractions, as well as to steady-state forearm blood flow during rhythmic handgrip exercise; 3) to determine whether vascular hyperpolarization via activation of KIR channels and Na+/K+-ATPase contributes to the observed blunting of sympathetically-mediated vasoconstriction that occurs during moderate intensity rhythmic forearm exercise; and 4) to determine whether vascular hyperpolarization via activation of KIR channels and Na+/K+-ATPase contributes to the observed reactive hyperaemia that occurs in the human forearm following release of temporary ischaemia. Our collective findings demonstrate a significant contribution of KIR channels and Na+/K+-ATPase activation to rapid vasodilatation following a single muscle contraction, the onset of exercise hyperaemia in response to repeated muscle contractions, steady-state muscle blood flow during rhythmic handgrip exercise and reactive hyperaemia following temporary ischaemia. In contrast to our hypothesis, we did not observe a significant contribution of KIR channels and Na+/K+-ATPase to the observed blunting of sympathetic α-adrenergic vasoconstriction that occurs during handgrip exercise. In all studies, any role of NO and PGs was modest, if present at all. Taken together, our findings indicate that during a variety of vasodilatory stimuli, there is a large contribution of pathways that are independent of NO and PGs, specifically activation of KIR channels and Na+/K+-ATPase. Hyperpolarization via activation of KIR channels and Na+/K+-ATPase represents a novel mechanistic pathway in the understanding of in vivo regulation of muscle blood flow in response to contractions and ischaemia. These findings may provide insight into understanding impaired vascular function in patient populations and as such, could represent a novel therapeutic target for reversing microvascular dysfunction.Item Open Access Super-resolution imaging reveals mechanisms of glutamate transporter localization near neuron-astrocyte contacts(Colorado State University. Libraries, 2021) Leek, Ashley N., author; Tamkun, Michael M., advisor; Hentges, Shane T., committee member; Tjalkens, Ronald B., committee member; Tsunoda, Susan, committee memberAstrocytes contact neurons at several locations, including somatic clusters of Kv2.1 potassium channels and synapses across the brain. A primary function of astrocytes at these locations is to limit the action of extracellular glutamate. Astrocytic glutamate transporters, such as Glt1, ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. Additionally, they act to limit glutamate induced hyperexcitability by preventing the spread of glutamate to extrasynaptic receptors. The role of Glt1 in limiting neuronal hyperactivity relies heavily on the localization and diffusion of the transporter in the membrane, however, little is known about the mechanisms governing these properties. The work presented in this dissertation examines the mechanisms of Glt1 localization near Kv2.1-mediated neuron-astrocyte contact sites. To that end, in Chapter 2, we used super-resolution imaging to analyze the localization of two splice forms of Glt1, Glt1a and Glt1b. In cultures of primary astrocytes, we find that Glt1a, but not Glt1b, is specifically localized over cortical actin filaments. We go on to discover that this localization is dependent on the Glt1a C-terminus, where Glt1a and Glt1b differ, as exogenous expression of the Glt1a C-terminus was able to prevent localization of Glt1a to cortical actin filaments. In the somatosensory cortex, astrocyte Glt1 forms net-like structures around neuronal Kv2.1 clusters, however the cause of this Glt1 localization pattern is unknown. In Chapter 3, using super-resolution imaging of mixed cultures of astrocytes and neurons, we replicate findings of astrocyte Glt1 in a net-like localization around neuronal Kv2.1 clusters. We discover that both astrocyte actin and ER were excluded from the region across from neuronal Kv2.1 clusters. The actin-Glt1a relationship discussed in Chapter 2 is likely responsible for the net-like appearance of Glt1, as astrocytic Glt1 and actin colocalize in nets around Kv2.1 clusters at points of neuron-astrocyte contact. Neuronal control over the astrocyte cytoskeleton appears central to this Glt1a localization, although the mechanism of this control is still unknown. Together, these data describe a novel interaction between the Glt1a C-terminus and cortical actin filaments, which localizes Glt1 near neuronal structures involved in detecting ischemic insult. Although the mechanism of neuronal control over the astrocyte cytoskeleton remains a mystery, presumably cell-cell contact has a major influence. Contacts between neurons and astrocytes at Kv2.1 clusters could be mediated by the Kv2.1 β-subunit, AMIGO, which acts a cell adhesion molecule. Only one member of the AMIGO family of proteins is known to be an auxiliary β-subunit for Kv2 channels and to modulate Kv2.1 electrical activity. However, the AMIGO family has two additional members of ∼50% similarity that have not yet been characterized as Kv2 β-subunits. In Chapter 4, we show that the surface trafficking and localization of all three AMIGOs are controlled by their interaction with both Kv2.1 and Kv2.2 channels. Additionally, assembly of each AMIGO with either Kv2 alters important electrophysiological properties of these channels. The coregulatory effects of Kv2s and AMIGOs likely fine-tune both electrical and cell adhesion properties of the neurons in which they are expressed. Altogether, the work presented in this dissertation further defines the composition of Kv2.1-induced neuron-astrocyte contact sites, representing the first significant addition to this field in more than a decade.Item Open Access Three guanylyl cyclases in the green shore crab, carcinus maenas: cDNA cloning and effects of molt stage on expression in molting gland (Y-organ)(Colorado State University. Libraries, 2010) Gunderson, Jennifer L., author; Mykles, Donald L., advisor; Reddy, Anireddy N., committee member; Tjalkens, Ronald B., committee memberYO ecdysteroidogensis is suppressed by molt-inhibiting hormone (MIH) and the pleiotropic neuropeptide crustacean hyperglycemic hormone (CHH) with these neuropeptides being produced in the X-organ of the eyestalk (ES) ganglia. CHH signaling is mediated by a membrane receptor guanylyl cyclase (GC), while MIH signaling may involve a soluble NO-sensitive GC. Here we report the cloning of cDNA sequences encompassing the catalytic domain of the~ subunit of a soluble NO-sensitive GC ( Cm-GC-I ß), membrane receptor GC ( Cm-GC-II), and soluble NO-insensitive GC (Cm-GC-III) from the European green crab, Carcinus maenas, using RT-PCR and RACE. Adult C. maenas occur as two color morphs that differ in growth traits; the "green" morphs molt more frequently than the "red" morphs. These data suggest that YOs in red morphs are less active than the YOs in green morphs, and that the red morphs are refractory to molt induction by eyestalk ablation (ESA). We tested the effects of ESA on the expression of NO synthase ( Cm-NOS), Cm-GC-I ß, Cm-GC-II, and Cm-GC-III in the YOs from green and red morphs using quantitative PCR. Elongation factor-2 (Cm- EF2) served as a constitutively expressed control. ESA caused a small, transient increase in hemolymph ecdysteroid titers in both morphs, with the increase occurring at 3 days post-ESA in green morphs and at 14 days post-ESA in red morphs. ESA had no significant effect on the expression of Cm-EF2, Cm-NOS, Cm-GC-II, and Cm-GC-III, which were not correlated with hemolymph ecdysteroid titers. ESA caused a significant decrease in Cm-GC-I ß expression in red morphs, but had no significant effect in green morphs. Expression was also quantified in green morphs undergoing spontaneous natural molting. There was no significant difference in the expression of Cm-EF2, Cm-NOS, Cm- GC-II, and Cm-GC-II in YOs from intermolt, premolt, and postmolt animals. By contrast, Cm-GC-III mRNA was about 2-fold greater in YOs from postmolt animals than in intermolt and premolt animals. The results show that both color morphs are resistant to ESA and that molting has little effect on NOS and GC expression.