A key issue in neuroscience is to determine the connection between neuronal circuits and behaviour. In the adult brain, all neuronal circuits include a glutamatergic component. Three proteins designated Vesicular glutamate transporter 1-3 (VGLUT1-3) possess the capability of packaging glutamate into presynaptic vesicles for release of glutamate at the nerve terminal. The present study aimed at determining the role of VGLUT2 in neuronal circuits of higher brain function, emotion, and reward-pocessing. A conditional knockout (cKO) strategy was utilised, and three different mouse lines were produced to delete VGLUT2 in specific neuronal circuits in a temporally and spatially controlled manner. First, we produced a cKO mouse in which Vglut2 was deleted in specific subpopulations of the cortex, amygdala and hippocampus from preadolescence. This resulted in blunted aspects in cognitive, emotional and social behaviour in a schizophrenia-related phenotype. Furthermore, we showed a downstream effect of the targeted deletion on the dopaminergic system. In a subsequent analysis of the same cKO mice, we showed that female cKO mice were more affected their male counterparts, and we also found that female schizophrenia patients, but not male patients, had increased Vglut2 levels in the cortex.  Second, we produced and analysed cKO mice in which Vglut2 was deleted in the cortex, amygdala and hippocampus already from midgestation, and could show that this deletion affected emotional, but not cognitive, function. Third, we addressed the role of VGLUT2 in midbrain dopamine neurons by targeting Vglut2 specifically in these neurons. These cKO mice showed a blunted activational response to the psychostimulant amphetamine and increased operant self-administration of both sugar and cocaine reinforcers. Further, the cKO mice displayed strongly enhanced cocaine-seeking in response to cocaine-associated cues, a behaviour of relevance for addiction in humans. In summary, this thesis work has addressed the role of the presynaptic glutamatergic neuron in different neuronal circuits and shown that the temporal and spatial distribution of VGLUT2 is of great significance for normal brain function.

Antibodies have the ability to influence the antibody response against the very antigen they are specific for, in a process called antibody feedback regulation. Depending on the nature of the antigen, the antibody response can be either enhanced or almost completely inhibited. This thesis focuses on the underlying mechanisms of antibody feedback regulation in vivo. Antigen-specific IgG can inhibit the antibody response to a particulate antigen. Based on its ability to inhibit B cell activation, the inhibitory FcγRIIB (low affinity receptor for IgG) has been suggested to be involved. Here we show that although FcγRIIB is required for efficient suppression in vitro, it is not required in vivo. Therefore, even though FcγRIIB can inhibit antibody responses, other mechanisms (such as epitope masking and enhanced antigen clearance) play a more dominant role in vivo. The antibody response to soluble antigen is greatly enhanced when it is introduced to the immune system in complex with antigen-specific IgG or IgE. We found that FcγRIIB attenuates the magnitude of IgG-mediated enhancement. In mice lacking FcγRIIB, IgG enhanced the antibody response much more efficiently than in normal mice. Since B cells require CD4+ T cell help in order to become antibody-producing cells, we examined the CD4+ T cell response to immune complexes in vivo. Using an adoptive transfer strategy with transgenic ovalbumin (OVA)-specific CD4+T cells, we could show that the enhanced OVA-specific IgG response to IgG2a/OVA and IgE/OVA complexes was preceded by a potent OVA-specific CD4+ T cell response. IgG2a-mediated enhancement was dependent on activating Fcγ receptors, whereas IgE-mediated enhancement was dependent on CD23, the low affinity receptor for IgE. We identified CD23+ B cells as the responsible effector cells for IgE-mediated enhancement in vivo. Taken together, these results show that Fc receptor-mediated antigen presentation is a major mechanism underlying antibody feedback enhancement.

Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These results indicate that Aire has an important function in peripheral tolerance by controlling the phenotype of myeloid-derived APCs and thereby regulating the activation of T and B lymphocytes. / Autoimmune diseases develop when the immune system fails to distinguish self from non-self or when the immune system is hypersensitive to endogenous or exogenous danger signals, or when a tissue erroneously sends a danger signal to the immune system. The education of the immune system to distinguish self from non-self is mainly carried out in the thymus and gives rise to central tolerance, whereas the ability to sense a danger or a healthy tissue constitutes peripheral tolerance. In these studies we have investigated the peripheral tolerance mechanisms controlled by the autoimmune regulator (Aire) gene in Aire deficient mice, an animal model of the monogenic disease autoimmune polyendocrine syndrome type I (APS I). Aire-/- mice displayed increased numbers of myeloid-derived antigen-presenting cells (APCs) in the spleen, lymph nodes and peritoneum as well as more blood monocytes and metallophilic macrophages in the spleen. Monocytes were also increased in the blood of APS I patients. Monocyte precursors displayed an accelerated development in the bone marrow of Aire-/- mice, and Aire-/- APCs had an altered phenotype that caused an increased immune response in several different contexts. Aire-/- splenic and lymph node dendritic cells had an increased ability to activate naive T cells, partly as a result of an upregulated expression of the costimulatory molecule VCAM-1. In Aire-/- mice increased activity of the metallophilic macrophages in the splenic marginal zone seems to be responsible both for the activated phenotype of marginal zone B cells and for the frequent development of marginal zone lymphoma with aging. In a TCR transgenic model Aire deficiency caused an increased superantigen-mediated TCR revision in the spleen, perhaps as a result of the altered phenotype of APCs in the spleen. Finally, Aire was shown to influence autoimmune disease development by a macrophage-dependent mechanism in diabetes induced with multiple low dose streptozotocin injections. These results indicate that Aire has an important function in peripheral tolerance by controlling the phenotype of myeloid-derived APCs and thereby regulating the activation of T and B lymphocytes.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

Fracture healing requires a series of events including inflammatory response and callus formation, callus remodeling and bone healing. Fracture healing is a complex process, there are several overlapping phases , including inflammation , cartilage formation and bone remodeling, there are many internal or external factors could impact on fracture healing, leading to delayed bone healing or non healing. The global androgen receptor knockout (GARKO) mice has been know to reduce bone mass in endochondral bone and osteoblast mineralization, but the impact for callus formation in fracture healing is still unclear. The goals of study is to investigate the role of androgen and androgen receptor in wild-type (WT) mice and GARKO mice after fracture healing during callus formation and bone mineralization and bone remodeling. Therefore, long-term animal experiments observed by micro-computed tomography to study the roles of androgen and androgen receptor on the process and mechanisms of fracture healing is necessary. We applied in vivo micro-computer tomography (Micro-CT) to build up the three-dimensional model images at different time points for wild-type mice and GARKO mice after fracture healing and observe the bone healing process of micro-structure of the development of callus during fracture healing. The callus tissue morphology observed by histological staining to study the proportion and position of collagen, fibrous tissue and bone. The results show that the healing of WT mice is better than GARKO mice. GARKO mice develop smaller callus size and less bone volume and show delayed healing. In general, orchiectomy (ORX) decreases callus size in WT mice but not in GARKO mice. However, the healing rate of elderly GARKO mice is not obvious in comparison with young GARKO mice. Together, our study demonstrated that the androgen and androgen receptor regulate fracture healing and play an important role in bone repair and healing. Our mouse model may be used for the therapeutic drug screening of bone fractures caused by osteoporosis.

The most active form of Vitamin D, 1alpha,25(OH)2D3, modulates cells via receptor mediated mechanisms. While studies have elucidated the pathway via the classical nuclear Vitamin D Receptor (VDR), little is known about the membrane-associated Vitamin D Receptor (ERp60). Caveolae and its characteristic protein Caveolin-1 have been involved in many signaling pathways due to its specific structure and physical configuration. Other studies have shown that many components of the Vitamin D pathway have been found in caveolae. This study hypothesizes that caveolae and Caveolin-1 are important for the effects of 1,25 Vitamin D signaling via ERp60. Research up to date have shown that in rat and mouse growth zone chondrocytes, cells deprived of intact caveolae either through disruption through beta-Cyclodextrin or genetic knockout do not exhibit the characteristic responses to Vitamin D through ERp60 when compared to chondrocytes with functional caveolae. Studies using immunofluorescence co-localization and caveolae fractionation have shown that ERp60 is localized in the caveolae domains. Cellular fractionation was also performed to examine the localization of the ERp60 receptor in lipid rafts and caveolae. Histology and transmission electron microscopy were also used to examine the physiological importance of caveolae and Caveolin-1 in growth plate morphology and cellular characteristics.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references (leaves 121-140).

Retinol (vitamin A) and its active metabolite, all-trans retinoic acid, signal through nuclear retinoic acid and retinoid X receptor (RAR/RXR) heterodimers. These complexes regulate the expression of genes involved in developmental processes such as limb development. In excess, retinoids are potent teratogens and cause marked reductive effects on the developing limb. The goal of this thesis was to elucidate the molecular mechanisms underlying retinoid-induced limb dysmorphogenesis. Specifically, using an in vitro limb culture system, I examined the involvement of one RAR isoform, RARgamma, in mediating retinoid insult. / My first objective was to examine how limbs deficient in RARgamma responded to exogenous retinoid exposure. I showed that RARgamma-null limbs (on an RARalpha1-null background) exhibited less severe limb defects following retinoid insult when compared to their wild-type counterparts. Additionally, the absence of RARgamma abolished the retinoid-induced misregulation of genes important for chondrogenesis (Sox9 and Col2a1 ) and limb outgrowth (Meis-1 and -2). / The next objective set out to determine how pharmacological activation of RARgamma affected limb development. The RARgamma-selective agonist (BMS-189961) caused limb dysmorphology (namely, effects on cartilage) that was comparable to pan-RAR activation with all-trans retinoic acid. A chondrogenesis-focused gene array analysis identified Mgp and Gdf10 as two RARgamma-responsive genes that may mediate retinoid-induced limb insult. / Subsequently, I assessed the functional involvement of Mgp in mediating retinoid teratogenicity. Limbs were treated with all- trans retinoic acid and warfarin (an inhibitor of MGP); warfarin co-treatment rescued limbs from retinoid-induced insult. / My final objective was to determine the importance of Gdf10 in mediating limb development. Recombinant human Gdf10-soaked beads were implanted into distal limb structures; ectopic overexpression of Gdf10 in the web (but not the digital ray) resulted in marked proximal limb malformations. / Collectively, these studies have illustrated the importance of RARgamma in retinoid teratology and have identified several potential mechanisms by which retinoids cause limb defects.

The development of T cells in the thymus is a tightly regulated process. Any defect in thymic differentiation could result in autoimmune disorders, inability to ward off infections or neoplasm. Early thymocyte development requires signals mediated through the preTCR complex by the associated CD3 chains (gamma, delta, epsilon, and zeta). Research conducted towards this project has revealed that signaling modules within the intracytoplasmic domain of CD3epsilon is absolutely required for this process. Interestingly, our results emphasized the importance of the proline-rich sequence motif in preTCR mediated signaling events, such as the proliferation of double negative thymocytes and the regulation of TCR surface expression on double positive thymocytes in a stage-specific manner. The outcomes of this project may provide a better understanding of the mechanism of preTCR-mediated thymocyte differentiation and the role of CD3 chains in these processes.

Distraction Osteogenesis (DO) is a technique for bone lengthening and filling of bone defects following trauma, infection or resection of tumors. DO consists of an osteotomy of the bone to be lengthened, followed by controlled distraction of the bone segments with an external fixator until the desired lengthening is obtained (distraction phase). This is followed by the consolidation phase, during which the external fixator is kept in place until the newly formed bone in the distracted zone consolidates. This phase is long and may cause numerous problems. Ongoing research aims at finding a method to accelerate the consolidation of the newly formed bone. / Fibroblast Growth Factors (FGF) play a significant role in bone development and repair. FGF 18 has been shown to be the only FGF member to be expressed throughout both the distraction and the consolidation phases of DO. It was also reported that FGF18 is the physiological ligand of FGFR3. Therefore, we hypothesized that FGF18 and FGFR3 may have an important role in DO. / To test this hypothesis, we investigated DO in FGFR3 deficient mice (FGFR3-/-). (FGF18 deficient mice are not viable). A miniaturized DO apparatus was applied to the tibia followed by an osteotomy. Distraction began after a 5-day latency period at a rate of 0.2 mm/12 hours for 12 days. / Samples were collected at 3 time points comparing the mutants (FGFR3-/-) to their wild type litter' sates: end of distraction (17 days post-surgery), mid-consolidation (34 days post-surgery), and end of consolidation (51 days post surgery). The samples were analyzed using X-ray, DEXA, microCT, histology, biomechanical testing and Real-Time PCR. / Our results revealed that FGFR3 deficient mice showed accelerated bone formation compared to the W.T. littermates at mid-consolidation where the parameters measured revealed increased bone mineral density, bone mineral content and trabecular number in the mutant tibial samples. The newly regenerated bone consolidated faster in the FGFR3 knock-out mice and the bone was of better quality as revealed by biomechanical tests in which more force was needed to break the mutant bone because it exhibited higher resistance than the age matched wild-type sample. The marker gene expression patterns revealed an up-regulation of chondrogenic markers that suggest that the knock-out mice follow the endochondral ossification pathway during DO. All results were statistically significant. / These results show that signaling through FGFR3 acts to decrease bone formation during DO. Consequently, blocking FGFR3 may lead to accelerated bone formation in DO. This may have important clinical implications in attempts to improve the functional outcome of DO by decreasing the long duration that the external fixator has to be kept on.