Psychedelics are a subset of hallucinogenic drugs that exert their characteristic effects through agonist activity at the serotonin receptor 2A (5-HT2A). In this study, I aimed to characterize the modulatory role of the metabotropic glutamate subtype 2 receptor (mGluR2) in the 5-HT2A-specific rodent model of hallucinogenic action, head-twitch response (HTR). Secondly, I aimed to explore if 5-HT2A agonist-induced deficits in prepulse inhibition (PPI) of the startle response, an additional model of hallucinogenic action, could be produced in mice. Though 5-HT2A agonist-induced PPI deficits, which represent interruptions in normal sensorimotor gating, have been described in both rats and humans, attempts to translate this behavior to mice are rare. In contrast to prior gene knockout studies suggesting the mGluR2 is necessary for 5-HT2A agonist-induced HTR, mGluR2 knockout (Grm2-/-) mice still displayed HTR upon administration of the psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI). Additionally, DOI and lysergic acid diethylamide (LSD) produced unexpected improvements in PPI in male 126S6/Sv wild-type mice, depending on the experimental protocol used and the origin of the animals. Sex differences were observed as DOI-induced improvements in PPI were present in female 129S6/Sv mice of the same origin and tested with the same protocol as their male counterparts; this effect in females was absent in 5-HT2A knockout (Htr2a-/-) mice. The results of this study shed light on issues with replicability and reproducibility in science, the importance of highlighting the origin and background of animal subjects, and potential sex differences in hallucinogenic drug action.

The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway.

Epstein-Barr virus (EBV) is associated with a spectrum of benign and malignant lymphoproliferative disorders, including acute infectious mononucleosis (IM), Burkitt's lymphoma (BL) and immunosuppression-associated B cell lymphoproliferative disease (LPD). Immunosurveillance mediated by virus-specific cytotoxic T lymphocytes is believed to protect immunocompetent hosts from EBV-associated lymphoma and LPD. Due to the lack of an adequate animal model, however, the precise immunologic mechanisms which provide this protection have not been directly demonstrated in vivo. Human peripheral blood mononuclear cell-reconstituted C.B.-17-scid/scid mice (hu-PBMC-SCID mice) develop EBV-positive LPD following intraperitoneal injection of PBMC from EBV-seropositive donors. The SCID mouse disease mirrors human EBV-associated LPD in morphology, presence of the EBV genome, clonality, and patterns of expression of latent viral cellular differentiation antigens. The hu-PBMC-SCID mouse provides a unique small animal model of EBV+ LPD, and it was used in this study to examine the role of CD8+ CTL in controlling LPD. Survival time increase significantly when EBV-specific cytotoxic T-cell lines (CTL) are adoptive transferred into hu-PBMC-SCID mice, demonstrating suppression of LPD in vivoby a CTL-mediated virus-specific mechanism. Survival time also increases significantly with administration of alloreactive CTL lines, suggesting that a non-virus-specific mechanism also contributes to control of EBV-associated LPD by CTL. NOD-SCID mice reconstituted with PBMC from donors with latent EBV infection develop EBV+ LPD with significantly less frequency than do C.B.17-SCID mice reconstituted with PBMC from the same donors. Administration of anti-CD8 mAb to these mice depletes human CD8+ cells and increases the incidence of LPD to 100%, demonstrating that CD8+ T cells are neccessary for protection from EBV-associated LPD. Adoptive transfer of human CD8+ T cells, but not CD4+ T cells, prevents LPD in CD8-depleted NOD-SCID mice. In vivo depletion of CD4+ T cells prevents engraftment of human T cells, and LPD does not develop in most mice after CD4+ cell depletion. These studies are the first to directly demonstrate both the protective role of CD8+ T cells and a requirement for CD4+ T cells in EBV -associated LPD in an in vivo model.

The Snail protein functions as a transcriptional regulator to establish early mesodermal cell fate in Drosophila. Later, in germ band-extended embryos, Snail is considered a pan-neural protein based on its extensive expression in neuroblasts. The evidence presented in thesis links snail expression and function in CNS. Cloning and functional characterization of a novel snail homologue, in Drosophila, are also described here. Cloning of this gene, worniu (Chinese for snail), revealed that the neural function of snail is masked by this and another closely related gene escargot. Both Escargot and Worniu contain zinc finger domains that are highly homologous to that of Snail. These three members of Snail protein family are redundantly required for CNS development. Although not affecting formation of neuroblasts, the loss of expression of these three members correlates with disruption of Nb asymmetry and division. Downstream targets of Snail protein family, in these processes, are inscuteable and string. In mutant embryos, which have the three genes deleted, the RNA expression of inscuteable and string is significantly lowered. Consistent with the gene expression defects, the mutant embryos have loss of asymmetric localization of prospero RNA in neuroblasts and nuclear localization of Prospero protein in ganglion mother cells. Transgenic expression of inscuteable and string together, in the snail family deletion mutant, efficiently restores the Prospero expression in GMC, demonstrating that the two genes are key targets of Snail in Nbs. Like in the mesoderm, in CNS Snail function depends on interaction with dCtBP co-repressor. These results suggest that Sna [Snail] family of proteins control both asymmetry and cell division of neuroblasts by activating, perhaps indirectly, the expression of inscuteable and string.

In vivo DNA is compacted tightly, via its association with histones and non-histone proteins, into higher-order chromatin structure. In this state, the DNA is refractory to the cellular factors that require access to DNA. The repressive nature of chromatin is alleviated in part by the action enzymes that modify chromatin structure. There are two major groups of chromatin modifying enzymes: those that post-translationally modify histones by the addition of small chemical moieties and those that utilize the energy derived from ATP hydrolysis to physically disrupt chromatin structure. The SWI/SNF enzyme belongs to this latter group. The SWI/SNF complex was identified originally in yeast. Several of its subunits are required for the expression of a subset of inducible genes. The ATPase activity is provided by the SWI2/SNF2 protein. In mammals, there are two biochemically separable SWI/SNF complexes that contain either BRG1 or BRM, both homologs of yeast SWI2/SNF2. The yeast and mammalian SWI/SNF complexes are able to disrupt the Dnase I digestion pattern of in vitro assembled mononucleosomes and arrays, as well as facilitate the accessibility of restriction nucleases and transcription factors. The mechanism by which SWI/SNF functions has yet to be elucidated. SNF5 is a component of the yeast SWI/SNF complex. It is required for sucrose fermentation and mating type switching. The mammalian homolog of Snf5 is SNF5/INI1. SNF5/INI1 was identified simultaneously by two groups as a protein that shares homology with Snf5 and via a yeast two hybrid assay as a protein that interacts with HIV integrase (INtegrase Interactor). INI1 is a component of all mammalian SWI/SNF complexes purified to date. In humans, mutations and/or deletions in INI1 are associated with a variety of cancers, including malignant rhabdoid tumors, choroid plexus carcinomas, medullablastomas, primitive neuralectodermal tumors, and some cases of leukemia. Furthermore, constitutional mutations within INI1in individuals presenting with these tumors support the role of INI1 as a tumor suppressor. In this thesis, we show that Ini1 also functions as a tumor suppressor in mice. Approximately 20% of mice heterozygous for Ini1 present with tumors. Most of these tumors are undifferentiated or poorly differentiated sarcomas with variable rhabdoid features. All tumors examined to date show loss of heterozygosity at the Ini1 locus. We also show that Ini1 is essential for embryonic development. Mice homozygous-null for Ini1die between days 4 and 5.5 post-fertilization due to an inability to adhere to their substratum, form trophectoderm, and expand their inner cell mass. We further characterize the function of Ini1 in tumor suppression by generating mice heterozygous for both Ini1 and either Rb or p53. While heterozygosity at the Ini1 locus appears to have no effect on the rate of tumorigenesis in Rb-heterozygous mice, many of the tumors arising in compound heterozygous mice present with an altered morphology. This finding suggests that Ini1 may contribute to tumor progression due to loss of Rb. In contrast, mice compound heterozygous for Ini1 and p53 show a marked reduction in the rate of tumorigenesis compared to p53-heterozygous mice. Furthermore, the tumor spectrum is altered in these compound heterozygous mice. These findings suggest that Ini1 may function normally to repress p53 activity. Lastly, we show that expression of the Ini1 tumor suppressor itself is regulated tightly. Tissues and cells heterozygous for Ini1 express roughly equivalent levels of Ini1 protein and mRNA as their wild-type counterparts. We further show that this compensation is mediated by an increase in the rate of transcription from the wild-type Ini1 allele. Moreover, when exogenous Ini1 is introduced into Ini1-heterozygous cells, expression from the Ini1 promoter is reduced. These data indicate that a compensatory mechanism exists to ensure that the steady-state levels of Ini1 are constant. In summary, research detailed in this thesis has contributed to our understanding of the regulation of Ini1 as well as the role this protein plays in mammalian development and tumor suppression.

The overall objective of this thesis work was to develop new insights into the etiology of scleroderma, a human systemic autoimmune disease, by analyzing the autoantibodies to centrosome antigens that develop during the disease. Centrosomes are perinuclear organelles that form microtubule arrays, including mitotic spindles that ensure the faithful segregation of chromosomes during mitosis. These studies used a novel methodology to determine the prevalence of anti-centrosome autoantibodies in patients with scleroderma. Recombinant centrosome antigens were used to determine the antigenic specificity of anti-centrosome antibody subsets by immunoblotting. Centrosome marker antibodies were used in indirect immunofluorescence assays to distinguish centrosomes within the polymorphic staining pattern frequently given by scleroderma sera. We found that 43% of patients are autoreactive to centrosomes, a prevalence higher than has been reported for any other scleroderma autoantigen. Half of the centrosome-positive patients also had autoantibodies against other antigens used in scleroderma diagnosis. However, in the remaining half of these patients, anti-centrosome antibodies represented the sole class of autoantibodies that was detectable. Anti-centrosome antibodies were detected in only a small percentage of normal individuals and patients with other connective tissue diseases. These data suggest that anti-centrosome autoantibodies may represent a new diagnostic tool in scleroderma. Upon examination of anti-centrosome autoantibody development in an animal model, it appeared that this autoantibody specificity may develop in mice as a consequence of an infection. An infectious agent was isolated by plaque-formation from carrier mice. Further characterization of the infectious agent was undertaken to obtain information on its physical, morphological and cytopathological properties. The infectious agent was identified by sequence and unique antigenic properties to be homologous to the pig pathogen Mycoplasma hyorhinis. When reintroduced into naive mice, the murine mycoplasma triggered anti-centrosome autoantibody development. While anti-centrosome autoantibodies of IgM isotype are part of the repertoire of naive unimmunized mice, mycoplasma infection specifically triggered the development of anti-centrosome IgG. Moreover, centrosome autoreactivity was prevented by antibiotic treatment. The autoantibody response evolved to recruit additional specificities, having IgM isotypes, reactive to endoplasmic reticulum-associated autoantigens.

Itk is a member of the Tec family of non-receptor tyrosine kinases. It is expressed in T cells, NK cells, and mast cells. The purpose of this study was to determine the role of Itk in T cell development. Previous work from our lab and others has demonstrated that Itk is involved in signaling downstream of the T cell receptor and initial analysis of Itk-deficient mice revealed that these mice had some defects in T cell development. There are two stages of T cell development, the pre-T cell stage and the CD4+ CD8+ double positive stage, at which signals downstream of the T cell receptor are important. At the CD4+ CD8+ double positive stage, these signals direct two concurrent, but distinct processes known as repertoire selection and CD4/CD8 lineage commitment/differentiation. I show that there are only slight defects in development at the pre-T cell stage, presumably due to reduced TCR signaling. However these results clearly demonstrate that Itk is not essential at this stage of development. In contrast, repertoire selection, in particular positive selection, is significantly affected by the absence of Itk. Similarly, I show that Itk plays a role in lineage differentiation, although commitment to the appropriate lineage occurs normally in the absence of Itk.

The antibody class switch is achieved by DNA recombination between the sequences called switch (S) regions located 5' to immunoglobulin (Ig) heavy chain constant (CH) region genes. This process can be induced in cultured B cells by polyclonal stimulation and switching can be directed to specific antibody classes by certain lymphokines. These stimuli may regulate the accessibility of CH genes and their S regions to a recombinase as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged (germ-line) CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germ-line CH genes is unknown. How stimuli regulate the accessibility of CHgenes is also unclear. I report in this dissertation the structure of the RNA transcribed from the unrearranged Cγ1 gene in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4 (rIL-4). I will also show that an 150-bp region upstream of the first initiation site of germ-line γ1 RNA contains promoter and enhancer elements responsible for basal level expression and inducibility by phorbol 12-myristate 13-acetate (PMA) and synergy with IL-4 in an IgM+ B cell line, L10A6.2, and an IgG2a+B cell line, A20.3. The germ-line γ1 RNA is initiated at multiple start sites 5' to the tandem repeats of the γ1 switch (Sγ1) region. As is true for analogous RNAs transcribed from other unrearranged genes, the germ-line γ1 RNA has an I exon transcribed from the region 5' to the Sγ1 region.. The Iγ1 exon is spliced at a unique site to the Cγ1 gene. The germ-line γ1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acids in length. Elements located within the 150 bp region 5' to the first initiation site of germ-line γ1 RNA are necessary and sufficient to confer inducibility by PMA and synergy with IL-4 to a minimal thymidine kinase (TK) promoter in L10A6.2 cells but are not sufficient to confer this inducibility in A20.3 cells. Linker-scanning mutations demonstrated that these multiple elements function in a mutually dependent manner as indicated by the fact that mutation of any single element will decrease constitutive expression and inducibility by PMA and PMA plus IL-4. This 150-bp region contains several consensus sequences that bind to known or putative transcription factors, including a C/EBP binding site/IL-4 response element (in the promoter for Ia Aαkgene), four CACCC boxes, a PU box, a TGFβ inhibitory element (TIE), an interferon-αβ response element (αβIRE), and an AP-3 site. My results begin to provide a description of the mechanism of regulation of the accessibility of unrearranged germ-line Sγ1-Cγ1 gene. By activating the germ-line γ1 promoter, IL-4 induces transcription of germ-line γ1 RNA, thereby inducing accessibility of the Sγ1-Cγ1 gene. By inhibiting expression of the germ-line γ1 promoter, IFNγ and TGFβ down-regulate transcription of germ-line γ1 RNA, thus reducing the accessibility of the Sγ1-Cγ1 gene. My results also suggest that signaling via the antigen receptor on B cells may be involved in induction of switch to IgG1. Furthermore, this is the first case reported in which multiple functionally interdependent elements are needed to respond to PMA.

The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

This thesis investigates the role of B cells as antigen-specific antigen-presenting cells (APC) in self tolerance to low concentrations of soluble self proteins and in acquired tolerance to low doses of soluble foreign protein antigens. Experiments were performed in normal and B cell-deficient animals, and tolerance induction was measured by T cell proliferation assays. T cell proliferation was reduced in B cell-deficient mice, indicating that B cells may be involved in efficient activation of naive T cells in response to protein antigen both in vivo and in vitro. To study acquired tolerance induced by low doses of soluble foreign protein antigen, normal and B cell-deficient adult mice were injected intravenously with repeated low doses (10 μg) of deaggregated ovalbumin (OVA), and then challenged with OVA in complete Freund's adjuvant. In animals treated with deaggregated OVA, the in vitro proliferative responses of LN T cells to OVA were significantly reduced, and production of the Th1 cytokine, IFN-γ, in response to OVA was lost. This occurred in both normal and B cell-deficient treated animals, indicating that B cell antigen presentation was not required for this phenomenon. B cells were also unnecessary for self tolerance of T cells to the transgenic self antigen, hen egg lysozyme (HEL), in a transgenic mouse strain with very low serum lysozyme concentration. Partial low zone tolerance induced by deaggregated, low-dose OVA was selective for the Th1 response, as measured by in vitro proliferation and IL-2 and IFN-γ production, because antibody responses of normal mice to this T cell-dependent antigen were largely unaffected. Both treated and untreated animals produced equivalent titers of anti-OVA antibodies, predominantly of the IgG1 and IgG2b isotypes, following challenge with OVA in complete Freund's adjuvant. Tolerance to low levels of the transgenic HEL self protein in mice expressing different MHC molecules was also addressed. Transgenic mice that were H-2b/b in the class II region were not tolerant to the transgenic self protein, whereas transgenic mice of the H-2b/k were tolerant.