IRF2BP2 is a corepressor of IRF2, a transcription factor involved in the immune response. IRF2BP2 is also a coactivator of the VGLL4/TEAD4 complex in muscle. Given its functional duality, we asked how IRF2BP2 deletion would affect mouse development and adult muscle regeneration. Most Irf2bp2-/- mice die prior to birth, those that survive develop lymphoma in adulthood. Microarray profiling of Irf2bp2 knockout liver, heart, and skeletal muscle revealed a shared program of upregulated genes involved in inflammation and immunity. The function of IRF2BP2 in adult skeletal muscle recovery from cardiotoxin-induced injury was evaluated. Compared to WT mice, mice with macrophage-specific ablation of IRF2BP2 (Irf2bp2flox/LysMCre) or muscle-specific ablation of Irf2bp2 (Irf2bp2flox/MckCre) mice showed increased inflammation and impaired muscle regeneration. Global deletion of Irf2bp2 in mice results predominantly in embryonic death or lymphoma in adults. Irf2bp2 suppresses genes that mediate inflammation in mouse liver, heart, and in skeletal muscle, where IRF2BP2 promotes regeneration.

Abstract: Folylpoly-γ-glutamate synthetase (FPGS) catalyzes the formation of polyglutamate forms of the reduced folates and antifolates such as methotrexate (MTX) and pemetrexed; this allows the retention of folates and antifolate cancer drugs inside the cell. The enzyme activity of FPGS is essential for cell proliferation and survival. The mouse fpgs gene contains two promoters spaced 10 kb apart which are activated in a tissue-specific manner. The upstream promoter (P1) and exons A1a and A1b are used in some differentiated tissues, mainly liver and kidney, whereas the downstream promoter (P2) and exon 1 are used in rapidly dividing cells. In contrast, the human fpgs gene expresses virtually all transcripts from the downstream promoter. In order to more faithfully mimic human folate metabolism in the mouse, we have deleted the upstream promoter and the associated two small exons of fpgs in the mouse genome by homologous recombination. Homozygous deletion mice survive embryonic development, grow to adulthood, and reproduce through several generations, they appear to be normal. The results of Q-RT-PCR analysis on RNA from adult mouse liver of three different genotypes (A1aA1b +/+, +/-, -/-) indicated that deletion of P1 results in the release of promoter interference of P2, and activation of the downstream P2 promoter is increased by 3-5 fold. Interestingly, the total FPGS mRNA expression in KO mouse liver is 20-100 fold lower than in liver from wild-type mice. However when the FPGS activity was measured using an FPGS enzyme assay, the liver of knockout mice appeared to have only 2 fold lower enzyme activity than liver from wild-type mice. In conclusion, we have successfully generated a mouse which reflects human folate metabolism much closer than seen in wild type mice. The FPGS- humanized mouse liver model would be an appropriate in vivo tool for the study of the antifolate drug toxicity and inhibition.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role. / May 2008

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role.

There are four mammalian SSB proteins (SSB-1 to -4), and these are characterized by a C-terminal SOCS box and central SPRY domain. The C-terminal SOCS box was first observed in proteins that were found to act as Suppressors of Cytokine Signalling and function by virtue of their SH2 domain. Other families containing the SOCS box motif were defined by the domains N-terminal to this, such as the ASBs (Ankyrin repeats), WSBs (WD40 repeats) and of course the SSBs (SPRY domains). This thesis describes a very broad investigation of the SSBs, a protein family about which very little was known. To begin with, functional investigation into the evolution of this family and analysis of murine SSB expression patterns was performed. This highlighted that the family was highly conserved and had differential expression in the mouse, suggestive of important, unique functional roles for the individual family members. The majority of work in the thesis then proceeds in three directions; (i) analysis of the SSB proteins in vivo, with genetic deletion of SSB-2 in the mouse, (ii) biochemically, with analysis of SSB binding partners, and (iii) structurally, with functional analysis of the structure of SSB-2.

The transcription factor DEAF-1 is the mammalian homologue of a critical Drosophila developmental gene and is essential for neonatal survival in mice. Haploinsufficiency of Deaf-1 in the testis of adult mice was initially thought to cause loss of spermatogenesis and disrupted morphology of the seminiferous tubules, and this heterozygosity was thought to be sufficient to disrupt epigenetic programming in the developing sperm to produce inheritance of testicular defects in both heterozygous and genotypically normal offspring. Although Deaf-1 knockout mice do display disrupted testis structure, infertility at advanced age, hyperproliferation of early germ cells, and abnormal staging of seminiferous tubules, this phenotype was also observed in normal mouse strains that were born in the SIUC vivarium. Mice ordered from a vendor and raised at SIUC did not show testicular defects. This suggests an environmental factor at the SIUC vivarium may act as an endocrine disruptor during embryonic testicular development. Deaf-1-/- mice die soon after birth, often as the result of exencephaly, a gross neural tube defect (NTD). Unlike many mouse models, exencephalic Deaf-1-/- mice do not display a higher incidence of NTDs in females as compared to their male littermates. DEAF-1 promotes Bax-mediated apoptosis; studies using terminal UTP nick-end labeling (TUNEL) suggest a global increase in apoptosis in both exencephalic and normal Deaf-1-/- fetuses during neurulation as compared to their Deaf-1+/+ littermates. This indicates that Deaf-1 is crucial for correct apoptotic patterning during development, which, in turn, is essential for neural tube closure. Finally, cDNA microarray comparison of e14.5 Deaf-1 knockout and wildtype fetuses reveals expression of translation initiation factor 4g3 (Eif4g3) to be downregulated in Deaf-1-/- fetuses. Electrophoretic mobility shift assay using recombinant DEAF-1, and chromatin immunoprecipitation assay of a human cell line confirmed DEAF-1 could bind the eIF4G3 promoter both in vitro and in vivo. Additionally, transcription of the Deaf-1 Antisense Transcript (Das) was found to be significantly downregulated in both e14.5 fetuses and e18.5 fetal brains from Deaf-1-/- mice, suggesting that either lack of Deaf-1 protein or lack of exons 2 through 5 in Deaf-1 knockout mice causes changes in levels of the noncoding RNA that shares Deaf-1's promoter in the mouse.

Dehydropyrrolizidine alkaloids (DHPAs) are a large group of globally important plant-derived pro-toxins that can contaminate or are naturally present in animal feed and the human food supply as well as herbal supplements. Their bioactive metabolites are potentially hepatotoxic, pneumotoxic, genotoxic and carcinogenic. Due to the difficulty in obtaining sufficient quantities of purified DHPAs, toxicity studies have largely relied on single intraperitoneal injections in rodent models, and carcinogenicity studies have been limited to a small handful of the hundreds of isolated DHPAs. To assess the relative toxicity of structurally diverse DHPAs in a more biologically relevant manner, male California White chicks were dosed orally with 0.01, 0.04, 0.13, or 0.26 mmol of seven different DHPAs and three DHPA N-oxides kg-1 bodyweight for 7 days. DHPAs were grouped in relation to their toxicity based on clinical, serum biochemical, and histopathological evaluations as well as tissue adduct accumulation rates. Using the same model, a reduced extract from comfrey, a commonly used DHPA containing herb, was compared to its two major constituent DHPAs, intermedine and lycopsamine. Based on the same parameters, the comfrey extract was more toxic than pure lycopsamine or intermedine. Addressing the need for a more sensitive carcinogenicity model, male heterozygous p53 knockout mice were treated with riddelliine 5, 15 or 45 mg kg–1 bodyweight day-1 by oral gavage for 14 days, or given a long-term treatment of riddelliine 1 mg kg-1 bodyweight day–1 in pelleted feed for 12 months. Exposure to riddelliine increased the odds of tumor development in a dose-responsive manner (odds ratio 2.05 and Wald 95% confidence limits between 1.2 and 3.4). The most common neoplasm was hepatic hemangiosarcoma, which is consistent with previously published lifetime rodent studies. The results of this research demonstrate that the California White chick model is sensitive for comparison of DHPA toxicity, and data obtained from this research can be used to validate previous DHPA toxicity research. It also demonstrates that comfrey toxicity may have been previously underestimated. The heterozygous p53 knockout mouse model is beneficial for further investigation of comparative carcinogenesis of structurally and toxicologically different DHPAs and their N-oxides.

Duchenne muscular dystrophy (DMD) is a severe and fatal muscle wasting disease characterized by a high mutation rate in the gene that encodes the membrane-associated protein dystrophin that results in absence of expressed protein. Although the primary genetic defect for DMD is known, the mechanisms that initiate the onset of DMD are not currently understood. This study tested the hypothesis that pathophysiological processes involved in DMD could be identified by the global expression of mRNA in maturing dystrophin- and utrophin-deficient mouse (mdx:utrn-/-) muscles. Two potential dystrophic onset mechanisms targeted for analysis were (1) disrupted expression of calcium handling proteins; and, (2) increased expression of immune response markers. An mRNA expression profile was developed following isolation of total RNA from control and mdx:utrn-/- triceps surae (TS) muscles at ages 9-10 and 20-21 days using Affymetrix® Mu74Av2 GeneChips®. Compared to control, the mRNA expression profile in mdx:utrn-/- muscles revealed there was a 3-fold increase in the number of gene transcripts differentially expressed more than 2-fold (53 transcripts at ages 9-10 days; 153 at ages 20-21 days). However, there were no changes in the mRNA transcripts for calcium handling proteins. In distinct contrast, there was up-regulation of transcripts that corresponded to an immune response (40 transcripts), extracellular matrix activity (14), and proteolysis (8). Up-regulation of several transcripts corresponded to cytokines and their receptors (11), chemokines and their receptors (5), and lymphoid and myeloid markers (16) suggesting that dystrophic muscle is susceptible to invasion by macrophages, leukocytes, B- and T-cells. These results are consistent with several reports (Spencer et al., 1997; Chen et al., 2000; Porter et al., 2002; Porter et al., 2003a; Porter et al., 2003b; Porter et al., 2004) that indicate the immune system may play an important role in the early pathophysiology of DMD. Understanding the functional aspects of an immune response in DMD onset should lead to more effective therapeutics. / Master of Science

Tobacco dependence is the leading cause of preventable death in the United States. Despite currently available smoking cessation therapies, there is a high rate of relapse in smoking among those attempting to quit. While the somatic signs of nicotine withdrawal (insomnia, increased appetite, weight gain) contribute to the continuation of smoking behavior, it has been hypothesized that the affective signs (depression, anxiety, craving, irritability) are greater motivators of relapse and continued tobacco use. There are few studies that assess the molecular and receptor-mediated mechanisms of nicotine withdrawal; therefore, our studies focus on identifying the nicotinic acetylcholine receptor (nAChR) subtypes and post-receptor calcium-dependent mechanisms involved in nicotine withdrawal behaviors. Using precipitated, spontaneous, and conditioned place aversion (CPA) models, we measured physical and affective signs of nicotine withdrawal in mice. Our data show that major nAChR subtypes have differential roles in nicotine withdrawal. Additionally, our results suggest a behavioral relevance for L-type calcium channels in physical nicotine withdrawal signs, while calcium/calmodulin dependent protein kinase II (CaMKII) appears to be involved in both physical and affective withdrawal behaviors. Additionally, we conducted biochemical studies in the ventral tegmental area (VTA) and nucleus accumbens (NAc) to examine the relationship between altered withdrawal behavioral responses and calcium-dependent molecular mechanisms that contribute to nicotine withdrawal behaviors. Our results suggest an important role for β2-containing nAChRs in nicotine-withdrawal induced decreases in CaMKII and synapsin I function in the NAc. Overall, our studies implicate a critical role for the α4α6β2* nAChR subtype in the behavioral and molecular aspects of nicotine withdrawal, thus aiding in the elucidation of nAChR subunits and mechanisms that contribute to nicotine withdrawal behaviors. The current studies are imperative for generating more successful smoking cessation therapies.