Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and renal failure in humans. The molecular pathways that lead to DN are not well known. This research investigates possible roles of several signal transducers and activators of transcription (STAT) proteins in this disease using a STAT5A/B knockout (SKO) mouse model. Based on previous observations of increased inflammation-related gene expression in the kidneys of diabetic SKO mice, the hypothesis of the current project was that the combination of the loss of STAT5 repression and increase of STAT3 activity escalates inflammation-related gene expression in the kidneys of diabetic SKO mice. In support of this hypothesis, an increase of IRF-1 RNA expression, reflective of the loss of STAT5 repression, was observed in the kidneys of diabetic SKO mice. Levels of phosphorylated STAT3 were also increased in the kidneys of diabetic SKO mice. These results suggest that STAT5 acts as a repressor of inflammation-related genes in DN and, in its absence, expression of these genes is no longer repressed, either due to direct loss of the STAT5 repression or due to increased STAT3 activity which could potentially increase their expression.

Microarray analyses were performed comparing mRNA expression levels in subcutaneous adipose tissue between mice with disruption in the growth hormone (GH) receptor gene (GHR-/- mice) and littermate controls. The data revealed that 87 genes were significantly upregulated and 72 genes were significantly downregulated in GHR-/- mice versus littermate controls. Among the 159 altered genes, a subgroup of five genes was chosen for confirmation by a second mRNA quantification method, real time reverse transcriptase polymerase chain reaction (RT-RT PCR). RT-RT PCR studies confirmed the altered gene expression for only three genes, angiotensinogen (Agt), adiponutrin (Adpn) and angiopoietin-like protein 4 (Angptl4). To better understand the role of GH in adipose specific expression of these three genes (Adpn, Agt, Angptl4), RT-RT PCR analysis was expanded to include an additional genotype of altered GH function, the bovine GH transgenic mice, an additional diet treatment high-fat diet, as well as two other adipose depots, the epididymal and retroperitoneal adipose depots. This thesis discusses in detail all gene alterations observed in the microarray analyses and the RT-RT PCR analyses for Adpn, Agt, Angptl4 in adipose tissue.

Growth Hormone (GH) is known as a diabetogenic molecule. In excess it can inhibit insulin's action resulting in insulin resistance and diabetes, which can be seen in the GH receptor knockout (GHR-/-) mice since the absence of GH action throughout the whole body improves insulin sensitivity and increases life span. Three tissues that are both insulin and GH sensitive are liver, adipose tissue, and muscle. To determine their individual contributions, three tissue specific GHR disrupted mouse lines have been independently produced: liver-specific, adipose-specific, and muscle-specific GHR-/- mice using the CRE-LOX system. Tissue specific promoters, albumin, aP2, MCK, were utilized to drive the CRE recombinase expression in liver, adipose, and muscle tissues separately. This thesis aimed to validate the gene deletion in specific tissues at the DNA level. The results confirm that liver and muscle specific GHR-/- mice had the deletion of the GHR gene in the liver and muscle tissues, respectively. However, in the adipose specific GHR-/- mice, all tissues, and not just adipose tissue, had evidence of GHR deletion. These findings need to be taken into account when evaluating the phenotype.

Both epidemiological and empirical data have demonstrated the association between long-term exposure to fine particulate matters (PM2.5) and the burden of atherosclerosis. Strawberry, a rich source of micronutrients and several bioactive phytochemicals, has been implicated in the prevention of cardiovascular diseases and metabolic syndromes. This study was designed to explore the effects of freeze-dried strawberry supplementation on PM2.5-potentiated atherosclerosis in apoE-/- mice, which is a spontaneous atherosclerotic model. Mice, fed with high-fat chow or high-fat chow supplemented with 10% strawberry powder, were exposed to either filtered air or concentrated ambient particles using “Ohio Air Pollution Exposure Systems for Interrogation of Systemic Effects” for 6 months. After PM2.5 exposure, plaque area and lipid area of aorta were evaluated using hematoxylin & eosin staining and oil-red O staining, respectively. In addition, total cholesterol, high-density lipoprotein and fasting glucose were measured using diagnostic kits. Furthermore, the levels of inflammatory cytokines in plasma were determined via ELISA kits, and the mRNA expressions of inflammatory cytokines within lung were quantitated using real-time PCR. Our data showed that long-term PM2.5 exposure potentiated atherosclerosis and inflammation in these mice; strawberries reduced inflammation and improved lipid profile, but did not attenuate atherosclerosis after PM2.5 exposure. These findings indicate that other mechanistic pathways, e.g. systemic oxidative stress, autonomic nervous system imbalance, and the direct toxic effects of particles, may diminish the effects of strawberries on PM2.5-potentiated atherosclerosis.

We investigated the interaction between prolactin (PRL) and the HPA axis. A physiologically relevant, hyperprolactinemic model, i.e. postpartum female rats, or PRL knockout mice were used. Animals housed with or separated from their pups for 1 or 24 hours or 8 days were acutely stressed. Circulating PRL and corticosterone (CORT) and PRL-R mRNA in the choroid plexus were determined. PRL returned to pre-pregnancy values by one hour after pup removal. The HPA axis response was restored after 24 hours of separation, although basal CORT levels remained elevated for 8 days after terminating lactation. PRL-R expression decreased 24 h after pup separation, but returned to pre-pregnancy levels by 8 days. Stress-induced HPA axis activation occurred only when PRL-R expression levels were similar to or lower than levels in virgins indicating PRL-R up-regulation contributes to an attenuated HPA response. The sustained, elevated basal CORT levels suggest the metabolic demands and/or stress of lactation persist in the absence of suckling. Additionally, differential effects of gender, diet and PRL on HPA axis activation were studied using wild-type (wt) and PRL knockout (KO) mice. Male and female mice fed normal chow (C) or high-fat diet (HFD) for 12 weeks were acutely stressed. CORT levels were quantified under basal conditions, during stress and at the time of sacrifice. Serial sampling acted as a non-specific stressor, regardless of gender, genotype or diet; PRL dampened this response in males only. HFD increased body weight and fat accumulation in males, but not in females, regardless of genotype. In chow fed animals, stress increased CORT in both males and females, regardless of genotype, indicating that PRL is not involved in activating the HPA axis. HFD increased PRL-R mRNA in wt and KO females, but only in wt males indicating factors other than PRL regulate PRL-R expression in females. HFD increased leptin levels only in wt males and females, suggesting PRL influences lepti (open full item for complete abstract)

Activation of the hypothalamic-pituitary-adrenocortical (HPA) axis plays a role in regulating basic biological functions in the basal state. During homeostatic perturbations, it potentiates and induces physiological adaptations that are designed to aid an organism in overcoming challenges and regain homeostatic equilibrium. However, prolonged HPA axis activation can be deleterious to long-term survival, so the HPA axis is subject to autoregulation via a negative feedback loop. The endpoint of HPA axis stimulation is corticosterone (CORT) release, which travels through the general circulation and inhibits further HPA axis activation at the levels of the pituitary, hypothalamus, and extra-hypothalamic structures such as the hippocampus. Central to HPA axis response and regulation is the glucocorticoid receptor (GR), a ligand dependent transcription factor that is richly expressed in limbic forebrain regions such as the hippocampus, medial prefrontal cortex, and amygdala. These same regions are differentially affected in several psychiatric disorders, and many of these disorders feature HPA axis dysregulation that includes changes in GR signaling. Females are differentially affected by some psychiatric disorders, such as depression, and there are well-established sex differences in HPA axis responsivity, suggesting a possible link between sex and stress regulation. Dysregulation of the HPA axis can also occur during the aging process. Previous studies using lesions and antagonists have implicated GR in feedback inhibition, but to date, the role of forebrain GR has not been directly tested. Here we present a series of studies investigating the role of forebrain GR in basal HPA axis regulation, and after acute and chronic stress, taking into account sex and aging as factors. We employ a forebrain specific GR knockout mouse model (FBGRKO), in which GR is developmentally disrupted via the loxP-Cre-Recombinase system. We show that forebrain GR is necessary for basal regulat (open full item for complete abstract)

Renal morphogenesis involves the reciprocal inductive interactions between the ureteric bud and metanephric mesenchyme forming the collecting ducts and nephrons within adult kidney. We applied microarray technology to the study of renal morphogenesis in order to better understand the molecular mechanisms underlying development. Additionally, the techniques employed in the expression analysis of the embryonic kidney were extended to the study of renal disease. Embryonic kidneys representing different stages of renal development were analyzed using expression microarrays. Renal developmental analysis revealed many novel genes and genetic pathways involved in renal development. In addition, the normal renal development data provides a baseline for the analysis of gene targeted mice possessing disruptions in renal morphogenesis. Microarray analysis was also performed on the Hoxa11/Hoxd11 compound null renal defect throughout renal development. In conclusion, these microarray studies greatly advance our knowledge of gene expression within the normal renal morphogenesis and identify possible downstream candidate genes regulated by the Hox11 genes. Wnt signaling is crucial for normal renal morphogenesis. In Drosophila, the pygopus gene encodes a transcriptional co-activator required for canonical Wnt signaling. The targeted deletion of the mammalian orthologs of pygopus, Pygo1 and Pygo2, in mice was investigated in renal development. A disruption in ureteric number tip and morphology was identified in Pygo1/Pygo2 compound null kidneys. Additionally, canonical Wnt signaling as measure by the Bat-gal transgene is reduced within the ureteric compartment in Pygo1/Pygo2 null kidneys. Overall, these experiments suggest that Pygo function is required for activation of canonical Wnt signaling in the ureteric compartment of the developing kidney. Focal segmental glomerulosclerosis (FSGS) is characterized by the segmental scarring of the glomerulus, ultimately resulting loss of neph (open full item for complete abstract)

miR-122, the most abundant liver-specific microRNA (miRNA), is involved in many different biological functions, including cholesterol metabolism, hepatitis C virus replication, and hepatocarcinogenesis. Previous studies have shown that downregulation of miR-122 in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Among the deregulated miRNAs in HCC, miR-122 is drastically reduced in HCC. Based on these observations, our hypothesis is that miR-122 is a liver-specific tumor suppressor and its loss may promote hepatocarcinogenesis. To test this hypothesis, we generated germ-line (KO) and liver-specific (LKO) miR-122 knockout mice. Both KO and LKO mice exhibited hepatic microsteatosis and hepatic inflammation at early adult stage. Lipid analysis showed accumulated hepatic triglyceride, which correlated with increased de novo triglyceride synthesis and reduced triglyceride secretion. By 6 month both KO and LKO mice develop hepatic steatosis, inflammation, and fibrosis. After twelve months, these mice produce spontaneous liver tumors resembling HCC. The HCC incidences were ~30% and ~50% in LKO and KO mice, respectively. Microarray and realtime RT-PCR analysis attributed these pathological phenotypes to dysregulated expression of signaling pathways involved in triglyceride synthesis, cytokine expression, and oncogenesis. Among the deregulated genes, Agpat1, Cidec and Mapre1 were identified for the first time as the direct targets of miR-122. Exploration of the mechanism leading to hepatic inflammation in KO and LKO mice led to the identification of CD11bhighGr-1+ subtype of inflammatory cells increased in the liver of KO mice. These cells were determined as the major source of high levels of IL-6 and TNF-α that accumulated in the livers of KO and LKO mice. Ccl2, a reported myeloid chemo-attractant, was induced in hepatocytes of KO and LKO mice and was inversely regulated by miR-122 in vitro. To establish further the tumor suppressor role of mi (open full item for complete abstract)

Social behavior is essential to an animal's survival and has been widely studied in a variety of species. All are regulated by the central nervous system and modulated by neuropeptides. One neuropeptide that is known to play a role in the regulation of social behavior is arginine vasopressin (Avp). Most of Avp's effects on behavior have been attributed to its action via its 1a receptor (Avpr1a). However, there is compelling evidence from knockout studies that the Avp 1b receptor (Avpr1b) also plays a significant role in the modulation of social behavior. Avpr1b knockout (-/-) mice show deficits in social behaviors, such as reduced aggression and impaired social recognition. The Avpr1b is more discretely distributed than the Avpr1a being found primarily in the CA2 region of the hippocampus by in situ hybridization. The presence of the Avpr1b within the CA2 region is of particular interest because animals with lesions to the hippocampus that include the CA2 region show social behavior deficits similar to that of Avpr1b -/- mice. This dissertation set out to study the role of the Avpr1b within the CA2 region of the hippocampus in the neural regulation of social behaviors, including aggression, social memory, and social motivation.

Oxytocin (Oxt) is a nonapeptide synthesized in the magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus and it is released to the periphery from axon terminals. Numerous studies indicate that Oxt plays an important role in cognition and social behavior. Abnormalities in the Oxt system have also been implicated in several neuropsychiatric disorders such as schizophrenia, obsessive compulsive disorder (OCD) and Tourette's syndrome. Patients diagnosed with these disorders show deficits in a fundamental form of information filtering known as sensorimotor gating. Sensorimotor gating can be measured across species using prepulse inhibition (PPI) of the startle reflex. PPI can be disrupted by treating animals with dopamine receptor agonists (e.g. apomorphine and amphetamine) and NMDA receptor antagonists (e.g. phencyclidine (PCP) and MK-801). Previous studies suggest that Oxt may act as a natural antipsychotic and our lab has shown that Oxt knockout mice (Oxt-/-) are more susceptible to the psychosis-related effects of PCP. So, to further characterize the role of the Oxt system in sensorimotor gating deficits, a rescue experiment was performed by pretreating mice with Oxt prior to administering PCP. We found that pretreatment with Oxt appears to cause a partial rescue of the PPI-disrupting effects of PCP in male Oxt-/- mice. As the actions of Oxt are mediated via binding to the Oxt receptor, pharmacologically disrupted PPI was examined in mice lacking the Oxt receptor (Oxtr-/-). We found that treatment with apomorphine, amphetamine and MK-801 equally disrupted PPI in wildtype and Oxtr-/- mice. Our data suggest that in Oxtr-/- mice the neurocircuitry involved in the regulation of PPI might be normal. Overall, these results indicate that the Oxt/Oxtr system plays an important role in social behavior and may have important implications in human behavioral disruptions.

Ski is the most studied member of a family of proteins all sharing a conserved Dachshund homology domain. It has been implicated in oncogenic transformation, myogenic conversion of avian embryo fibroblasts and also many aspects of vertebrate development, especially myogenesis. Ski-/- mice exhibit severe defects in skeletal muscle and die at birth, yet little is know about either the underlying mechanisms or the role of Ski in adult muscle regeneration. In these studies, I used Ski knockout mice and C2C12 myoblast cultures to address these issues, respectively. Detailed analysis of Ski-/- embryos revealed dramatically reduced hypaxial muscles but less affected epaxial muscles. The reduced number of myogenic regulatory factor positive cells in Ski-/- mice suggested an insufficient myogenic cell pool to support muscle formation. However, both the dermomyotomal hypaxial progenitors and myotomal epaxial progenitors formed and committed to myogenic fate appropriately. The hypaxial muscle defect in Ski-/- mice was not caused by abnormal proliferation, terminal differentiation or apoptosis of the myogenic cells either, but due to impaired migration of embryonic hypaxial progenitors. Surprisingly, the normal distribution of fetal/postnatal myogenic progenitors in Ski-/- mice suggested different effects of Ski on the behaviors of embryonic and fetal/postnatal myogenic progenitors. In addition, although not affecting the terminal differentiation of embryonic myogenic cells, Ski was necessary for that of adult satellite-cell derived C2C12 myoblasts as evidenced by impaired myotube formation and reduced induction of genes essential for myogenic differentiation in the absence of Ski. This function was mainly mediated by Ski's ability to form a complex with Six1 and Eya3 and activate Myog transcription through a MEF3 site. It is important in the future to further study mechanisms underlying the contrasting effects of Ski on embryonic, fetal and adult muscle development, to investi (open full item for complete abstract)