The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring's phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease.

Chronic alcohol use results in accelerated liver injury, leading to alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. However, due to the complex nature of this disease process, a central, druggable mechanism has remained elusive. microRNAs are potent post-transcriptional regulators of gene expression. A single miRNA has the ability to regulate hundreds of pathways simultaneously, defining cellular fate and function. microRNA-122 (miR-122), the most abundant miRNA in hepatocytes, has a demonstrated role as an tumor suppressor, regulator of hepatocyte metabolism, and hepatic differentiation. In this dissertation I demonstrate the role of miR-122 on alcoholic liver disease (ALD) pathogenesis over four parts. In chapter II, I will demonstrate chronic alcoholic patients, free of neoplastic changes, have a reduction of miR-122 and that this miRNA regulates HIF-1α, a determinant of ALD pathogenesis. In chapter III, using hepatocytetropic adeno-associated virus 8 (AAV8) vector, I demonstrate that miR-122 inhibition mimics ALD pathogenesis, and furthermore, using hepatocyte-specific HIF-1α-null (HIF1hepKO) mice that this phenomenon is HIF-1α dependent. Given this finding, in chapter IV, I demonstrate that ectopic expression of miR-122 in vivo can reverse alcoholinduced liver damage, steatosis, and inflammation by directly targeting HIF-1α. Finally, in chapter V, I present evidence that alcohol-induced dysregulation of grainyhead-like proteins 1 and 2 (GRHL2), mediate the inhibition of miR-122 at the transcriptional level. These findings dissect a novel mechanistic regulatory axis of miR-122 and indicate a potential opportunity for restoration of miR-122 as a therapy in early ALD.

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset progressive neurodegenerative disease that causes degeneration in both upper and lower motor neurons. ALS progresses relentlessly after the onset of the disease, with most patients die within 3-5 years of diagnosis, largely due to respiratory failure. Since SOD1 became the first gene whose mutations were associated with ALS in 1993, more than 17 ALS causative genes have been identified. Among them, TAR DNA-binding protein (TARDBP) lies in the central of ALS pathology mechanism study, because TDP43 proteinopathy is observed not only in familial ALS cases carrying TARDBP mutations, but also in most of the sporadic ALS cases, which account for 90% of the whole ALS population. Several TDP43 overexpression mouse models have been successfully generated to study the gain-of-toxicity mechanism of TDP43 in ALS development, while the investigation of loss-of-function mechanism which could also contribute to ALS still awaits a proper mouse model. The major difficulty in generating TARDBP knock out mouse model lies in the fact that TARDBP is a development essential gene and complete depletion of TDP43 function causes embryonic lethality. In chapter I, I reviewed the recent advances in ALS study. Emphasis was given to ALS mouse models, especially TARDBP ALS mouse model. In Chapter II, I made a Tet-responsive construct that contains mCherry, a fluorescent protein, as an indicator for the expression of the artificial miRNA (amiTDP) residing in the 3’UTR of mCherry and targeting TARDBP. The construct was tested in NSC34 cells and TRE-mCherry-amiTDP43 transgenic mouse was generated with this construct. Crossing TRE-mCherry-amiTDP43 mouse with mPrp-tTA mouse, mCherry expression was successfully induced in mouse forebrain and cerebellum, but not in other tissues including spinal cord. By quantitative real-time PCR, amiTDP43 expression was confirmed to be coupled with mCherry expression. Fluorescent immunostaining revealed that mCherry was expressed in neurons, but not in astrocytes or microglia cells, and that in mCherry positive cells, TDP43 was significantly knocked down. Results from Nissl staining and GFAP immunostaining suggested that decrease of TDP43 in forebrain neuron only was not sufficient to cause neurodegeneration and neuron loss. In chapter III, I investigated the function of Armadillo Containing Protein 4 (ARMC4), which was originally considered ALS causative gene. Our study of the function of CG5155, the possible homolog of ARMC4 in Drosophila, indicated that CG5155 is a male fertility gene that is involved in spermatogenesis. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with Bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 is a subfamily of Armadillo-repeat containing proteins with an evolutionarily conserved function in spermatogenesis.

Switches play an important regulatory role at all levels of biology, from molecular switches triggering signaling cascades to cellular switches regulating cell maturation and apoptosis. Medical therapies are often designed to toggle a system from one state to another, achieving a specified health outcome. For instance, small doses of subpathologic viruses activate the immune system’s production of antibodies. Electrical stimulation revert cardiac arrhythmias back to normal sinus rhythm. In all of these examples, a major challenge is finding the optimal stimulus waveform necessary to cause the switch to flip. This thesis develops, validates, and applies a novel model-independent stochastic algorithm, the Extrema Distortion Algorithm (EDA), towards finding the optimal stimulus. We validate the EDA’s performance for the Hodgkin-Huxley model (an empirically validated ionic model of neuronal excitability), the FitzHugh-Nagumo model (an abstract model applied to a wide range of biological systems that that exhibit an oscillatory state and a quiescent state), and the genetic toggle switch (a model of bistable gene expression). We show that the EDA is able to not only find the optimal solution, but also in some cases excel beyond the traditional analytic approaches. Finally, we have computed novel optimal stimulus waveforms for aborting epileptic seizures using the EDA in cellular and network models of epilepsy. This work represents a first step in developing a new class of adaptive algorithms and devices that flip biological switches, revealing basic mechanistic insights and therapeutic applications for a broad range of disorders.

Systemic glucose regulation is essential for human survival as low or chronically high glucose levels can be detrimental to the health of an individual. Glucose levels are highly regulated via inter-organ communication networks that alter metabolic function to maintain euglycemia. For example, when nutrient levels are low, pancreatic α-cells secrete glucagon, which signals to the liver to promote glycogen breakdown and glucose production. In times of excess nutrient intake, pancreatic β-cells release insulin. Insulin signals to the liver to suppress hepatic glucose production, and signals to the adipose tissue and the skeletal muscle to take up excess glucose via insulin-regulated glucose transporters. Defects in this inter-organ communication network including insulin resistance can result in glucose deregulation and ultimately the onset of type-2 diabetes (T2D). To identify novel regulators of insulin-mediated glucose transport, our laboratory performed an siRNA-mediated gene-silencing screen in cultured adipocytes and measured insulin-mediated glucose transport. Gene silencing of Mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a Sterile-20-related serine/threonine protein kinase, enhanced insulin-stimulated glucose transport, suggesting Map4k4 inhibits insulin action and glucose transport. Thus, for the first part of my thesis, I explore the role of Map4k4 in cultured adipose cells and show that Map4k4 also represses lipid synthesis independent of its effects on glucose transport. Map4k4 inhibits lipid synthesis in a Mechanistic target of rapamycin complex 1 (mTORC1)- and Sterol regulatory element-binding transcription factor 1 (Srebp-1)-dependent mechanism and not via a c-Jun NH2-terminal kinase (Jnk)-dependent mechanism. For the second part of my thesis, I explore the metabolic function of Map4k4 in vivo. Using mice with loxP sites flanking the Map4k4 allele and a ubiquitously expressed tamoxifen-activated Cre, we inducibly ablated Map4k4 expression in adult mice and found significant improvements in metabolic health indicated by improved fasting glucose and whole-body insulin action. To assess the role of Map4k4 in specific metabolic tissues responsible for systemic glucose regulation, we employed tissue-specific knockout mice to deplete Map4k4 in adipose tissue using an adiponectin-cre transgene, liver using an albumin-cre transgene, and skeletal muscle using a Myf5-cre transgene. Ablation of Map4k4 expression in adipose tissue or liver had no impact on whole body glucose homeostasis or insulin resistance. However, we surprisingly found that Map4k4 depletion in Myf5-positive tissues, which include skeletal muscles, largely recapitulates the metabolic phenotypes observed in systemic Map4k4 knockout mice, restoring obesity-induced glucose intolerance and insulin resistance. Furthermore these metabolic changes were associated with enhanced insulin signaling to Akt in the visceral adipose tissue, a tissue that is nearly devoid of Myf5-positive cells and does not display changes in Map4k4 expression. Thus, these results indicate that Map4k4 in Myf5-positive cells, most likely skeletal muscle cells, inhibits whole-body insulin action and these effects may be mediated via an indirect effect on the visceral adipose tissue. The results presented here provide evidence for Map4k4 as a potential therapeutic target for the treatment of insulin resistance and T2D.

XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.

Teleost fishes incorporate renal ammonia excretion as part of a greater acid-base regulatory system. However, the transport mechanisms employed by the renal epithelium to excrete ammonia are relatively unknown. I hypothesized that, under metabolic acidosis, increased renal ammonia excretion would be the product of tubular secretion and involve a Na+/NH4+ exchange metabolon mediated through Rhesus (Rh) glycoproteins. To induce metabolic acidosis, goldfish (Carassius auratus) were exposed to a low pH environment (pH 4.0; 48-h). There was a clear signal of metabolic acidosis: a reduction in both plasma [HCO3-] and blood pH with no influence on plasma PCO2. Goldfish demonstrated an elevation in total plasma [ammonia] with a reduction in PNH3 under acidosis. Metabolic acidosis induced higher rates of urinary excretion of acidic equivalents in the form of both NH4+ and titratable acidity-HCO3- (TA-HCO3-) excretion. Urinary Na+ excretion was not affected by acidosis and urine [Na+] did not correlate with urinary [ammonia]. Alanine aminotransferase activity in the kidney was higher in acidotic goldfish. Glomerular filtration rate and urine flow rate were not affected by acidosis. Increased renal NH4+ excretion was due to increased secretion, and not increased filtration, of ammonia. There was a corresponding elevation in Rhcg1b mRNA expression but no change in renal Na+ reabsorption. My data support a secretion-based mechanism of teleost renal ammonia transport. This system is Na+ independent and is likely mediated by Rh glycoproteins and H+ ATPase, involving a parallel H+/NH3 secretion mechanism. To investigate effects of metabolic acidosis on elasmobranch fish, Pacific spiny dogfish (Squalus acanthias suckleyi) were infused with an acidic saline (125 mM HCl/375 mM NaCl; 3 ml/kg/h; 24-h). The results are preliminary, with no marked effects of HCl infusion on plasma acid-base or N-status, but increased branchial NHE2 and lower renal NHE3 protein expressions. These data are summarized in an Appendix. / Thesis / Master of Science (MSc)

Functional vasodilation in arterioles is impaired with chronic ischemia. We sought to examine the impact of chronic ischemia and age on skeletal muscle resistance artery function. To examine the impact of chronic ischemia, the femoral artery was resected from young (2-3mo) and adult (6-7mo) mice and the profunda femoris artery diameter was measured at rest and following gracilis muscle contraction 14 days later using intravital microscopy. Functional vasodilation was significantly impaired in ischemic mice (14.4±4.6% vs. 137.8±14.3%, p<0.0001 n=8) and non-ischemic adult mice (103.0±9.4% vs. 137.8±14.3%, p=0.05 n=10). In order to analyze the cellular mechanisms of the impairment, a protocol was developed to apply pharmacological agents to the experimental preparation while maintaining tissue homeostasis. Endothelial and smooth muscle dependent vasodilation were impaired with ischemia, 39.6 ± 13.6% vs. 80.5 ± 11.4% and 43.0 ± 11.7% vs. 85.1 ± 10.5%, respectively. From this data, it can be supported that smooth muscle dysfunction is the reason for the observed impairment in arterial vasodilation.

Previous research regarding aquatic acidification has examined the protonation of the carbonate and does not consider calcium to be a limiting factor. This is the first study to suggest that pH may affect the uptake of calcium in crustacean gills. This project describes ion transport mechanisms present in the cell membranes of white river shrimp Litopenaeus setiferus gill epithelium, and the effects of pH on the uptake of calcium by these means. Partially purified membrane vesicles (PPMV) of shrimp gills were prepared through a homogenization process that has been used previously to define ion transport in crab and lobster gill tissues. In the current study, shrimp gill PPMV calcium uptake at 50 µM, and 250 µM was greatest at pH 7.0 (p=0.01, p=0.0001). A valinomycin/K+ induced membrane potential (PD) at pH 7.0 significantly increased (p=0.003) calcium uptake from that observed in the absence of a PD. An induced PD at pH 8.0 significantly increased (p=0.003) calcium uptake from that observed in the absence of a PD, however, was not significantly greater than uptake at pH 7.0 in the presence of a PD (p=0.05). Amiloride (2mM) treatments, and amiloride (2mM) + verapamil (100µM) cocktail treatments showed significant decrease in calcium uptake from the control (p=0.03), however, they were not different from each other. This indicates an electrogenic carrier with two driving forces: calcium concentration, and asymmetric exchange stoichiometry.

The field of stem cell research has been growing more because of the interest in using stem cells to cure diseases and heal injuries. Human embryonic stem cells, because of the controversy surrounding them—and subsequently the difficulties in acquiring samples of the existing aging cell lines—can only be used in limited capacities. While the development of induced pluripotent stem cells in the last decade has allowed the field to progress closer to medical treatments, the low efficiency of reprogramming a somatic cell to a pluripotent state, and the vast molecular and genomic differences between human embryonic stem cells and human induced pluripotent stem cells is still an issue. Therefore, the goal is to discover methods, chemicals, and factors that can reduce these differences and increase the efficiency of inducing pluripotency. This proposal aims to look at the effects of the protein ECAT1 in inducing pluripotency in human somatic cells. Little is known about ECAT1, otherwise known as Embryonic Stem Cell-Associated Transcript 1, beyond its presence in human embryonic stem cells and oocytes and its absence in differentiated cells. While originally considered by scientists during the development of the reprogramming technique, ECAT1's effects have not been tested in humans. Therefore, a series of experiments will be performed in which ECAT1 will be used in conjunction with OSKM to induce pluripotency in adult human dermal fibroblasts, which will then be differentiated into spinal motor neurons. The three stages of this proposal--inducing pluripotency, comparing pluripotencies in the reprogrammed cells and embryonic stem cells, and differentiating the stem cells--should answer questions about ECAT1 and the reprogramming process. It is predicted that ECAT1 should reduce the genomic and molecular differences between embryonic stem cells and induced pluripotent stem cells. ECAT1's presence should also increase the efficiency of reprogramming as well as successful differentiation to other cell types.