Charles E. Schmidt College of Medicine

Recently, there has been an increase in marine mammal mortalities, most commonly Atlantic bottlenose dolphins, Tursiops truncatus, which is an alarming indication of the health status of the marine ecosystem. Studies have demonstrated that some free-ranging dolphins exhibit a suppressed immune system possibly because of exposure to contaminants or infectious microorganisms. However, this research has been limited due to a lack of commercially available marine-specific antibodies. Therefore, the first chapter of this thesis aims to identify cross-reactive terrestrial-specific antibodies that could be used to phenotype and compare the immune cell populations of dolphins under human care and free-ranging dolphins. The second chapter aims to utilize terrestrial-specific growth factors and dendritic cell (DC) surface markers to generate, characterize, and compare ex vivo DCs from peripheral blood mononuclear cells (PBMCs) of dolphins under human care and free-ranging dolphins. In summary, I have identified differences within the PBMCs and ex vivo generated DCs of dolphins under human care and free-ranging dolphins that could potentially shed light on the impact of environmental contaminants and infectious microorganisms on immune cells which could lead to increased morbidity and mortality.

Restrictive cardiomyopathy (RCM) is represented in part by left ventricular stiffness
and diastolic dysfunction. Missense mutations of the cardiac troponin I (cTnI) gene cause
idiopathic RCM. These mutations are located in the C-terminus of cTnI and affect cardiac
relaxation. Transgenic mouse models presenting the pathology observed in clinical
patients with RCM have been generated previously and express the mutant cTnI in their
hearts. RCM-linked mutations increase cardiac myofilament Ca2+ sensitivity and promote
diastolic dysfunction in the heart. Previous studies using double transgenic mice
(cTnI/R193H/ND) showed that ventricular relaxation is enhanced in the cTnI/R193H
transgenic mice. In this study, another double transgenic mouse model,
(cTnI/R193H/ND/KO), provides an avenue to investigate its rescuing effects on RCMlinked
mutations in the cTnI /R193H/KO mouse. Use of molecular biological techniques,
transgenic animal developments and murine echocardiography in this study has
culminated into a greater understanding of RCM and diastolic dysfunction.

This research focuses on obesity and other major risk factors for chronic diseases
such as Type II Diabetes Mellitus, Heart Disease, and Stroke. Worksite wellness
programs have been successful in this realm of health promotion and disease prevention
for heart disease and stroke, but their effectiveness in treating diabetes has been uncertain
partially due to poor patient compliance, lack of stress reduction strategies, poor diet and
lack of persuasive health education on the risk of being obese. Published peer-reviewed
articles were reviewed, coded and analyzed to determine best practices, using a modified
systematic review approach. The findings from these studies yield results that were used
to develop a new employer-sponsored wellness program that is in accordance with the
recently passed Affordable Care Act.

A naturally-occurring recessive lethal mutation in axolotls, Ambystoma mexicanum, is an intriguing model for studying tropomyosin expression regulation. Homozygous embryos(c/c) form hearts that are deficient in tropomyosin, lack organized myofibrils and fail to beat. Previous studies have shown that a non-coding RNA gene, MIR (Myofibril Inducing RNA), is sufficient to rescue the non-beating homozygous recessive mutant hearts by promoting sarcomeric tropomyosin expression that leads to formation of organized myofibrils and beating hearts. Real time RT-PCR reveals that mutant hearts express the same level mRNA of the alpha-tropomyosin and TM4 type tropomyosin (ATmC-3) gene as normal embryonic hearts. These genes show no differences with regard to the splicing patterns of normal and mutant. Using protease inhibitor LLnL and E-64d treatments and two-dimensional Western blots of normal and mutant hearts, it is found that mutant hearts express all tropomyosin protein isoforms as normal hearts but protein expression are at low levels. These studies suggest that there is a failure in the translational or posttranslational control mechanisms for tropomyosin protein synthesis in cardiac mutant axolotl hearts during development.

Exoribonucleases degrade RNA and are important in RNA metabolism and gene expression. Mycoplasma genitalium, a bacterium with the smallest genome known, has only one identified exoribonuclease, RNase R (MgR). In this work RNA degradation properties of purified MgR were examined. As observed in Escherichia coli RNase R (EcR) studies, MgR degrades poly(A), rRNA, and oligoribonucleotides in 3'--->5' direction, though its substrate specificity and optimal activity requirements vary. Interestingly, MgR is sensitive to 2-O-methylation stopping downstream of such modifications in native rRNA and synthetic oligoribonucleotides. MgR removes the 3' trailer sequence from a tRNA precursor of M. genitalium and generates products equal to the mature tRNA, demonstrating a role of MgR in tRNA maturation. The 3' terminal CCA sequence and the acceptor stem of tRNA play a role in determining the formation of such products by MgR. These results suggest multiple functions of RNase R in RNA metabolism in Mycoplasma.

Troponin I (TnI) plays an important role in cardiac muscle contraction. Two TnI genes (cardiac and slow skeletal TnI) are predominantly expressed in the heart. In cTnI knockout mice, myocardial TnI deficiency results in a diastolic dysfunction and a sudden death in homozygous mutants. In the present studies, energy metabolism has been analyzed in myocardial cells from cTnI null hearts. Our results have demonstrated that damaged relaxation and increased Ca2+-independent force production in cTnI null hearts stimulated myofibril MgATPase activities accompanied by the increase of mitochondria quantity and ATPase activities. In addition, an increase of ssTnI phosphorylation level has been observed in cTnI null hearts. The results indicate that TnI deficiency can cause the disturbance of energy metabolism and some protein overphosphorylation.

Soluble form of Junctional adhesion molecule C (sJAM-C) has been identified to cause angiogenesis as well as chemotaxis in endothelial cells. However, the role of sJAM-C in the context of cancer has not been elucidated. Our atomic force microscopy (AFM) stiffness measurements of normal mammary epithelial cells (MCF 10A) have shown a two-fold decrease in cell's stiffness in response to sJAM-C. Changes in cell stiffness are indicative of modulations in a cell's mechanical properties. Our results indicated that sJAM-C increased the MCF 10A cell migration about two-fold and also promoted a three-fold increase in chemotaxis. Additionally, sJAM-C treatment resulted in considerable filamentous-actin loss and peripheral actin ring breakage. We also found activation of Rho signaling pathway to be the main mechanism behind sJAM-C mediated alterations in MCF 10A cell cytoskeleton and motility. Our data present for the first time that sJAM-C is a pro metastatic mediator for normal mammary epithelial cells.

Restrictive cardiomyopathy (RCM) is a heart muscle disease, characterized by diastolic dysfunction. The present dissertation is to understand the mechanisms underlyijng the initiation of diastolic dysfunction and the fast disease progression to early death in a RCM mouse model, the transgenic cTnI193His mouse... These data showed that myocardial ischemia occurred after diastolic dysfunction and before systolic dysfunction which proceeded congestive heart failure. The results demonstrate that myocardial ischemia causing cardiomycete death is a link between the initial diastolic dysfunction and late-stage systolic dysfunction, and accelerates the disease progression to fatal heart failure in the early age.

We attempted to understand the molecular regulators that impact inflammation using a rat model of human sensation-seeking/risk-taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress. We found that HRs have a pro-inflammatory phenotype as indicated by increased protein expression of the inflammatory cytokine TNF-(Sa(B. Furthermore, we found that HRs have a lower gene expression of the glucocorticoid receptor and histone deacetylase 2 which are known to play an immunosuppressive role. Autophagy (macroautophagy) is a homeostatic process needed for cell maintenance, growth and proliferation and known to assist in tumor survival. FYVE and coiled-coil domain containing 1 (FYCO1) is a novel protein implicated to assist in the plus-end directed trafficking and fusion of autophagosomes. In these studies, we show that FYCO1 gene expression among human breast cell lines of varying degrees of malignancy.

The lens is an avascular organ that focuses light onto the retina where neural signals are transmitted to the brain and translated into images. Lens transparency is vital for maintaining function. The lens is formed through a transition from organelle-rich epithelial cells to organelle-free fiber cells. Lens cell differentiation, leading to the lack of organelles, provides an environment optimal for minimizing light scatter and maximizing the ability to focus light onto the retina. The process responsible for orchestrating lens cell differentiation has yet to be elucidated. In recent years, data has emerged that led our lab to hypothesize that autophagy is likely involved in lens cell maintenance, cell differentiation, and maintenance of lens transparency. As a first step towards testing this hypothesis, we used RT-PCR, western blot analysis, immunohistochemistry, confocal microscopy, and next generation RNA-Sequencing (RNA-Seq) to examine autophagy genes expressed by the lens to begin mapping their lens function.