<p> Understanding the biophysical processes underlying biological and biotechnological processes is a prerequisite for therapeutic treatments and technological innovation. With the exponential growth of computational processing speed, experimental findings in these fields have been complemented by dynamic simulations of developmental signaling and genetic interactions. Models provide means to evaluate "emergent" properties of systems sometimes inaccessible by reductionist approaches, making them test beds for biological inference and technological refinement.</p><p> The complexity and interconnectedness of biological processes pose special challenges to modelers; biological models typically possess a large number of unknown parameters relative to their counterparts in other physical sciences. Estimating these parameter values requires iterative testing of parameter values to find values that produce low error between model and data. This is a task whose length grows exponentially with the number of unknown parameters. Many biological systems require spatial representation <i>(i.e.,</i> they are not well-mixed systems and change over space and time). Adding spatial dimensions complicates parameter estimation by increasing computational time for each model evaluation. Defining error for model-data comparison is also complicated on spatial domains. Different metrics compare different features of data and simulation, and the desired features are dependent on the underlying research question.</p><p> This dissertation documents the modeling, parameter estimation, and simulation of two spatiotemporal modeling studies. Each study addresses an unanswered research question in the respective experimental system. The former is a 3D model of a nanoscale amperometric glucose biosensor; the model was used to optimize the sensor's design for improved sensitivity to glucose. The latter is a 3D model of the developmental gap gene system that helps establish the bodyplan of <i>Drosophila melanogaster;</i> I wished to determine if the embryo's geometry alone was capable of accounting for observed spatial distributions of gap gene products and to infer feasible genetic regulatory networks (GRNs) via parameter estimation of the GRN interaction terms. Simulation of the biosensor successfully predicted an optimal electrode density on the biosensor surface, allowing us to fabricate improved biosensors. Simulation of the gap gene system on 1D and 3D embryonic demonstrated that geometric effects were insufficient to produce observed distributions when simulated with previously reported GRNs. Noting the effects of the error definition on the outcome of parameter estimation, I conclude with a characterization of assorted error definitions (objective functions), describe data characteristics to which they are sensitive, and end with a suggested procedure for objective function selection. Choice of objective function is important in parameter estimation of spatiotemporal system models in varied biological and biotechnological disciplines.</p>

<p> In the past five years, Single Molecule Real Time (SMRT) sequencing technology has been found to be a reliable indicator of certain epigenetic modifications in bacterial genomes. The genome of the model organism <i>Saccharomyces cerevisiae</i> has long been thought to be free of DNA level modification, but literature surrounding this subject is conflicting. Additionally, the mitochondria of <i>S. cerevisiae</i> control the transition between three distinct chronological life phases &ndash; exponential, postdiauxic, and stationary - as defined by their main metabolic processes. This study attempted to identify base modifications to mtDNA using PacBio sequencing while additionally establishing gene expression changes as a result of altered mitochondrial metabolic capabilities. PacBio results showed intriguing results but statistical analysis proved experimentation with improved protocols were necessary. Multiple genes with unknown or uncharacterized function were also shown to have significant differential expression between metabolic life phases.</p>

<p> Ring canals are made from arrested cleavage furrows, and provide direct cytoplasmic connections among sibling cells. They are well documented for their participation in Drosophila oogenesis, but little is known about their role in several somatic tissues in which they are also found. Using a variety of genetic tools in live and fixed tissue, I demonstrate that the &sim;250 nm diameter somatic ring canals permit rapid intercellular exchange through somatic ring canals by diffusion. Additionally, intercellular diffusion of protein was observed between cells with highly disparate levels of mRNA transcript, suggesting a possible role for ring canals in smoothing gene expression within a tissue. I also used a novel combination of markers to evaluate the extent of protein movement within mitotic clones and across clone boundaries in ovarian follicle cells and imaginal discs, providing evidence of robust movement of GFP between the two sides of mitotic clones and frequently into non-recombined cells. These data suggest that, depending on the experimental setup and proteins of interest, inter-clonal diffusion of protein may alter the interpretation of clonal data in follicle cells. Our work illustrates the lack of cytoplasmic autonomy in these tissues and suggests a role for somatic ring canals in promoting homogeneous protein expression within the tissue.</p>

<p> Refractive development has been shown to be influenced by optical defocus in the eye and the interpretation of this signal appears to be localized in the retina. Optical defocus is not uniform across the retina and has been suggested as a potential cause of myopia development. Specifically hyperopic focus, i.e. focusing light behind the retina, may signal the eye to elongate, causing myopia. This non-uniform hyperopic signal appears to be due to the retinal shape. Ultimately, these signals are detected by the retina in an as yet undetermined manner. The purpose of this thesis is to examine the retinal profile using a novel method developed at Indiana University and then to examine retinal structural changes across the retina associated with myopia. </p><p> Myopes exhibited more prolate retinas than hyperopes/emmetropes using the SD OCT. Using the SD OCT, this profile difference was detectable starting at 5 degrees from the fovea, which was closer than previously reported in the literature. These results agreed significantly with results found from peripheral refraction and peripheral axial length at 10 degrees. Overall, the total retina was thinner for myopes than hyperopes/emmetropes. It was also statistically significantly thinner for the Outer Nuclear Layer (ONL), Inner Nuclear Layer (INL) and Outer Plexiform Layer (OPL) but not for other retinal layers such as the Ganglion Layer. Thinning generally occurred outside of 5 degrees. </p><p> The SD OCT method provided a nearly 10 fold increase in sensitivity which allowed for detection of profile changes closer to the fovea. The location of the retinal changes may be interesting as the layers that showed significant differences in thickness are also layers that contain cells believed to be associated with refractive development (amacrine, bipolar, and photoreceptor cells.) The reason for the retinal changes cannot be determined with this study, but possible theories include stretch due to axial elongation, neural remodeling due to blur, and/or direct influence on refractive development due to neural cell densities.</p>

<p> Cellular DNA is condensed and stored with the help of proteins, especially histones. This mix of DNA and proteins is termed chromatin. Manipulations of chromatin include post-translational modifications (PTMs) of histone proteins, such as the addition of methyl or acetyl groups, and the movement of histones by chromatin remodeling complexes to control how tightly or which regions of DNA are condensed or exposed. These actions can impact gene expression and thus influence the differentiation of tissues during development. I investigated if the enzyme protein arginine methyltransferase 5 (PRMT5), which mono- or symmetrically dimethylates arginine, is acting on histones during early development in Xenopus laevis. I also investigated if Williams syndrome transcription factor (WSTF) occurs within chromatin remodeling complexes during early development in X. laevis. WSTF interacts with the protein ISWI in early embryos, indicating it is part of a chromatin remodeling complex during this period of development. </p>

Introduction: Hirschsprung’s Disease (HD) is the congenital absence of ganglion cells (GCs) within the distal intestine. Our objectives are to determine if apoptosis of myenteric GCs occurs during human development and to determine if myenteric GC apoptosis or injury contributes to HD. Materials and Methods: Apoptosis of myenteric GCs was assessed in archived fetal intestinal tissue (n = 4; 15-41 weeks gestational age) and in HD at the transition zone (TZ) (n = 6) using anti-cleaved caspase-3. Immunohistochemistry for GFAP, CD68, HLA-DR and APP was used to assess the presence of enteric reactive changes. Results: No activated caspase-3 expression was present in the myenteric GCs of the developing human intestine or the TZ of HD. No significant increase in GFAP, CD68, HLA-DR or APP expression was present. Conclusions: Apoptosis does not appear to occur during the development of the human myenteric plexus or, in conjunction with GC injury, in HD.

<p> To maintain human health, the lymphatic system requires a structurally and functionally sound network of lymph vessels to absorb lipid-based nutrients, preserve extracellular fluid homeostasis, and mediate immune responses. Aside from lymphedema, investigations in the past few decades have found that impairment of the lymphatic vasculature is also involved in processes such as inflammation, tumor metastasis, fat metabolism, and obesity. However, despite a long history of study and rekindled vigor in the field of lymphatic vascular research, our knowledge of lymph vessel development and physiology is still quite limited. Recently, mutations in a protein family known as connexins (Cxs) were identified as the cause of lymphatic dysfunction in some cases of inherited lymphedema. This dissertation explores the role of primarily two specific connexins, Cx37 and Cx43, and the transcription factor Foxc2 in the morphogenesis and function of the lymphatic vasculature in mice. To accomplish this, phenotypic characterization of mice with genetic deficiencies (knockout mice) in Cx37, Cx43, and/or Foxc2 was performed principally via necropsy, histological techniques (immuno-fluorescence microscopy and H&amp;E staining), and Evans blue dye (EBD) injections. Developmental abnormalities were found in lymphatic vascular growth, patterning, and remodeling in mice lacking Cx37, Cx43, Foxc2 or a combined deficiency of these proteins. Reductions or complete loss of lymphatic valves were a common finding in mice lacking one or more of these proteins. These valve deficits underlay lymphatic insufficiencies that resulted in lymphedema and chylothorax in some genotypes. Foxc2 was found to be a regulator of Cx37 expression. Moreover, Foxc2 was also dependent on Cx37 function for proper morphogenesis of lymph vessels. These findings pertaining to the expression of connexins in the lymphatic vasculature, their role in lymphatic valvulogenesis, and the interdependence of Cx37 and Foxc2 during lymph-vascular development represent my original contributions to human knowledge.</p>

In aging Brown Norway rats, there is an activation of the immune system represented by a striking increase in the number of halo cells. As the blood-epididymis barrier should protect from immunological attack, we hypothesized that there would be changes in the structure and function of this barrier with age. Immunocytochemical localization of occludin, ZO-1 and E-cadherin, as well as lanthanum nitrate permeability of the blood-epididymis barrier, were done using the epididymides of rats aged 3, 18, and 24 months. Occludin, ZO-1, and E-cadherin immunostaining was observed at the apico-lateral junction between principal cells in the initial segment of 3-month-old animals; with increasing age, occludin and ZO-1 reactivity decreased, while E-cadherin staining increased along the lateral membrane between principal cells. The most dramatic changes were seen in the corpus epididymidis with age; the intense E-cadherin cytoplasmic staining that was observed at 3 months was absent by 24 months and no occludin or ZO-1 reactivity was observed in older animals. (Abstract shortened by UMI.)

To evaluate why discordance may occur between regression coefficients from cross sectional and longitudinal analyses when ventilatory function is related to ageing, a population was created by computer, and modelled to simulate functional change during life. The program incorporated the effects of many factors known to influence lung function measurements, which were adjusted experimentally so that their contributions to any discordance could be assessed. Regression analyses showed that significant discordance could be induced if the oldest birth cohort failed to reach the same maximal level of function as the youngest; if a quadratic ageing term was excluded from the independent regression variables; or if the effects of certain confounders were present. Discordance occurred additionally if cross sectional estimates became imprecise, but then the differences (often marked) from longitudinal estimates were not significant. It is concluded that discordance may be fundamental and unavoidable (though explicable), or merely a consequence of imprecision.

We studied the consequences of providing rat dams with elevated levels of dietary fat during lactation on stress responses in the offspring at different stages of development. High-fat feeding increased total milk lipid levels, and led to increased lipid deposition and plasma leptin levels in pups. Ten-day-old neonates from high-fat fed mothers had reduced stress responsiveness compared to controls. In contrast, 35-day-old pups from mothers fed high-fat diets showed greater stress-induced ACTH secretion. These findings indicate that maternal diet has effects on hypothalamic-pituitary-adrenal (HPA) responsiveness, which can persist after weaning to a normal rat chow diet. We also investigated macronutrient selection in the offspring, since differences in nutrient preferences may produce long-lasting consequences of lactational diet. However, our results did not support an involvement of maternal diet in postweaning nutrient selection. Furthermore, HPA stress responses were no longer related to maternal diet once offspring reached maturity.