<p> Polycystic Ovarian Syndrome (PCOS) is a metabolic and endocrine disorder that is the most common cause of infertility. PCOS can manifest itself as a long and short term disability and is characterized by insulin resistance (IR), hyperandrogenism, anovulation, hyperinsulinaemia and polycystic ovaries. Our lack of understanding of this disorder and its long term effects has complicated the treatment of the disorder; yet, it is clear that PCOS involves the intricate interaction between genetics, environments and behaviors. To study this disease, scientists have used various animal models. Since the <i> Drosophila</i> model for PCOS has only been postulated,in this work, we determined whether starvation along with the addition of steroid hormones would induce a PCOS-like disorder in <i>D. melanogaster</i> after 24 hour exposure. </p><p> In women with PCOS, testosterone levels and the expression of the androgen receptor are elevated. In fruit flies, ecdysone (E) and its &ldquo;active&rdquo; form, 20-hydroxyecdysone (20E), are homologous to the human testosterone and 20-hydroxytestosterone, respectively. This hormone is required for circadian cycles, molting, and maturation in insects. More specifically, this hormone is also located in ovarian tissue and aids in follicular development. The receptor for ecdysone is the ecdysone receptor (EcR). In this work, we examined the expression of the ecdysone receptor (EcR) upon starvation for up to 24 hours by immunofluorescence microcopy. Using qRT-PCR, we determined the levels of expression of genes usually associated with inflammation. Ovarian dysfunction was examined by measuring the fecundity of the females. Starvation increases the expression of the EcR and pro-inflammatory gene expression and decreases fecundity, suggesting that <i>Drosophila melanogaster</i> is a potentially useful model organism in the study of PCOS.</p>

Thesis (Ph.D.) -- University of Texas at Arlington, 2009.

<p> <i>Myo-inositol</i> is a six-carbon sugar alcohol. It is essential as a precursor of the phospholipid membrane component phosphatidylinositol (PI) and the phosphoinositide signaling pathway in all eukaryotes. It aids in cellular metabolism, osmoregulation, and plays an important role in fertilization and diseases such as diabetes, bipolar disorder, and Alzheimer&rsquo;s disease. <i> Myo</i>-inositol metabolism is comprised of synthesis, transport, catabolism, and recycling. <i>Myo</i>-inositol synthesis is catalyzed by myo-inositol-3-phosphate synthase (MIPS). Surprisingly, synthesis of <i>myo</i>-inositol and its role in fertilization has not yet been studied in the model organism <span style="text-decoration:overline"> Drosophila melanogaster</span> (fruit fly). We hypothesize that MIPS expression is essential for growth and development of <i>D. melanogaster.</i> In this study, a precise deletion of the entire MIPS gene was generated and confirmed through PCR amplification and sequencing of the resultant DNA fragments. The lack of the MIPS transcript in homozygous MIPS deletion flies was confirmed by RT-PCR. During that experiment, two additional isoforms of MIPS were identified in wild-type flies (CS). Supplementation of chemically defined food with 0.5mM inositol was required to sustain all homozygous MIPS deletion fly strains. Fully-grown homozygous deletion flies could live without additional inositol in the food, but newly emerged larvae only survived to the first instar larval stage. However, even while on rich media supplemented with 170mM inositol, a homozygous MIPS deletion stock was unable to produce viable offspring. Homozygous MIPS deletion strains were identified as male-sterile, incapable of producing offspring when mated to any strain of females (including wild-type). Homozygous female MIPS deletion flies were fertile and maintained a high fecundity rate when mated to any strain (with an exception of homozygous male MIPS deletion flies). The male-sterility was complemented with the addition of a wild-type MIPS gene to chromosome 3. Testes dissections of homozygous male MIPS deletion flies revealed improper progression of spermatogenesis, specifically during sperm individualization. These studies contribute to the understanding of the role of inositol synthesis in growth, development, and fertilization.</p>

<p> Celsr1 is an atypical cadherin central to the asymmetric cell-cell complexes that define planar cell polarity (PCP). Previous work has shown that Celsr1 undergoes bulk endocytosis during cell division in the basal layer of the mouse skin. Here, we report the unexpected finding that Celsr1-mediated intercellular complexes remain intact during mitotic internalization, resulting in uptake of Celsr1 and associated PCP components into dividing cells from their neighbors in a process known as trans-endocytosis. Our observations suggest that the bulk of this internalized pool of Celsr1 is targeted for degradation. Furthermore, Celsr1 internalized from neighboring cells carries with it additional core PCP proteins, including the posteriorly-enriched Fzd6 and anteriorly-enriched Vangl2. However surprisingly, Vangl2 originating from the dividing cell is excluded from mitotic endosomes and remains associated with the membrane. Overexpression of Vangl2 in vitro is sufficient to interfere with Celsr1 internalization, and mitotic internalization of Celsr1 within the skin is delayed at anterior cell surfaces. We propose that Vangl2 stabilizes Celsr1 at the membrane and its dissociation from Celsr1 is a prerequisite for Celsr1 turnover. Together our results indicate that mitotic turnover of Celsr1 depends on the displacement of Vangl2 from PCP complexes and results in the non-autonomous turnover of PCP proteins from neighboring cells.</p><p>

<p> Early embryonic cells in many species polarize radially by distinguishing their contacted and contact-free surfaces. Radial polarization is a critical patterning event driven by cell-cell contact and is required for developmental processes, such as the first differentiation event in the early mammalian embryo. The homophilic adhesion protein E-cadherin is required for contact-induced polarity in many cells. However, it is not clear whether E-cadherin functions instructively as a spatial cue, or permissively by ensuring adequate adhesion so that cells can sense other contact signals. In <i>C. elegans,</i> radial polarity begins at the four-cell stage, when cell contacts restrict the PAR polarity proteins to contact-free surfaces. We previously identified the RhoGAP PAC-1 as an upstream regulator that is required to exclude PAR proteins from contacted surfaces of early embryonic cells. PAC-1 is recruited specifically to sites of cell contact and directs PAR protein asymmetries by inhibiting the Rho GTPase CDC-42. How PAC-1 is able to sense where contacts are located and localize to these sites is unknown. We show that HMR-1/E-cadherin, which is dispensable for adhesion, functions together with HMP-1/&alpha;-catenin, JAC-1/p120 catenin, and the previously uncharacterized linker PICC-1/CCDC85/DIPA to bind PAC-1 and recruit it to contacts. Furthermore, we show that ectopically localizing the intracellular domain of HMR-1/E-cadherin to contact-free surfaces of cells recruits PAC-1 and depolarizes cells, demonstrating that HMR-1/E-cadherin plays an instructive role in polarization. Furthermore, we show that radial polarity is defective in embryos lacking HMR-1/E-cadherin. Our findings identify an E-cadherin-mediated pathway that translates cell contacts into cortical polarity by directly recruiting a symmetry-breaking factor to the adjacent cortex.</p>

<p> Agents are of central importance to cognitive science, but research usually takes them as pre-given and proceeds to study some of their particular aspects, often without awareness of or a definite answer to the question, &ldquo;what is an agent?&rdquo; The conceptual framework of autopoiesis and enaction provides a foundation that defines agents as emergent individuals that act in an environment to fulfill their physiological needs. </p><p> To establish this definition in concrete examples, this dissertation introduces computational models and analyses that demonstrate several properties of agents. It examines an artificial chemistry that supports the emergence of minimal protocells. These protocells have a metabolism made of autocatalytic components, and an external boundary that self-assembles and encapsulates the metabolism, keeping it from diffusing into the environment. Metabolism and boundary are mutually enabling processes, which together counter the effects of diffusion and decay. When their symbiosis is broken, the protocell disintegrates. </p><p> Systematic analysis reveals the rich consequences of protocellular organizations. Ontogenies are mapped as network structures, with the networks' nodes as reachable protocell morphologies and its edges as the transitions between morphologies. Analyses of these networks reveal properties such as irreversibility (some changes cannot be reversed under any circumstance) and branching (unfolding down one ontogeny excludes morphologies accessible by other ontogeny). Viability is quantified as expected lifespan, and measured across different protocell configurations. This provides a basis for measuring agents' basic goal of adaptivity &mdash; to increase their viability in a given environment through internal restructuring or environmental change. </p><p> The cellular Potts model (CPM) framework is examined to study structural coupling (the bi-directional interactions between an agent and environment). The network-based methodology for analyzing ontogenies is extended to incorporate a local environmental state and is demonstrated in a CPM. This reveals several interesting features, such as a divergence in the space of possible ontogenies when placed in different environments, and that niche construction can increase an individual's viability.</p>

<p> Recent advances regarding commensals in the gastrointestinal tract point to an intimate &ldquo;accessory&rdquo; organ status. To study the cross-talk that an accessory organ must have, the Piletz laboratory began in 2014 developing a three-dimensional (3D) <i>in vitro</i> co-culture model system, whereby two differentiated cell lines are juxtaposed along with &ldquo;luminal&rdquo; contents. The model uses differentiated C2BBe1 cell line enterocytes grown to confluency on polycarbonate filters with 0.4 &micro;m pores over-layered atop SH-SY5Y cell line neurons to study cross-talk from either the lumen-side or the neuron-side. The focus is on an endogenous molecule, agmatine (1-amino-4-guanidobutane), made by gut bacteria at millimolar concentrations in the mucosa of the small intestine&mdash;yet in the brain known to be a neurotransmitter. Starting with each individual cell line in standard mono-cultures, agmatine was added at varying doses and varying times to replicate what is essentially dogma to the agmatine field, that of being anti-proliferative to all mammalian cells. Above 1 mM agmatine, the predicted anti-proliferative response was realized as a non-toxic, non-divisional state sustained for at least 4 days from single dosing. Moving to the 3D co-culture system, wherein the C2BBe1 cells were differentiated as per high transepithelial electrical resistance (TEER) over a 24-hour equilibration period, it was expected that agmatine would again be <i>anti-proliferative</i>. Yet, apical agmatine appeared to exert a <i>pro-proliferative</i> effect starting as low as 0.002 mM. A parallel decline in metabolism per SH-SY5Y cell was found using the color dye reaction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was therefore hypothesized that apical agmatine had caused the C2BBe1 cells to secrete a growth signal(s) impacting the underlying SH-SY5Y cells; and to test this, conditioned basal media collected from just C2BBe1 cells grown 4 days in the presence of apical 2 mM agmatine was taken to replace the media of na&iuml;ve SH-SY5Y cells growing in log phase mono-cultures. The expectation was that growth factors would be carried over, but to the contrary, an anti-proliferative response emerged from the conditioned media, mirroring the earlier studies with agmatine in mono-cultures. Cellular lysates were also prepared from treated cells exposed for 24 h to 2 mM agmatine, and these were probed on immune-blots to assess if any of 32 common receptor tyrosine kinases had phosphorylated /activated post-addition of apical mM agmatine. No evidence was obtained that agmatine (mM apical) had elicited such flags of cell activation. Next, the 3D co-culture condition was re-run for longer periods and with more controls, and from this came the realization that the model had hidden the existence of an anti-proliferative response from the C2BBe1 cells before agmatine was even added. In short, the starting hypothesis was disproven, but in doing so it was realized that micromolar apical agmatine is able to rejuvenate a cytostasis rendered by the C2BBe1 co-culturing. Two fundamentally different mechanisms must be invoked by agmatine, because the concentrations of agmatine at which these two processes occurred were 500-fold different (0.002 mM for the reversal of cytostasis vs. 1 mM for anti-proliferative, respectively). In summary, any microbial dysbiosis involving agmatine-producing bacteria is likely to act through two molecular signaling mechanisms from the &ldquo;accessory&rdquo; organ bacteria to enteric nervous system.</p><p>

<p>Fibroblast growth factors (FGFs) and their receptors (FGFRs) play a vital part in the proper development and maintenance of the brain. FGFR1, which is one of four FGFRs total and one of three found in the brain (FGFR1-3), has been shown to be important in cellular proliferation, cellular migration, synaptogenesis, cellular morphology, and has also been implicated in multiple neuropsychiatric disorders. Understating the role FGFR1plays in these and other cellular processes is vital to our understanding of the human body and in the prevention and treatment of some neuropsychiatric and developmental disorders. Although previous studies have produced groundbreaking findings in the field, they have fallen short in the accurate identification of which cell type express Fgfr1. Therefore, to validate the use of a transgenic mouse line in the accurate and efficient study of Fgfr1 expression during experimental manipulations and cellular localization, we utilized the tgFGFR1- EGFPGP338Gsat BAC mouse line (tgFgfr1-EGFP+) obtained from the GENSAT project. By utilizing the tgFgfr1-EGFP+ mouse line, we were able to accurately identify which cell types in the embryonic and perinatal mouse brain express Fgfr1. Furthermore, we were able to measure relative changes in Fgfr1 expression via GFP fluorescence as a proxy after both exposure to chronic stress and the chemical demyelinator, cuprizone. The combination of our results lead us to conclude that the tgFgfr1-EGFP+ mouse line is a very useful tool in the study of FGFR1 and may aid in the identification of potential targets for therapeutic treatment of the many disorders associated with FGFR1 signaling.

<p> Group I Paks are serine/threonine kinases that function as major effectors of the small GTPases Rac1 and Cdc42. They regulate many cellular functions, including cell polarity, cytoskeletal dynamics, and transcription. Pak1 and Pak2 are redundantly essential for embryonic skeletal myoblast fusion in <i> Drosophila</i>, with Pak2 playing the more important role. Both are expressed in mammalian skeletal muscle, but little is known as to their function in myogenesis. We find that Pak1 and Pak2 are expressed in mammalian myoblasts and are activated specifically during differentiation. Individual genetic deletions of <i>Pak1</i> and <i>Pak2</i> in mice show no overt defects in muscle development or regeneration. However, young adult mice with muscle-specific deletion of <i>Pak1</i> and <i>Pak2 </i> together (dKO mice) present with reduced muscle mass and a higher proportion of myofibers with smaller cross-sectional area compared to controls. This phenotype is exacerbated after repair to acute injury. Primary myoblasts from dKO animals show delayed differentiation, with lower expression of myogenic markers and inefficient myotube formation. Additionally, with age, dKO mice develop a chronic myopathy. Histological analyses of resting muscle show the presence of central nuclei in the majority of fibers, as well as significant fibrosis, inflammation, necrosis, and hypertrophy with fiber splitting. Ultrastructural analysis revealed grossly elongated and branched intermyofibrillar mitochondria, known as megaconial mitochondria, along with occasional accumulation of subsarcolemmal mitochondria. Moreover, dKO mice show impaired mitochondrial function, with significantly reduced Complex I and II activity. These characteristics are absent in control animals. We conclude that the role of Pak1 and Pak2 in embryonic myoblast fusion, first identified in the fly, is not conserved in mammals. Rather, our data demonstrate that Pak1 and Pak2 function redundantly in regulating myoblast differentiation, thereby impacting overall postnatal muscle size. Furthermore, their major function appears to be in muscle homeostasis. Few protein kinases have been implicated in muscle disease. Group I Paks have wide roles in cell regulation, and the generation of dKO mice provides a genetic system to gain new mechanistic insights into muscle maintenance, as well as to discover the substrates of Paks that regulate this process.</p><p>

<p>Over the course of development, cells and tissues of the embryo must take on the correct fates and morphologies to produce a functioning organism. The patterning events and morphogenetic processes that accomplish this task have been the subject of decades of research, the consequence of which has been a detailed comprehension of the molecular mechanisms that regulate each. Equally important is an understanding of the mechanisms that coordinate patterning with morphogenesis, such that they occur with the correct relative spatiotemporal dynamics. My thesis work sought to characterize such co-regulation in the context of two developmental events in a vertebrate model, the African clawed frog Xenopus laevis: induction of bottle cell formation at the onset of gastrulation after germ layer induction, and regulation of the morphogenetic movements of neurulation in relation to neural plate patterning. Chapter 1 of this dissertation provides a general introduction to the patterning and morphogenetic events of early development relevant to my thesis. Chapter 2 presents a discussion of my work to characterize the function of two signaling pathways, namely Nodal signaling and Wnt/Planar Cell Polarity, in the induction of bottle cells. My experiments confirm the requirement for Nodal signaling in bottle cell induction, but do not support a role for the individual transcriptional targets of Nodal signaling tested here or for Wnt/PCP. Chapter 3 summarizes my work to address the function of two transcription factor-encoding genes, sall1 and sall4, in neural development, including their roles in anteroposterior neural patterning, neural tube morphogenesis, and neural differentiation. My work shows that both sall1 and sall4 are required for all three processes, and supports the hypothesis that their key role in this context is to transcriptionally repress stem cell factors of the pou5f3 family, allowing progression through neural development. As a whole, this work summarizes my research to characterize molecules that co-regulate early patterning and morphogenetic events in the X. laevis embryo.