No description available.

<p><strong>Abstract</strong></p> <p>RanBPM (Ran-Binding Protein in the Microtubule Organizing Center) is an evolutionarily conserved protein presumed to function as a scaffold in various signal transduction pathways. Null mutations in the <em>Drosophila</em> <em>RanBPM</em> gene cause a pleiotropic effect as seen by the late larval lethality, impaired growth, reduced CNS proliferation and disruption in feeding and response to light behaviours (Scantelbury et al., 2010). We report that<strong> </strong><em>RanBPM</em> mutants display impaired expression and secretion of one of the major <em>Drosophila</em> insulin like peptides, DILP2. Consistent with these findings<strong> </strong><em>RanBPM</em> mutants showed reduced membrane localization of the Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) reporter TGPH (GFP-pleckstrin homology domain (PH) fusion regulated by the Tubulin promoter) and reduced activation of the serine threonine kinase AKT, both read outs of insulin signalling (InS). Ubiquitous overexpression of <em>DILP2</em> partially rescued the reduced larval growth, CNS proliferation and feeding phenotypes caused by <em>RanBPM</em> mutations. Over expression of DILP6 in the glia rescued the reduced CNS proliferation but not the reduced larval growth and feeding behaviour phenotypes. Targeted expression of <em>RanBPM</em> in the fat body suppressed all <em>RanBPM</em> mutant phenotypes except for lethality. In <em>Drosophila</em>, the fat body acts a nutritional sensor involved in coordinating neuroblast proliferation by a TOR- dependent mechanism. Our results present evidence for the role of AKT in the fat body of the <em>RanBPM</em> mutants for the regulation of CNS proliferation, DILP2 secretion, overall growth and feeding</p> / Master of Science (MSc)

The Hedgehog (Hh) signaling pathway is vital for vertebrate embryogenesis and aberrant activation of the pathway can cause tumorigenesis in humans. In this study, we used a phosphatome RNAi screen for regulators of Hh signaling to identify a member of the Eyes Absent protein family, Eya1, as a positive regulator of Hh signal transduction. Eya1 is both a phosphatase and transcriptional regulator. Eya family members have been implicated in tumor biology, and Eya1 is highly expressed in a particular subtype of medulloblastoma (MB). Here we show that RNAi-mediated knock-down of Eya1, as well as knock-down of its co-factor, Six1, blocks Hh signaling as assessed by induction of Hh response genes. Utilizing small molecule agonists, RNAi, and protein over-expression methods, we place the influence of Eya1 and Six1 within the Hh signaling pathway downstream of Smoothened (Smo) and at or above the level of Suppressor of Fused (Sufu). Interestingly, Eya1 appears to be specifically required for Hh-responsive gene activation mediated by Gli transcriptional activators but not for Hh-mediated transcriptional de-repression mediated by the inhibition of Gli transcriptional repressors. Furthermore, we find that Eya1 and Six1 regulate the expression of Neuropilin1 (Nrp1) and Neuropilin2 (Nrp2), known positive regulators of Hh signaling, providing a mechanism by which Eya1 and Six1 exert their effects. Based on these data, we investigated a role of Eya1 in Hh signaling in vivo. We obtained Eya1-/- mice and focused our attention on the developing cerebellum, where Sonic Hedgehog (Shh) is a major factor promoting neural precursor proliferation. In the Eya1-/- cerebellum, we find a striking reduction in neural precursor proliferation. In addition, we surveyed several other locations where Shh and/or Eya1 are known to be important for development. These include the embryonic otic vesicle, neural tube, and lung. In the developing inner ear we find Eya1-/- mice display reduced Hh signaling in vivo and a genetic interaction between Eya1 and Hh signaling. In lung tissue, Eya1-/- mice have reduced levels of Nrp expression. Together, these data further our understanding of the Hh signaling pathway and provide evidence for a role of Eya1 in Hh signal transduction.

<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.

The mechanisms by which epithelial progenitor cells integrate local signals to balance proliferation with differentiation and regulate patterning during lung organogenesis are still poorly understood. The Hippo pathway and its transcription co-activator Yap have recently emerged as major regulators of progenitor cell expansion and differentiation in development and cancer. Here we investigated the role of Yap signaling in the cellular and molecular events associated with lung epithelial morphogenesis and differentiation. We provide evidence that when airway epithelial tubules are forming and branching, a nuclear to cytoplasmic shift of Yap marks the boundary between the progenitors of the distal lung and the airway compartment. At this transition zone, Yap specifies a transcriptional program that controls the expression of Sox2, restricting distal gene expression and initiating an airway progenitor cell program key to generate the airway epithelium and its branched tubular structures. In Yap deficient mice, epithelial progenitors are unable to properly respond to local Tgf beta-induced cues to control levels and distribution of Sox2, resulting in expansion of the distal epithelial compartment and inability to form airways. Moreover, we show that Yap levels and phosphorylation status play a major role in regulating differentiation of airway progenitors later in development and in adult life. Analysis of YAP-interacting partners in adult airway progenitors by Mass Spectroscopy suggests phosphorylated Yap interactions with ciliome proteins. Our study reveals a crucial role for Yap in specification and differentiation of airway progenitors likely to be also relevant in regeneration-repair of the adult airway epithelium.

Embryonic stem (ES) cells are cell lines isolated from the embryo at a time just prior to implantation into the uterus. In the right cocktail of medium and cytokines, these cell lines can be maintained indefinitely in vitro in a self-renewing state. Initially it was assumed that these cells represented a homogeneous population however, more recently it has been shown that there are a great number of genes that are expressed heterogeneously. ES cell cultures are therefore a mix of different subpopulations, some of which have distinct functional properties including a bias or ‘lineage priming’ towards a particular cell fate. These populations are also dynamic in nature, converting from one state to another with fairly rapid kinetics. The main focus of this thesis was to gain a more in depth understanding of the mechanisms regulating heterogeneity and lineage priming in murine ES cells by asking which signalling pathways play a role in this phenomenon and how the switch between states is regulated at a transcriptional level. These questions were asked using an ES cell line containing a sensitive reporter for the endoderm marker Hex. This reporter, developed by a previous lab member, allowed the identification and separation of a population of ES cells primed towards a primitive endoderm fate. Primarily, I assessed the effect of a defined culture system (2i) on the Hex-expressing population. This culture system contains inhibitors that block FGF signalling and the Wnt pathway component GSK3. Culturing ES cells in 2i has been suggested to generate a more homogeneous culture. Here, I have shown that culturing ES cells or pre-implantation embryos in 2i did not eliminate heterogeneity but maintained them in an early state prior to lineage segregation. When ES cells were cultured in standard serum-containing medium, Hex was expressed in a mutually exclusive manner with the embryonic marker NANOG, while in 2i a subpopulation of cells coexpressed both Hex and NANOG. This population was functionally primed towards extraembryonic endoderm and trophoblast. Furthermore, these ES cells could efficiently contribute to 2-cell embryos in chimaera assays. LIF signalling promoted this population through the JAK/STAT pathway. I then asked how transcription was regulated during the switch between unprimed ES cells to those primed towards a primitive endoderm fate, as well as how regulation changes during further differentiation. To ask this, Hex positive (primed) and negative (unprimed) ES cell populations were sorted as well as a Hex positive differentiated sample. These samples were analysed by GRO-seq to determine the location, density and orientation of RNA-polymerase throughout the genome. Changes in gene expression between primed and unprimed states were regulated primarily through elongation whereas genes upregulated during differentiation were regulated at the point of de novo initiation.

No description available.

No description available.

A comparison between germicidal (254 nm) UV and simulated sunlight (above 300 nm) radiation emitted from a solar simulator has been made using five mammalian cell lines. The cell lines used included normal and photosensitive human skin fibroblasts and one African green monkey kidney cell line. The ratio of 10% survival by exposure to 254 nm radiation divided by 10% survival by simulated sunlight exposure derived from the above comparison was used as a criterion for showing that the above cell lines are statistically significantly different in their relative response to simulated sunlight.

Five-week-old male BALB/c mice were anesthetized and incised mid-dorsally to expose the fascia over the right kidney. The kidney was then palpated up and an inoculum of 0.05 ml containing approximately 100,000 viable Asperaillus flavus spores in phosphate-buffered saline was injected into the cortex. A second group of mice underwent similar surgical procedures, but were injected with phosphate-buffered saline only into the right kidney. This group was designated the sham-operated control mice. All mice, including controls, were bled for differential blood counts 24 and 48 h after surgical procedures and subsequently on an every-other-day basis for the duration of the experiment. For Experiment 1, groups consisting of three infected mice each were killed at intervals of 1, 2, 4, 6, 8, 10, 13 and 16 d. For Experiment 2, three infected animals plus one sham-operated and a control mouse were killed at corresponding intervals as in Experiment 1 and also on day 21. Both kidneys were removed from all animals for weighing and measuring and then the right kidney was processed for histological study. Differential blood counts showed an increase in polymorphonuclear neutrophils in both infected and sham-operated animals, suggesting that the primary change in white blood cell counts was the result of the surgical procedure rather than the infection. Histological studies demonstrated a chronological progression of the development of the fungus in the pelvic area of the kidney, its infiltration into the surrounding tissues, and its effect on tissues in the cortex region of the kidney. The histopathological symptoms of pyelonephritis were present from day four of the infection and continued to be enhanced through the duration of the experiment. Other cellular changes such as fibrotic tissue formations around abscesses, stromal fibrous replacement, and epithelial proliferation also occurred at various stages of the infection.