Slits and their Roundabout (Robo) receptors were identified based on their role in regulating axon guidance, but are known to play multiple roles in development, including regulating heart development and myoblast migration. There are 3 vertebrate Slits (Slit1 – 3) and 4 Robos (Robo1 – 4), and previous work has demonstrated expression of Slit and Robo family members in and around developing joints where their function is unclear. Mutations in human Robo3 have been linked to degenerative joint disorders, such as scoliosis and rheumatoid arthritis. Misregulation of other members of the Slit/Robo signalling pathway is also reported in cells from arthritic joints. This suggests that Slit/Robo signalling is required for normal joint development and/or maintenance, though our understanding of their roles in these processes is rudimentary. The central question of my thesis is to determine the role/s of Slit/Robo signalling in limb and joint development. In situ hybridisation confirmed strong expression of Slits and Robos throughout mouse limb and joint development, though no expression of Slit1 or Robo3 was detected. Analysis of Slit1/2, Slit3 and Robo1 mutant (loss-of-function) mice revealed normal limb development, however misexpression of dominant-negative Robo2 during chicken limb development caused shortening of cartilage elements. To begin to identify molecular changes that may compensate for the loss of Slit/Robo signalling I demonstrated members of the Sema3/PlexinA/Nrp axon guidance family are expressed in patterns comparable to those of Robo1, Robo2 and Slit3. I discovered that PlexinA1 is downregulated in Slit3 mutant mouse limbs. My results suggest the role for Silt/Robo signalling may be more complex than previously thought and do not define a clear role for signalling during limb development. My results suggest the role for Silt/Robo signalling may be more complex than previously thought and do not define a clear role for signalling during limb development. Previous work has linked Slit/Robo signalling to development of degenerative joint disorders, and I propose some hypotheses as to how Slit/Robo signalling could cause bone and joint defects.

Amino acid deprivation of Escherichia coli results in the accumulation of guanosine 5'-triphosphate 3'-diphosphate and guanosine 3', 5'-bispyrophosphate, collectively designated (p)ppGpp. These nucleotides are synthesized by a ribosome-associated enzyme encoded by the relA gene and are thought to represent starvation stress signal molecules. They may mediate the global reorganization of cellular metabolism, known as the stringent response, that is characteristic of starving bacteria and which apparently represents a survival strategy. In this dissertation, the following aspects of the stringent response are characterized: (i) the temperature phenotypes of relA mutants; (ii) the C-terminal domain of RelA; and (iii) the role of RelC (ribosomal protein L11) in the regulation of RelA. All three of the commonly used relA mutant alleles of E. coli, relA1, relA2, and ∆relA251::kan, conferred temperature-sensitive (ts) phenotypes. The temperature sensitivity was associated with decreased thermotolerance, and relA mutants were killed at temperatures as low as 42°C. The ts phenotypes were suppressed by increasing the osmolarity of growth media and by certain mutant alleles of rpoB, the gene encoding the β-subunit of RNA polymerase, suggesting a defect in transcription. DNA in heat-shocked wild type bacteria was initially relaxed but the normal level of negative supercoiling was restored within 10 min after heat shock. In contrast, DNA in heat-shocked relA mutants remained relaxed. This relA-associated defect in DNA negative supercoiling was suppressed by increased medium osmolarity. Furthermore, the re/A-mediated ts phenotype was suppressed by low concentrations of novobiocin, a specific inhibitor of the B subunit of DNA gyrase. Moreover, low concentrations of novobiocin restored DNA negative supercoiling in the relA mutant at high temperature. Based on previous reports, it is proposed that low concentrations of novobiocin induce the synthesis of the DNA gyrase A and B subunits, and the resulting increase in DNA gyrase activity restores normal supercoiling at high temperature. Collectively, the data suggest that relA mutants are unable to efficiently transcribe key genes required for thermotolerance, and this defect is related to their inability to restore negative supercoiling of DNA at higher temperatures. In addition, the proposed defect in transcription may be related to the observation that ppGpp binds to the p-subunit of RNA polymerase. The portion of relA encoding the C-terminal half of RelA (starting at amino acid 455), designated 'RelA, was subcloned. Overexpression of 'RelA relaxed the stringent response by inhibiting (p)ppGpp synthesis during amino acid deprivation. 'RelA represented the ribosome-binding domain, and when overexpressed, 'RelA somehow replaced RelA on ribosomes. The 'RelA ribosome-binding domain was further localized to a region between amino acids 455 to 682 with the main binding activity in a fragment extending from amino acids 560 to 682. Several criteria were used to establish the fact that 'RelA also mediated the formation of homodimers. These included co-purification of RelA and 'RelA, glutaraldehyde protein crosslinking, and analysis by nondenaturing polyacrylamide gel electrophoresis. The dimerization domain overlapped with the ribosome-binding domain. Affinity blotting assays using 'RelA as a probe revealed RelA and 'RelA as the only proteins in crude cell extracts that bound 'RelA. Therefore, these studies failed to identify the ribosomal components that interact with RelA. Amino add-deprived rplK (previously known as relC) mutants of E. coli cannot activate ribosome-bound RelA and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. The overexpression of derivatives of rplK that contained short N-terminal deletions that eliminated the proline-rich helix resulted in relaxed phenotypes. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The L11 mutant proteins were incorporated into ribosomes. A derivative in which Pro22 was changed to Leu22 was constructed by site-directed mutagenesis. This amino add substitution was sufficient to confer a relaxed phenotype when it was overexpressed. A variety of methods were used in attempts to demonstrate a direct interaction between L11 and RelA, but all yielded negative results. These results indicate that the N-terminal proline-rich helix, and Pro22 in particular, is directly involved in activating RelA activity during amino acid deprivation. The mechanism apparently does not involve a direct interaction between RelA and L11 and is presumably mediated by another ribosomal component. / Graduate

The cellular regulation of acidification and intracellular ph (pHi) was studied in the integument of Rana pipiens, a model renal epithelium. Acidification was enhanced by : (1) chronic metabolic acidosis, (2) high salinity adaptation, and (3) ibuprofen treatment.

The purpose of these studies was to determine whether lactogen-dependent Nb2-11c cells and lactogen-independent Nb2-SP cells differ with respect to morphology and autocrine growth control. To this end, the ultrastructural and surface morphology of both Nb2 cell lines was analyzed and the autocrine growth modulatory activity of Nb2 cell conditioned medium (Nb2-CM) was determined. The autocrine growth inhibitory activity of Nb2-CM was biologically characterized and attempts were made to biochemically characterize and purify the Nb2 cell autocrine growth inhibitor as well as to determine its mechanism of action. Quantitative analysis of transmission electron micrographs reveals that the ultrastructural morphology of lactogen-dependent Nb2-11c cells differs from that of lactogen-independent Nb2-SP cells. Nb2-11c cells exhibit a greater incidence and volume density of nuclear pockets, whereas the incidence and volume density of lipid droplets is greater in the Nb2-SP cell line. Surface feature of Nb2-11c and Nb2-SP cells, as examined with scanning electron microscopy, and indistinguishable. Nb2-11c and Nb2-SP cells share a common mode of growth control in the form of constitutive secretion of an autocrine inhibitory factor. Medium conditioned by either Nb2-11c or Nb2-SP cells inhibits the growth of both cell lines. Nb2-CM-mediated growth inhibition is dose-dependent and reversible. Nb2-CM does not induce quiescence or cell death, but rather, causes a delay in the progression of cells through all phases of the cell cycle. Nb2 cell proliferation stimulated by a variety of mitogens is inhibited by Nb2-CM. Nb2-CM also has the ability to inhibit the growth of normal rat splenocytes as well as MCF-7 human breast cancer cells. Biochemical analysis of Nb2-CM was equivocal; however, indirect evidence suggests that the autocrine growth inhibitory factor produced by Nb2 cells may be a prostaglandin or another arachadonic acid metabolite since the growth inhibitory activity of Nb2-CM is reduced when CM is prepared in the presence of indomethacin. Interestingly, levels of prostaglandin F₁(α) are elevated in CM-treated culture supernatants. Examination of other signal transduction systems in Nb2 cells suggests that neither cAMP activation, polyamine biosynthesis, nor protein kinase C activation mediate or influence the inhibitory effect of Nb2-CM.

Receptor-activated phosphatidylinositol (PtdIns) hydrolysis was examined in AR42J rat pancreatic acini. Cholecystokinin-octapeptide (CCK₈) and bombesin induced a dose-dependent accumulation of [³H] inositol monophosphate ([³H]InsP₁). Manganese (Mn²⁺), a known calcium channel blocker, did not alter basal PtdIns hydrolysis. In contrast, when added 5 minutes prior to the addition of agonists for 60 minutes, Mn²⁺ markedly inhibited secretagogue-mediated [³H]InsP1 formation. Mn²⁺ also attenuated the CCK₈-mediated increase in biologically active inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. These inhibitory effects of Mn²⁺ were mimicked by lanthanum and by EGTA. Addition of calcium to EGTA-treated cells abolished the inhibitory effects of extracellular calcium depletion. Mn²⁺, La³⁺ and EGTA exerted similar inhibitory effects on PtdIns hydrolysis in pancreatic acini. These findings suggest that receptor-activated calcium influx is required for full activation of the CCK₈-mediated signal transduction pathway that is coupled to PtdIns hydrolysis.

Mammalian neurons exhibit extreme sensitivity to oxygen deprivation and undergo rapid and irreversible degeneration when oxygen supply is curtailed. Though several neuroprotective pathways are activated during oxygen deprivation, their analyses are masked by the complex series of pathological events which are triggered simultaneously. Such events can be analyzed in the anoxia tolerant fresh water turtle, which can inherently survive the conditions of oxygen deprivation and post-anoxic reoxygenation without brain damage. It is likely in such a model that modulation of a particular molecular pathway is adaptive rather than pathological. The major objective behind this study was to analyze the intracellular signaling pathways mediating the protective effects of adenosine, a potential neuromodulator, and its effect on cell survival by influencing the key prosurvival proteins that prevent apoptosis. In vivo and in vitro studies have shown that adenosine acts as a neuroprotective metabolite and its action can be duplicated or abrogated using specific agonist and antagonists. Stimulating the adenosine receptors using selective A1 receptor agonist N6-cyclopentyladenosine (CPA) activated the presumed prosurvival ERK and P13-K/AKT cascade promoting cell survival, and suppression of the receptor using the selective antagonist DPCPX (8- cyclopentyl-1,3-dipropylxanthine) activated the prodeath JNK and P38 pathways. The complex regulation of the MAPK's/AKT signaling cascades was also analyzed using their specific inhibitors. The inhibiton of the ERK and AKT pathway increased cell death, indicating a prosurvival role, whereas inhibiton of the JNK and p38 pathway increased cell survival in this model. In vitro studies have also shown a high Bcl-2/BAX ratio during anoxia and reoxygenation, indicating a strong resistance to cell death via apoptosis. / Silencing of the anti-apoptotic Bcl-2 gene using specific siRNA upregulated levels of prodeath BAX, thus altering the Bcl-2/BAX ratio and elevating cleaved Caspase-3 levels leading to increased cell death. Another promising neuroprotective target which we analyzed was Neuroglobin, which was induced during oxygen crisis and silencing this gene indicated that its plays a major role in modulation of ROS. This study strongly emphasizes the advantages of an alternate animal model in elucidating neuroprotective mechanisms and revealing novel therapeutic targets which could eventually help clinicians to design new stroke therapies based on naturally tolerant organisms. / by Gauri Nayak. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

The ubiquitin ligase Highwire is responsible for cell-autonomously promoting synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects in synaptic function and extra branching at the axon terminal, corresponding to transient branching that occur in the course of giant synapse formation during metamorphosis. The MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner, suggesting different molecular mechanisms downstream of the MAP kinase pathway govern function and pruning. A novel role for Highwire is revealed, regulating the giant fiber axon’s ability to respond to external cues regulated by Fos. When expression of the transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

New neurons are continuously generated in the olfactory system of adult mice, including olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) and interneurons, produced in the subventricular zone (SVZ) and migrated toward olfactory bulb (OB) along rostral migratory stream (RMS). The present study observed the effects of target neuron loss on the life-span and maturation of adult-born OSNs in the OE and on the proliferation, migration and differentiation of SVZ stem cells in the forebrain after eliminating bulb neurons. We found the life-span of newborn neurons in the absence of synaptic targets was shortened, but the timing of maturation was not delayed. In addition, SVZ cells continued to divide and migrate to the damaged bulb, and the migration of newborn cells in the RMS on the contralateral side was delayed at 2 weeks post-BrdU. Also, the proliferation of cells in dentate gyrus of the hippocampus was not affected by OB damage at 3 weeks post-lesion, though lesion affects occurred in the adult SVZ/RMS. / by Huan Liu. / Thesis (M.S.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

The primary purpose of this study was to examine the impact of acute highintensity interval Exercise (HIIE) on plasma cfDNA and IL-6 responses in obese and normal-weight subjects. Fifteen subjects (8 obese and 7 normal-weight) were recruited to participate in an acute HIIE protocol. Our results demonstrated a significant elevation across time in plasma cfDNA and IL-6 immediately following acute HIIE, with no difference between obese and normal-weight subjects. Furthermore, cfDNA was not correlated with IL-6 in response to acute HIIE in either group. These findings indicate that the obese state does not further exacerbate the release of acute HIIE-induced inflammatory mediators (cfDNA and IL-6), which suggests that HIIE training may serve as a time-effective exercise strategy to improve obesity-associated inflammation. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

The rodent hippocampus is critical for processing spatial memory but its contribution to non-spatial, specifically object memory is debated. The cognitive map theory of hippocampal function states that the hippocampus stores relationships of goal locations (places) to discrete items (objects) encountered within environments. Dorsal CA1 place cells were recorded in male C57BL/6J mice performing three variations of the novel object recognition paradigm to define "object-in-context" representation of hippocampal neuronal activity that may support object memory. Results indicate, (i) that place field stability is higher when polarizing environmental cues are provided during object recognition; (ii) hippocampal place fields remain stable throughout the novel object recognition testing without a polarizing cue; and (iii) time dependent effects on stability when objects were dissociated from the context. These data indirectly support that the rodent hippocampus processes object memory, and challenge the view that "object-in-context" representations are formed when mice perform novel object recognition task. / by Herborg Nanna âAsgeirsdâottir. / Thesis (M.A.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.