Intersections of distinct biological pathways in cells allow for nodes of metabolic regulation. This work describes the discovery of the intersection of two pathways in yeast mitochondria: RNA processing and fatty acid synthesis and attachment. Analysis of the components of the pathways is presented here along with a model illustrating the connection as a potential mode of regulation of mitochondrial gene expression.A genome-wide screen of respiratory-deficient <italic>Saccharomyces cerevisiae</italic> deletion strains for defects in mitochondrial RNA processing revealed that two novel genes affect processing of mitochondrial tRNAs by RNase P. One gene encodes Htd2, an enzyme in the type II mitochondrial fatty acid synthesis pathway (FAS II). The other gene is described here as encoding Lip3, an enzyme involved in the synthesis and attachment of the co-factor lipoic acid, which is synthesized from a product of the FAS II pathway.RPM1 is the mitochondrial-encoded RNA subunit of mitochondrial RNase P. The multigenic transcription unit containing RPM1 also contains tRNA<super>pro</super>. Maturation of RPM1 necessitates processing of the tRNA by RNase P. Thus, RNase P is required for maturation of its own RNA component, constituting a positive feedback cycle. The present work demonstrates that a product of the FAS II pathway is necessary for the assembly or activity of RNase P, as deletion of any gene encoding an FAS II enzyme results in inefficient processing of tRNApro from the transcript.Analysis of the enzymes involved in the synthesis and attachment of lipoic acid to target proteins is also described here. Disruption of any of these enzymes affects protein lipoylation and tRNA processing. Gcv3, a target of lipoylation, was found to be required for lipoylation as well as for efficient tRNA processing.A second feedback cycle controlling pyruvate dehydrogenase activity and fatty acid synthesis may be functional under certain conditions. Pyruvate dehydrogenase, which provides acetyl-CoA for the FAS II pathway, requires lipoic acid for its activity. It is hypothesized that the two feedback cycles and the role of Gcv3 may provide switch-like regulation of mitochondrial gene expression in response to the nutritional state of the cell.

The experiments reported here describe mechanisms involved in the establishment of the dorsal-ventral axis in the mud snail, Ilyanassa obsoleta. Ilyanassa and other spiralians utilize an embryonic organizer to induce dorsal identity, and thus establish the bilateral axis. The D macromere embryonic organizer in Ilyanassa is specified at the four-cell stage by the inheritance of the polar lobe, but does not function as an inductive center until the 24-cell stage. Previously it was assumed that the D macromere of Ilyanassa functioned autonomously through its inheritance of the polar lobe. I have found this is not the case. Rather, I describe the role that the micromeres play in the activation of the D macromere organizer. Specifically, I have found that micromeres of the first and second quartet are necessary for at least three known characteristics of the D macromere: the activation of MAPK in the D macromere, the division of the D macromere, and the inductive capacity of the D macromere. Thus, while the polar lobe is necessary for D macromere function, its inheritance does not provide the D macromere with functional autonomy.The localized activation of MAPK was the first molecular component of dorsal-ventral patterning to be identified in Ilyanassa and other spiralians. In addition to being activated in the D macromere organizer, MAPK is also activated in the micromeres that are induced by the D macromere. I undertook a pharmacological screen to identify other components involved in dorsal-ventral patterning. I have found that a member of the Protein Kinase C (PKC) family is also involved in the establishment of the dorsal-ventral axis in Ilyanassa. Inhibition of PKC disrupts patterning, resulting in a radialized animal. In addition, I have found that PKC functions in the same path as MAPK. PKC is necessary for the proper activation of MAPK in the D macromere organizer and the micromeres. These results suggest that either the same transduction pathway is used repeatedly in the establishment of the dorsal-ventral axis or that patterning is the result of one global signal. These results drastically change our view of dorsal-ventral patterning during spiralian development.

The polyamines positively charged molecules that are able to affect nucleic acid structure. Investigation of polyamine and RNA interaction shows that the two are able to form aggregates and these aggregates may be responsible for the inhibition of RNA decapping that is seen in the presence of polyamines. The polyamines are also responsible for a post-translational modification of a protein, eukaryotic initiation factor 5a (eIF5A). This protein is known to affect translation and RNA decay. This investigation shows that eIF5A is able to affect the formation of foci by Dhh1 but does not affect DCP1 or DCP2 foci formation. This investigation also shows that eIF5A mutation leads to a depression of polysome profiles and 35S-met incorporation and therefore eIF5A may truly be a translation initiation factor. This study also describes unique interactions of eIF5A with Dhh1p and eIF5A-independent effects of the polyamines on gene expression. The inhibition of eIF5A's hypusine modification leads to an increase in phoshorylation of eIF2&amp;#945; and this may contribute the induction of apoptosis and inhibition of protein synthesis associated with inhibition of eIF5A's hypusine modification.

There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function.

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The ability to obtain complex global behaviour from simple local rules makes cellular automata an interesting platform for massively parallel computation. However, manually designing a cellular automaton to perform a given computation can be extremely tedious, and automated design techniques such as genetic programming have their limitations because of the absence of human intuition. In this thesis, we propose elements of a framework whose goal is to make the manual synthesis of cellular automata rules exhibiting desired global characteristics more programmer-friendly, while maintaining the simplicity of local processing elements. We also demonstrate the power of that framework by using it to provide intuitive yet effective solutions to the two-dimensional majority classification problem, the convex hull of disconnected points problem, and various problems pertaining to node placement in wireless sensor networks. / Thesis (Master, Computing) -- Queen's University, 2007-12-05 10:26:09.591

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<p>Soft tissue sarcomas are rare malignancies that derive from connective tissue. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, while undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the cell(s) of origin of these sarcomas in the myogenic lineage, I used the tamoxifen-inducible CreERT2-loxP system in vitro and in vivo. Pax7-CreERT2 and MyoD-CreERT2 mice were utilized to transform Pax7+ and MyoD+ myogenic progenitors by expressing oncogenic K-rasG12D and deleting p53 in vivo. After injection of systemic tamoxifen into Pax7-CreERT2 and MyoD-CreERT2 mice, primary myogenic sarcomas including mouse rhabdomyosarcoma (mRMS) and mouse UPS (mUPS) developed within 2 to 6 months at various anatomical sites. Using unsupervised gene expression analysis, mRMS from Pax7+ myogenic progenitors clustered separately from the mUPS generated from the Pax7+ myogenic progenitors, as well as the mUPS generated by MyoD+ myogenic progenitors. These results suggest that Pax7+ and MyoD+ myogenic progenitor cells are tumor-initiating cells mUPS and that Pax7+MyoD- progenitors are tumor initiating cells for mRMS. These results demonstrate that mRMS and mUPS lie along a continuum. Furthermore, by comparing these tumors to their cell of origin, we find that Hedgehog signaling is dysregulated by increased expression of activated Gli3 in the sarcomas. Knockdown of Gli3 in cell lines derived from mouse and human sarcomas blocks tumor cell proliferation. I have established two novel mouse models of sarcoma with rapid onset and high penetrance, which may be useful for identifying novel therapies in sarcoma.</p> / Dissertation

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