Adult mammalian central nervous system (CNS) neurons are unable to extend axons after injury, partially owing to the inhibitory myelin and chondroitin sulfate proteoglycans (CSPGs) present in the environment. A neuron's intrinsic state is also important for determining its regenerative potential. Peripheral nervous system (PNS) neurons, unlike their CNS counterparts, have increased ability to regrow their axons after injury, even in the presence of inhibitory molecules. With the goal of discovering novel regeneration associated genes, we have isolated the genes differentially expressed by PNS neurons. We then developed a high throughput neuronal transfection method to test whether these genes were sufficient to modify neurite growth in vitro. Using high content screening, we measured the ability of cerebellar neurons to initiate neurite outgrowth on inhibitory and permissive substrates. This combination of technologies (subtractive hybridization, microarray, high throughput electroporation and high content screening) allowed phenotypic examination of neurons after the overexpression of over a thousand genes. Additionally, kinases and phosphatases were assayed for their ability to modify neurite outgrowth in hippocampal neurons. Results from both of these large unbiased screens confirmed many of the existing candidates for neurite growth during development and regeneration. We also discovered many novel genes which promoted neurite outgrowth such as GPX3, EIF2B5, RBMX, CHKA, IRF6, and PKN2. To accurately interpret the large volume of data, new methods of analysis were performed. Finally, we developed novel techniques that took advantage of public databases to cluster genes and determine whether those clusters produced robust changes in neurite growth. In summary, we have provided a vast repository of functional data to study axon development and regeneration after injury as well as developing the tools needed to interpret that data.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 188-225).

Pik1p, one of three phosphatidylinositol 4-kinases in the fission yeast, <i>Schizosaccharomyces pombe</i>, was found previously to interact with Cdc4p, a myosin essential light chain that is required for cytokinesis. The involvement of pik1 in cell cycle control was investigated. A fluorescently tagged Pik1p fusion protein was associated with Golgi throughout the cycle, and was found at the medial division plane of the cell during late cytokinesis. This latter distribution has not been reported previously. Gene deletion in diploid cells and tetrad analysis revealed that pik1 is essential for cell viability and is required for spore germination. The terminal phenotype of a temperature-sensitive, loss-of-function allele (pik1-td) indicated that pik1 is involved in cytokinesis: particularly for suppression of secondary septum material deposition, for suppression of initiation of supernumerary septa, and for cell separation. Contractile ring formation was normal in pik1-td cells at the restrictive temperature although the pattern of F-actin patches was disrupted. The F-actin patches were dispersed throughout the cytoplasm. Accumulation of extra inner membranous or vesicle-like structures was observed in these cells. The <i>S. pombe</i> nmt1 promoter and attenuated versions of it were found to be useful for complementation studies in <i>S. cerevisiae</i>. Heterologous expression of <i>S. pombe</i> pik1 complemented the essential functions of a temperature-sensitive allele (pik1﷓101) of its orthologue in <i>Saccharomyces cerevisiae</i> that were lost at the restrictive temperature. A residue required for <i>S. pombe</i> Pik1p lipid kinase activity, D709, was also required for this complementation. A residue, R838, which is required for interactions between Pik1p and Cdc4p was not required for this complementation. The timing and localization of Pik1p to the division plane of the cell late in cytokinesis combined with analysis of the terminal phenotype of a loss-of-function allele, indicate that Pik1p and/or its derived phosphoinositides are required for regulation of septation and cell separation. Pik1p may be involved in the transport, possibly via vesicular transport, of enzymes required for hydrolysis of the primary septum. It may be involved in signaling pathways that lead to the initiation of septation and to the cessation of the deposition of secondary septum material.

Pik1p, one of three phosphatidylinositol 4-kinases in the fission yeast, <i>Schizosaccharomyces pombe</i>, was found previously to interact with Cdc4p, a myosin essential light chain that is required for cytokinesis. The involvement of pik1 in cell cycle control was investigated. A fluorescently tagged Pik1p fusion protein was associated with Golgi throughout the cycle, and was found at the medial division plane of the cell during late cytokinesis. This latter distribution has not been reported previously. Gene deletion in diploid cells and tetrad analysis revealed that pik1 is essential for cell viability and is required for spore germination. The terminal phenotype of a temperature-sensitive, loss-of-function allele (pik1-td) indicated that pik1 is involved in cytokinesis: particularly for suppression of secondary septum material deposition, for suppression of initiation of supernumerary septa, and for cell separation. Contractile ring formation was normal in pik1-td cells at the restrictive temperature although the pattern of F-actin patches was disrupted. The F-actin patches were dispersed throughout the cytoplasm. Accumulation of extra inner membranous or vesicle-like structures was observed in these cells. The <i>S. pombe</i> nmt1 promoter and attenuated versions of it were found to be useful for complementation studies in <i>S. cerevisiae</i>. Heterologous expression of <i>S. pombe</i> pik1 complemented the essential functions of a temperature-sensitive allele (pik1﷓101) of its orthologue in <i>Saccharomyces cerevisiae</i> that were lost at the restrictive temperature. A residue required for <i>S. pombe</i> Pik1p lipid kinase activity, D709, was also required for this complementation. A residue, R838, which is required for interactions between Pik1p and Cdc4p was not required for this complementation. The timing and localization of Pik1p to the division plane of the cell late in cytokinesis combined with analysis of the terminal phenotype of a loss-of-function allele, indicate that Pik1p and/or its derived phosphoinositides are required for regulation of septation and cell separation. Pik1p may be involved in the transport, possibly via vesicular transport, of enzymes required for hydrolysis of the primary septum. It may be involved in signaling pathways that lead to the initiation of septation and to the cessation of the deposition of secondary septum material.

Diatoms are unicellular photosynthetic algae that display intricately patterned cell walls made of amorphous silicon dioxide (silica). Long-chain polyamines and highly phosphorylated proteins, silaffins and silacidins, are believed to play an important role in biosilica formation. The phosphate moieties on silaffins and silacidins play a significant role in biomineral formation, yet no kinase has been identified that phosphorylates these biomineral forming proteins. This dissertation describes the characterization of a novel kinase from the diatom Thalassiosira pseudonana, tpSTK1, which is upregulated during silica formation. A recombinantly expressed histidine-tagged version of tpSTK1 was capable of phosphorylating recombinant silaffins but not recombinant silacidin in vitro. Through establishing methods for subcellular fraction of T. pseudonana membranes in combination with antibody inhibition assay, it was discovered that native tpSTK1 phosphorylates silaffins but not silacidins in vitro (i.e. it exhibits the same substrate specificity as recombinant tpSTK1). As tpSTK1 is an abundant protein in the ER lumen (~ 0.5 % of total ER protein) it seems highly likely to function as a silaffin kinase in vivo. TpSTK1 lacks clear sequence homologs in non-diatom organisms and is the first molecularly characterized kinase that appears to be involved in biomineralization. The predicted kinase domain (KD) of tpSTK2, the only T. pseudonana homolog of tpSTK1, was recombinantly expressed and tested for phosphorylation activity. Recombinant tpSTK2-KD and native tpSTK2 exhibited detectable activity with myelin basic protein, but did not phosphorylate silaffins or silacidins in vitro. Western blot analysis demonstrated that native tpSTK2 was not present in the ER, but associated with the cytosol and Golgi membrane containing subcellular fractions.

Thèse doctorat : Sciences et Techniques du Déchet : Villeurbanne, INSA : 2004. / Titre provenant de l'écran-titre. Bibliogr. p. 161-171.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 143-156). Also available on the Internet.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 82-99). Also available in electronic version. Access restricted to campus users.

Thesis (Ph.D)--Earth and Atmospheric Sciences, Georgia Institute of Technology, 2010. / Committee Chair: Taillefert, Martial; Committee Member: DiChristina, Thomas; Committee Member: Sobecky, Patricia; Committee Member: Van Cappellen, Philippe; Committee Member: Webb, Samuel. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The reversible phosphorylation of proteins plays a key role in nearly every aspect of cell life. This essential post-translational modification controls a myriad of cellular events from cell survival, differentiation, and migration to apoptosis. Two classes of enzymes, kinases and phosphatases, tightly control all phosphorylation events. Perturbation in the activity of any member of these classes of enzymes has been linked to numerous diseases including cancer, metabolic disorders, immune disorders and neurological disorders. Therefore, there is a great interest among the scientific community to develop methods to selectively modulate the activity of kinases and phosphatases not only for therapeutic purposes but also to understand the fundamental role of these enzymes in signaling events. The more than 500 kinases encoded in the human genome share a common catalytic fold and most inhibitors target the ATP binding site. Therefore selective targeting of a single kinase by an inhibitor at the highly conserved ATP binding site is one of the main concerns for designing probes or drugs. Our group has taken advantage of the potency and possible selectivity imparted by bivalent inhibitors and developed an in vitro selection approach to discover bivalent ligands. The strategy involves the use of an ATP-competitive small molecule warhead and a library of cyclic peptides displayed on phage that interact with the kinase of interest in a dynamic selection. The selection for a kinase binding peptide is carried out until consensus peptides are found and bivalent ligands are constructed by linking the selected cyclic peptide with the small molecule warhead through a synthetic linker. Using this approach a potent and selective bivalent inhibitor was found for PKA, a serine/threonine kinase. To interrogate the generality of this approach, a kinase closely related to PKA (PRKX) was used for which a very potent and selective bivalent ligand was found. The same selection strategy was further extended to the two kinases Lyn and Brk, which belong to the tyrosine kinase family. Though peptides were isolated that bound the desired kinase, potent bivalent inhibitors were not discovered. More generally, these experiments in sum are building a library of information regarding how to best design selections of potent and selective bivalent inhibitors. We further explored modulation of the activity of kinases and phosphatases by employing a ligand-gated split-protein approach. The small molecule gated spatial and temporal control of these enzymes should allow the study of signaling events in a controlled manner. The strategy employed consists in the identification of possible fragmentation sites within the catalytic domain of kinases and phosphatases by a sequence dissimilarity approach. Loop insertion mutants at the selected sites were tested for catalytic activity. Successful insertion mutants were further split into two catalytically inactive fragments, which were appended to two conditionally interacting protein domains. Upon addition of a small molecule, the two conditionally interacting domains reassemble the catalytic domain of the enzyme and restore catalytic activity. Using this approach we were able to modulate the activity of the tyrosine kinases Lyn, Fak and Src and the AGC kinase PKA. We also extended the approach to gate the activity of tyrosine phosphatases PTP1B, SHP1 and PTPH1. Finally, these ligand-gated split-kinases and phosphatases were validated in-cellulo. Thus, this work resulted in a new method for designing split-proteins and provided a palette of kinases and phosphatases that can be turned-on by small molecules. In total, these efforts describe two alternative routes that can be used to modulate phosphorylation events in a selective and controlled manner.