Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 123-159).

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and ubiquitous post-translational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC translocation observed by others is related to the O-GlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agents followed by analysis of cytosol and membrane concentrations of PKC-epsilon, -alpha, -betaII, and -iota. Of the four PKC isoforms analyzed, PKC-epsilon showed a significant reduction in its membrane associated levels in response to all agents tested whereas PKC-alpha showed reductions in response to only two agents. Investigation of the mechanism for the reductions in membrane associated PKC-epsilon and -alpha indicate that the increased O-GlcNAc levels did not disrupt the activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKC-epsilon suggesting that the decreases in membrane associated PKC-epsilon may be a result of increased phosphatase or protease activity. Additional studies revealed that decreases in membrane bound PKC-epsilon and PKC-alpha, both of which act as anti-apoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage -- a well characterized hallmark of apoptosis. In addition to PKC, the effects of increased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investigation of the effects of increased O-GlcNAc modification of Akt activation using glucosamine or streptozotocin revealed a relatively large, short-term increase in Akt phosphorylation in response to these treatments. However, further analysis with other O-GlcNAc modulators indicated that this activation was not related to O-GlcNAc protein modification. Furthermore, this activation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize the novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.

Human cytomegalovirus (HCMV), a beta herpesvirus, persists indefinitely in the human host through a life-long, latent infection. HCMV is associated with life threatening pathologies in the immune naïve or compromised and, therefore, understanding of the mechanisms of viral persistence is imperative to human health. The ULb' region of the HCMV genome is selectively lost in high-passage strains of the virus, yet retained in low-passage strains. As such, the ULb' is hypothesized to play a role in immune evasion, pathogenesis, latency, and dissemination. ULb' encoded viral products are poorly characterized, hindering a mechanistic understanding of HCMV persistence. We previously defined a 3.6-kb locus spanning UL133-UL138 within the ULb' region important to viral latency. UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and translation mechanisms. We mapped the origins of each isoform through advanced bacterial artificial chromosome recombineering, where each ATG was disrupted and the resulting UL136 recombinant virus was screened for altered expression of the pUL136 isoforms. Remarkably, 8 of the 11 potential translation initiation sites encoded within the ORF are utilized to create the pUL136 isoforms. The pUL136 isoforms have distinct localization and trafficking patterns within the cell, including varying degrees of Golgi association, suggesting each isoform may interface with different cellular components and pathways. Further characterization of UL136 recombinant viruses revealed a complex, antagonistic relationship between the pUL136 isoforms. In endothelial cells, which are important to viral persistence and dissemination due to their ability to maintain a slow, "smoldering" infection, the 33- and 26-kDa isoforms promote replication, while the 25-kDa isoform enhances their combined activity, and the 23-/19-kDa isoforms repress the activity of the 25-kDa isoform. The pUL136 isoforms are also required for virus maturation in endothelial cells, where the 33-kDa is required both for virion envelopment and efficient formation of the perinuclear viral assembly compartment. In both an in vitro CD34⁺ cell culture model of latency and an in vivo NOD-scid IL2Rɣc^(null) humanized mouse model, a virus lacking the 23-/19-kDa isoforms fails to establish latency, instead replicating and disseminating with increased efficiency while viruses lacking the 33- and 26-kDa isoforms fail to efficiently reactivate or disseminate. Our data suggest that the interplay between the pUL136 isoforms maintains an intricate balance of infection that governs replication, latency, and virus dissemination, which ultimately contributes to the role of the UL133/8 locus in mediating outcomes of HCMV infection.

Peripherin is a type III intermediate filament protein that is predominately expressed in the peripheral nervous system and in subsets of efferent projections in the central nervous systems. While the exact role of peripherin remains unclear, it is found upregulated after traumatic neuronal injury and in the devastating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Interestingly, peripherin overexpressing transgenic mice succumb to motor neuron disease with pathological hallmarks reminiscent of those found in ALS. Pathological peripherin abnormalities occur with high frequency in both familial and sporadic forms of ALS, with peripherin found associated with the majority intracellular inclusions present within degenerating motor neuron populations. The findings of peripherin mutations in sporadic ALS have reinforced the importance of peripherin as a prospective etiological or propagative factor of disease pathogenesis. Surprisingly, inherited peripherin gene mutations have not been identified; as such, understanding the post-transcriptional mechanism at which peripherin imparts its effect(s) is considered a key goal and represents a pathological point-of-convergence for an otherwise complex, multifaceted disease. Prior to the commencement of this work, our group identified the presence of an abnormal peripherin alternative splice variant upregulated in ALS. In doing so, we consistently observed the presence of a second peripherin species of ~45 kDa on immunoblots of cell lysates derived from full-length peripherin transfections. Here, we identified this protein as a constitutively expressed isoform, termed Per-45, that arises from alternative translation and that is required for normal filament assembly: changes to the normal isoform expression pattern are associated with malformed filaments and intracellular inclusions. In lieu of the possibility of distinct peripherin intra-isoform associations, we identified isoform-specific expression and ratio changes in traumatic neuronal injury, in mouse models of motor neuron disease, and in ALS. Finally, we explored the interrelationships between peripherin isoform expression, protein aggregation, and neuritic outgrowth by linking these phenotypes with major pathogenic features associated with ALS, including in vitro models of oxidation, glutamate excitotoxicity, and neuroinflammation. Overall, this thesis provides exciting new insight into our knowledge of basic IF biology and the role of peripherin isoforms in injury and in motor neuron disease.

The class I myosins are an ubiquitous family of non-filamentous, single-headed actin-binding motor proteins. The objective of this study was to identify the light chain composition of the short-tailed Dictyostelium class I myosins, MyoIA and MyoIE. Flag-tagged MyoIA head-neck and MyoIE head-neck constructs were generated and expressed in Dicyostelium discoidem. The MyoIA and MyoIE head-neck constructs both co-purified with a 17-kDa protein that reacted with an anti-calmodulin antibody and exhibited a mobility shift on SDS gels in the presence of calcium. Mass spectrometry analysis confirmed that the light chain bound to MyoIA and MyoIE was calmodulin. The finding that the short-tailed class I Dictyostelium myosins use the generic calcium-binding protein calmodulin as a light chain contrasts with previous work showing that the long-tailed Dictyostelium class I myosins MyoIB, MyoIC, and MyoID each bind a unique, specialized light chain called MlcB, MlcC, and MlcD, respectively. Despite having a calmodulin light chain, calcium did not affect the actin-activated Mg-ATPase activities of MyoIA or MyoIE. The p21-activated kinases (PAKs) are serine-threonine protein kinases that are activated by the small GTPases Cdc42 and Rac. PAKs phosphorylate a site in the motor domain of Dictyostelium class I myosins that is required for myosin activity. Studies were carried out to determine whether Dictyostelium RacB, which is known to bind to and activate Dictyostelium PAKs, promotes the phosphorylation of MyoID in vivo. A vector that expresses a constitutively active RacB under the control of a doxycycline-inducible promoter was created and transformed into Dictyostelium cells. Immunostaining demonstrated that the constitutively active RacB increased actin filament formation in AX3 cells by ~3-fold but by only ~1.5-fold in PakB-null cells. A rabbit polyclonal antibody against the MyoID tail was made. An anti-phospho antibody raised against a phosphorylated peptide corresponding to the MyoID TEDS site was tested and found to specifically recognize purified phosphorylation MyoIA and MyoID. The anti-phospho antibody did not detect phosphorylated MyoIA or MyoID in crude Dictyostelium cell extracts or in immunoprecipitates prepared using the anti-MyoID antibody. Further work is needed to improve the specificity of the anti-phospho MyoID antibody. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-24 19:51:55.032

My study investigated the evolution of oxidative metabolism in fishes. While intense selection for, or against, non-synonymous point mutations in coding sequence drives the evolution of mitochondrial OXPHOS genes, genome-specific mechanisms such as gene duplication events can play major roles in the evolution of nuclear OXPHOS genes. My thesis focused on the mitochondrial enzyme cytochrome c oxidase (COX), principally in fish because of their evolutionary origins and functional diversity in terms of energy metabolism. In the first part of my thesis, I examined a highly aerobic group of fishes (billfishes and tunas) to study the evolution of mitochondrial COX genes. Though the study began as a structure-function analysis of COX, my approach changed when my preliminary results called into question the accepted phylogenetic relationships of my species of interest. We generated a robust multigene phylogeny of this group to interpret data in a phylogenetically informative context. Phylogenetic analyses in this group provided us with a framework to study the evolution of mitochondrial OXPHOS genes, but unexpectedly revealed that: 1) billfishes are only distantly related to tunas, and share greater evolutionary affinities with flatfishes (Pleuronectiformes) and jacks (Carangidae), and 2) regional endothermy has evolved in a non-scombroid suborder in teleosts. These results collectively imply that regional endothermy has evolved independently at least twice within teleost fish. The second part of my thesis explored the evolution of the nuclear COX subunits, focusing on their origins in fish. Isoform transcription profiles coupled with phylogenetic analyses for each subunit show that vertebrate isoforms arose from a combination of early whole-genome duplications in basal vertebrates or specific lineages (e.g. teleosts), and more recent single gene duplication events. While there is evidence for retained function of some COX orthologues across fishes and mammals, others appear to have diverged in function since their earlier radiation, possibly contributing novel evolutionary functions. Together these two studies provide insight into the evolutionary forces facilitating adaptive change in mitochondrial and nuclear OXPHOS genes. / Thesis (Master, Biology) -- Queen's University, 2009-09-11 11:00:12.562

Peripherin is a type III intermediate filament protein that is predominately expressed in the peripheral nervous system and in subsets of efferent projections in the central nervous systems. While the exact role of peripherin remains unclear, it is found upregulated after traumatic neuronal injury and in the devastating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Interestingly, peripherin overexpressing transgenic mice succumb to motor neuron disease with pathological hallmarks reminiscent of those found in ALS. Pathological peripherin abnormalities occur with high frequency in both familial and sporadic forms of ALS, with peripherin found associated with the majority intracellular inclusions present within degenerating motor neuron populations. The findings of peripherin mutations in sporadic ALS have reinforced the importance of peripherin as a prospective etiological or propagative factor of disease pathogenesis. Surprisingly, inherited peripherin gene mutations have not been identified; as such, understanding the post-transcriptional mechanism at which peripherin imparts its effect(s) is considered a key goal and represents a pathological point-of-convergence for an otherwise complex, multifaceted disease. Prior to the commencement of this work, our group identified the presence of an abnormal peripherin alternative splice variant upregulated in ALS. In doing so, we consistently observed the presence of a second peripherin species of ~45 kDa on immunoblots of cell lysates derived from full-length peripherin transfections. Here, we identified this protein as a constitutively expressed isoform, termed Per-45, that arises from alternative translation and that is required for normal filament assembly: changes to the normal isoform expression pattern are associated with malformed filaments and intracellular inclusions. In lieu of the possibility of distinct peripherin intra-isoform associations, we identified isoform-specific expression and ratio changes in traumatic neuronal injury, in mouse models of motor neuron disease, and in ALS. Finally, we explored the interrelationships between peripherin isoform expression, protein aggregation, and neuritic outgrowth by linking these phenotypes with major pathogenic features associated with ALS, including in vitro models of oxidation, glutamate excitotoxicity, and neuroinflammation. Overall, this thesis provides exciting new insight into our knowledge of basic IF biology and the role of peripherin isoforms in injury and in motor neuron disease.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references

Determination of quantitative expression levels of alternatively spliced isoforms provides an important approach to the understanding of the functional significance of each isoform. Real-time PCR using exon junction overlapping primers has been shown to allow specific detection of each isoform. However, this design often suffers from severe cross amplification of sequences with high homology at the exon junctions. We used human GFRα2b as a model to evaluate the specificity of primers substituted with locked nucleic acids (LNAs). We demonstrate here that single LNA substitutions at different positions of 3’ terminus could improve the discrimination of the primers against GFRα2a template, a highly homologous isoform. While LNA substitutions of GFRα2b primer at the residues possessing different sequences as GFRα2a has limited improvement in specificity, two consecutive LNA substitutions preceding the different sequences has dramatically improved the discrimination by greater than 100,000-fold compared to the non-substituted primer. Thus, LNA when substituted at certain residues can allow the discrimination of highly homologous sequences. / Singapore-MIT Alliance (SMA)