Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

Background: The enhancement of quality of life (QOL) is a principal health care objective. Surprisingly, few studies have investigated this outcome in ANCA associated vasculitis (AAV), a complex chronic disease. Existing studies have, however, identified fatigue as a specific problem amongst this population. Although its aetiology is unknown, there is evidence, from other populations, to support a neural basis for this symptom. Aims: This study aimed to characterise QOL and its determinants amongst patients with AAV. A secondary study examined the association of AAV related fatigue with alterations of the brain. Methods: An AAV case-control study was conducted, incorporating a comparison and within-case analysis, using two groups of population and chronic disease controls. All participants completed a questionnaire comprising measures of QOL and putative determinants of QOL impairment. Concurrently, putative clinical determinants were collected from cases. The secondary study recruited AAV cases based on fatigue status. A further group with idiopathic fatigue was recruited from the general population. All subjects underwent magnetic resonance (MR) brain scanning incorporating structural and physiological imaging. Results: Compared to population controls, cases were substantially more likely to report low QOL and levels were equable to disease controls. Potentially modifiable biological and psycho-social factors were independently associated with poor QOL, of which fatigue was found to be of principal importance. In the secondary study, structural and physiological differences were observed between AAV patients with and without fatigue, as well as fatigued population subjects. Conclusions: AAV patients experienced significant QOL impairment. A bio-psychosocial approach to AAV health care is likely to improve QOL outcomes, although a better understanding of specific mechanisms is necessary to fully manage these problems. MR techniques have suggested a neural basis for AAV related fatigue. In the future they may help delineate the mechanisms of fatigue and consequently improve QOL in AAV.

Eukaryotic cells face a considerable challenge organizing a complicated interior with spatial and temporal precision. They do so, in part, through the deployment of the microtubule- based molecular motors kinesin and dynein, which translate chemo-mechanical force production into the movement of diverse cargo. Many aspects of kinesin’s motility mechanism are now known in detail, whereas fundamental aspects of dynein’s motility mechanism remain unclear. An important unresolved question is how dynein couples rounds of ATP binding and hydrolysis to changes in affinity for its track, a requisite for a protein that takes steps. Here we report a sub- nanometer cryo-EM reconstruction of the high affinity state of dynein’s microtubule binding domain in complex with the microtubule. Using molecular dynamics flexible fitting, we determined a pseudoatomic model of the high affinity state. When compared to previously reported crystal structure of the free microtubule binding domain, our model revealed the conformational changes underlying changes in affinity. Surprisingly, our simulations suggested that specific residues within the microtubule binding domain may tune dynein’s affinity for the microtubule. We confirmed this observation by directly measuring dynein’s motile properties using in vitro single molecule motility assays, which demonstrated that single point mutations of these residues dramatically enhance dynein’s processivity. We then sought to understand why dynein has been selected to be a restrained motor, and found that dynein-driven nuclear oscillations in budding yeast are defective in the context of highly processive mutants. Together, these results provide a mechanism for the coupling of ATPase activity to microtubule binding and release by dynein, and the degree to which evolution has fine-tuned this mechanism. I conclude with a roadmap of future approaches to gain further insight into dynein’s motility mechanism, and describe our work developing materials and methods towards this goal.

The ability of cells to respond to their microenvironment is controlled by a complex communication system. Cell signaling utilizes a series of post-translational events to regulate and coordinate cellular activities. Although phosphorylation is thought to be the key regulator of these events, recent findings implicate the O-GlcNAc modification as an additional control mechanism. Modulation of signal transduction requires compartmentalization of the kinases and phosphatases. Based on the evidence of subcellular localization of OGT isoforms, the diversity of O-GlcNAcylated proteins upon stimulation, and its role during insulin signaling, it can be hypothesized that O-GlcNAc is involved and regulates signal transduction in a compartmentalized manner. To investigate the spatio-temporal dynamics of O-GlcNAc in cell signaling, we have generated a series of genetically encoded O-GlcNAc reporters based on fluorescence resonance energy transfer (FRET). These reporters and localized variants have allowed compartment specific visualization of O-GlcNAc activity in the nucleus, cytoplasm and plasma membrane. Herein we describe these reporters and their use to examine O-GlcNAc dynamics in signaling using serum stimulation and environmentally relevant concentrations of arsenite. Acute exposure to arsenite through drinking water has become an environmental health concern worldwide. Our results imply a complex regulation of O-GlcNAc on a fast timescale that is tied to more canonical kinase pathways. / text

Previous studies have shown that the mitochondrial orf224/ atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus, and that the effects of nuclear fertility restoration on orf224/ atp6 transcripts co-segregate with the pol restorer gene Rfp in genetic crosses. Results presented in this thesis indicate that the pol CMS restorer gene Rfp acts in an organ-specific manner to promote the processing of primary, dicistronic orf224/atp6 transcripts into new restorer-specific, monocistronic transcripts. The single 1.1 kb atp6 transcript of nap cytoplasm and some orfB transcripts of nap and pol cytoplasm B. napus mitochondria are shown to arise by removal of sequences from the 5' end of a longer precursor. Specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may thus represent a common mechanism for tailoring transcripts in plant mitochondria. Northern analysis of a segregating F2 population indicates that the recessive rfp allele at the restorer gene locus, or a very tightly linked gene, acts as a dominant gene designated Mmt (modifier of mitochondrial transcripts). Mmt controls the presence of additional, smaller transcripts of the nad4 and ccl1-like (ccl1-l) genes. These results suggest that Rfp/Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes. One of these genes, ccl1-l, is split in the B. napus mitochondrial genome into two widely separated and independently transcribed parts, contrasting with the situation in other plants where this gene is present as an uninterrupted ORF. Although transcripts of both parts are edited, no "trans-spliced" transcripts spanning both parts of the split ORF, that could be translated into a full sized protein product, were detected. Moreover, an antiserum directed against the product of t

No description available.

Cytoplasmic male sterility (CMS) is a widespread, maternally inherited trait that results in an inability of plants to produce functional pollen. The Ogura CMS system originated in radish, but has since been transferred to, and confers male sterility on, plants in the related genus Brassica . A gene which restores male fertility is needed for the Ogura CMS system to be exploited commercially for hybrid seed production in oilseed species such as Brassica napus. The restorer gene Rfo is a dominant radish nuclear gene that restores the male fertility to plants with Ogura cytoplasm. This gene has been transferred into Brassica napus through intergeneric crosses; however the introgressed segment of radish DNA contains an unknown number of genes, some of which confer undesirable traits, such as an elevated content of seed glucosinolates, antinutritive compounds that render the seed meal unusable as animal feed. A fine scale linkage map of the region in radish containing Rfo was constructed, and a map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687 amino acid protein with a predicted mitochondrial targeting presequence was found to confer male fertility upon transformation into Ogura CMS B. napus . This gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. A transcript representing a non-functional allele (rfo) was detected in sterile radish plants. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis , although a smaller and related PPR gene is found about 40 kb from this site.

Spiders are host to a plethora of heritable endosymbiotic bacteria. Broad-taxa screening studies indicate that endosymbionts are particularly common among spiders, however, little is known about how these bacteria affect their spider hosts. In insects these bacteria ensure vertical transmission by either conveying a benefit to the host or manipulating host reproduction to eliminate males that serve as evolutionary dead-ends for maternally-inherited bacteria. Common modes of reproductive manipulation include parthenogenesis, male killing, feminization, and cytoplasmic incompatibility. Screening an assemblage of Mermessus genus spiders, I detected a high frequency and diversity of endosymbiont infection. Within a single species, M. fradeorum, I detected three endosymbionts in multiple combinations. Rearing two natural infection types of M. fradeorum demonstrated two distinct endosymbiotic reproductive manipulations. Mothers infected with Rickettsia and Wolbachia produced extremely female-biased offspring, and antibiotic elimination of the symbionts successfully restored the sex ratio to the expected 1:1 in subsequent generations. A two-way factorial mating assay detected strong cytoplasmic incompatibility induced by a different strain of Wolbachia: cured females mated with infected males produced 70% fewer offspring than all other pairings. These results show that M. fradeorum is subject to multiple layers of reproductive manipulation that likely drive host evolution and ecology.

Cytoplasrnic male sterility (CMS) is a maternally inherited defect in the production of pollen, the male gamete of the flower. This sterility can be suppressed by nuclear Restorer of Fertility (Rf) genes that normally downregulate the expression of the CMS-associated novel mitochondrial gene. In Brassica napus, nap CMS and pol CMS are associated with related chimeric mitochondrial genes orf222 and orf224, respectively. CMS in both nap and pol is associated with a polar loss of locule development, loss of synchronous locule development and clumping of sporogenous tissue away from the tapetal cell layer, as well as secondary effects on petal and bud formation. In nap CMS, early accumulation of orf222 transcripts in the locule regions of developing anthers is associated with sterility, while the absence of orf222 transcripts from the locules is associated with fertility restoration. Accumulation of novel antisense transcripts of atp6 in a cell specific manner which matches that of sense transcripts of orf222 and atp6 in nap CMS anthers may be indicative of a post-transcriptional regulatory mechanism associated with CMS in flower buds. / Restoration of fertility in Brassica napus nap and pol CMS is associated with nuclearly encoded genes Rfn and Rfp, respectively. These restorers are very closely linked to one another, and may be allelic. Further efforts to isolate Rfp have narrowed the genomic region to approximately 105 kb of a syntenic region in Arabidopsis thaliana. Cosmid clones isolated from a library of Brassica rapa genomic DNA introgressed with Rfp have been successfully sorted into contigs through the application of the amplified fragment length polymorphism technique. The region to which Rfp is mapped is syntenic to a region of Arabidopsis DNA that is a duplication of a second location at the 23 megabase region of chromosome 1 of that genome. This region contains pentatricopeptide (PPR) motif-encoding genes that are highly related to other restorers of fertility of other species. By inference, Rfp from Brassica napus may encode PPR motifs. The PPR genes related to these previously characterized restorers of fertility are often found alongside the restorer genes existing as mini-clusters of several PPR-encoding genes. This is likely caused by selective pressure acting on PPR-encoding genes that resulted in diversification and multiplication of these genes. In addition, the PPR genes of this duplicated region are not syntenically located, whereas the non-PPR-encoding genes maintain their syntenic locations. The same is true for orthologous comparisons between Arabidopsis and other plant species. PPR genes are therefore malleable and capable of alteration in response to changing environmental pressures, such as the evolution of sterility inducing genes.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.