Master of Science

thesisProper assembly of Cytochrome c Oxidase (CcO) is vital to mitochondrial respiration. The metallation and incorporation of CcO subunit 2 (Cox2) are important assembly steps that remain poorly understood. The Cox2 assembly factor Cox20 may provide a unique window on CcO assembly. Cox20 is known to bind Cox2 before the latter is incorporated into the larger CcO assembly in S. cerevisiae. Conservation of Cox20 in higher organisms and the lethality of its deletion in Drosophila melanogaster may suggest Cox20 plays a conserved role in CcO assembly. In an attempt to understand the nature of the hypothesized conserved role of Cox20, conserved residues within Cox20 were mutated and the resulting mutants studied. Mutation of a conserved cysteine pair impacted mitochondrial respiration, but did not block CcO assembly. The mutant Cox20 C87A forms a mixed disulfide species. Identifying the partner molecule may shed light on the role of Cox20. The present study finds Cox20, a 23 kD protein, in high molecular weight complexes that are unlikely to be homo-oligomeric. Identifying any other proteins in these complexes may shed light on the role Cox20 plays in CcO assembly. To this end, Cox20 was purified and analyzed by mass spectrometry (MS) to identify any accompanying proteins. Small quantities of the known CcO assembly factors Mss2, Coa1, and Mss51 were identified

Impact of slurry application method on phosphorus loss in runoff from grassland soils during periods of high soil moisture content

peer-reviewedPrevious studies have reported that the trailing shoe application technique reduces phosphorus (P) in the runoff postslurry application when compared to the traditional splash-plate application technique. However, the effectiveness of the trailing-shoe technique as a means of reducing P losses has not been evaluated when slurry is applied during periods of high soil moisture levels and lower herbage covers. To address this issue, three treatments were examined in a 3 × 4 factorial design split-plot experiment, with treatments comprising three slurry treatments: control (no slurry), splashplate and trailing-shoe, and four slurry application dates: 7 December, 18 January, 1 March and 10 April. Dairy cow slurry was applied at a rate of 20 m3/ha, while simulated runoff was generated 2, 9 and 16 days later and analysed for a range of P fractions. Dissolved reactive P concentrations in runoff at day two was 41% lower when slurry was applied using the trailing-shoe technique, compared to the splash-plate technique (P < 0.05). In addition, P concentrations in runoff were higher (P < 0.05) from slurry applied in December and March compared to slurry applied in January or April, coinciding with periods of higher soil moisture contents. While the latter highlights that ‘calendar’-based non-spreading periods might not always achieve the desired consequences, the study demonstrated that further field-scale investigations into the trailing shoe as a mitigation measure to reduced P loss from agricultural soils is warranted

Impact of slurry application method on phosphorus loss in runoff from grassland soils during periods of high soil moisture content

Abstract Previous studies have reported that the trailing shoe application technique reduces phosphorus (P) in the runoff postslurry application when compared to the traditional splash-plate application technique. However, the effectiveness of the trailing-shoe technique as a means of reducing P losses has not been evaluated when slurry is applied during periods of high soil moisture levels and lower herbage covers. To address this issue, three treatments were examined in a 3 × 4 factorial design split-plot experiment, with treatments comprising three slurry treatments: control (no slurry), splashplate and trailing-shoe, and four slurry application dates: 7 December, 18 January, 1 March and 10 April. Dairy cow slurry was applied at a rate of 20 m3/ha, while simulated runoff was generated 2, 9 and 16 days later and analysed for a range of P fractions. Dissolved reactive P concentrations in runoff at day two was 41% lower when slurry was applied using the trailing-shoe technique, compared to the splash-plate technique (P &lt; 0.05). In addition, P concentrations in runoff were higher (P &lt; 0.05) from slurry applied in December and March compared to slurry applied in January or April, coinciding with periods of higher soil moisture contents. While the latter highlights that ‘calendar’-based non-spreading periods might not always achieve the desired consequences, the study demonstrated that further field-scale investigations into the trailing shoe as a mitigation measure to reduced P loss from agricultural soils is warranted.</jats:p

Impact of slurry application method on phosphorus loss in runoff from grassland soils during periods of high soil moisture content

Previous studies have reported that the trailing shoe application technique reduces phosphorus (P) in the runoff postslurry application when compared to the traditional splash-plate application technique. However, the effectiveness of the trailing-shoe technique as a means of reducing P losses has not been evaluated when slurry is applied during periods of high soil moisture levels and lower herbage covers. To address this issue, three treatments were examined in a 3 × 4 factorial design split-plot experiment, with treatments comprising three slurry treatments: control (no slurry), splashplate and trailing-shoe, and four slurry application dates: 7 December, 18 January, 1 March and 10 April. Dairy cow slurry was applied at a rate of 20 m3/ha, while simulated runoff was generated 2, 9 and 16 days later and analysed for a range of P fractions. Dissolved reactive P concentrations in runoff at day two was 41% lower when slurry was applied using the trailing-shoe technique, compared to the splash-plate technique (P &lt; 0.05). In addition, P concentrations in runoff were higher (P &lt; 0.05) from slurry applied in December and March compared to slurry applied in January or April, coinciding with periods of higher soil moisture contents. While the latter highlights that ‘calendar’-based non-spreading periods might not always achieve the desired consequences, the study demonstrated that further field-scale investigations into the trailing shoe as a mitigation measure to reduced P loss from agricultural soils is warranted.</p

Selective Oma1 Protease-mediated Proteolysis of Cox1 Subunit of Cytochrome Oxidase in Assembly Mutants

Background: Yeast lacking Coa2 are deficient in cytochrome c oxidase due to Cox1 degradation. Results: Oma1 mediates Cox1 degradation in coa2∆ cells but not other mutants stalled in oxidase biogenesis. Conclusion: Impaired hemylation of Cox1 in coa2∆ cells leads to misfolding and facile degradation by Oma1. Significance: Oma1 functions in quality control of cytochrome oxidase assembly

The efficacy of a web-based gambling intervention program for high school students: A preliminary randomized study

Early onset in adolescent gambling involvement can be a precipitator of later gambling problems. The aim of the present study was to test the preliminary efficacy of a web-based gambling intervention program for students within a high school-based setting. Students attending a high school in Italy (N=168) participated in the present study (58% male–age, M=15.01; SD=0.60). Twelve classes were randomly assigned to one of two conditions: intervention ( N=6; 95 students) and control group (N=6; 73 students). Both groups received personalized feedback and then the intervention group received online training (interactive activities) for three weeks. At a two-month follow-up, students in the intervention group reported a reduction in gambling problems relative to those in the control group. However, there were no differences in gambling frequency, gambling expenditure, and attitudes toward the profitability of gambling between the two groups. In addition, frequent gamblers (i.e., those that gambled at least once a week at baseline) showed reductions in gambling problems and gambling frequency post- intervention. Frequent gamblers that only received personalized feedback showed significantly less realistic attitudes toward the profitability of gambling post-intervention. The present study is the first controlled study to test the preliminary efficacy of a web-based gambling intervention program for students within a high school-based setting. The results indicate that a brief web-based intervention delivered in the school setting may be a potentially promising strategy for a low-threshold, low-cost, preventive tool for at-risk gambling high school students

Functional visual fields: relationship of visual field areas to self-reported function

Purpose: The aim of this study is to relate areas of the visual field to functional difficulties to inform the development of a binocular visual field assessment that can reflect the functional consequences of visual field loss. Methods: Fifty-two participants with peripheral visual field loss undertook binocular assessment of visual fields using the 30-2 and 60-4 SITA Fast programs on the Humphrey Field Analyser, and mean thresholds were derived. Binocular visual acuity, contrast sensitivity and near reading performance were also determined. Self-reported overall and mobility function were assessed using the Dutch ICF Activity Inventory. Results: Greater visual field loss (0–60°) was associated with worse self-reported function both overall (R2 = 0.50; p < 0.0001), and for mobility (R2 = 0.64; p < 0.0001). Central (0–30°) and peripheral (30–60°) visual field areas were similarly related to mobility function (R2 = 0.61, p < 0.0001 and R2 = 0.63, p < 0.0001 respectively), although the peripheral (30–60°) visual field was the best predictor of mobility self-reported function in multiple regression analyses. Superior and inferior visual field areas related similarly to mobility function (R2 = 0.56, p < 0.0001 and R2 = 0.67, p < 0.0001 respectively). The inferior field was found to be the best predictor of mobility function in multiple regression analysis. Conclusion: Mean threshold of the binocular visual field to 60° eccentricity is a good predictor of self-reported function overall, and particularly of mobility function. Both the central (0–30°) and peripheral (30–60°) mean threshold are good predictors of self-reported function, but the peripheral (30–0°) field is a slightly better predictor of mobility function, and should not be ignored when considering functional consequences of field loss. The inferior visual field is a slightly stronger predictor of perceived overall and mobility function than the superior field

Effects of adult aging on reading filtered text: evidence from eye movements

Objectives. Sensitivity to spatial frequencies changes with age and this may have profound effects on reading. But how the actual contributions to reading performance made by the spatial frequency content of text differs between young (18–30 years) and older (65+ years) adults remains to be fully determined. Accordingly, we manipulated the spatial frequency content of text and used eye movement measures to assess the effects on reading performance in both age groups. Method. Sentences were displayed as normal or filtered to contain only very low, low, medium, high, or very high spatial frequencies. Reading time and eye movements were recorded as participants read each sentence. Results. Both age groups showed good overall reading ability and high levels of comprehension. However, for young adults, normal performance was impaired only by low and very low spatial frequencies, whereas normal performance for older adults was impaired by all spatial frequencies but least of all by medium. Conclusion. While both young and older adults read and comprehended well, reading ability was supported by different spatial frequencies in each age group. Thus, although spatial frequency sensitivity can change with age, adaptive responses to this change can help maintain reading performance in later life

Processing of a Multiple Membrane Spanning Epstein-Barr Virus Protein for Cd8+T Cell Recognition Reveals a Proteasome-Dependent, Transporter Associated with Antigen Processing–Independent Pathway

Epstein-Barr virus (EBV) latent membrane protein (LMP)2 is a multiple membrane spanning molecule which lacks ectodomains projecting into the lumen of the endoplasmic reticulum (ER). Human CD8+ cytotoxic T lymphocytes (CTL)s recognize a number of epitopes within LMP2. Assays with epitope-specific CTLs in two different cell backgrounds lacking the transporter associated with antigen processing (TAP) consistently show that some, but not all, LMP2 epitopes are presented in a TAP-independent manner. However, unlike published examples of TAP-independent processing from endogenously expressed antigens, presentation of TAP-independent LMP2 epitopes was abrogated by inhibition of proteasomal activity. We found a clear correlation between hydrophobicity of the LMP2 epitope sequence and TAP independence, and experiments with vaccinia minigene constructs expressing cytosolic epitope peptides confirmed that these more hydrophobic peptides were selectively able to access the HLA class I pathway in TAP-negative cells. Furthermore, the TAP-independent phenotype of particular epitope sequences did not require membrane location of the source antigen since (i) TAP-independent LMP2 epitopes inserted into an EBV nuclear antigen and (ii) hydrophobic epitope sequences native to EBV nuclear antigens were both presented in TAP-negative cells. We infer that there is a proteasome-dependent, TAP-independent pathway of antigen presentation which hydrophobic epitopes can selectively access

Mucociliary and long-term particle clearance in airways of patients with immotile cilia

Spherical monodisperse ferromagnetic iron oxide particles of 1.9 μm geometric and 4.2 μm aerodynamic diameter were inhaled by seven patients with primary ciliary dyskinesia (PCD) using the shallow bolus technique, and compared to 13 healthy non-smokers (NS) from a previous study. The bolus penetration front depth was limiting to the phase1 dead space volume. In PCD patients deposition was 58+/-8 % after 8 s breath holding time. Particle retention was measured by the magnetopneumographic method over a period of nine months. Particle clearance from the airways showed a fast and a slow phase. In PCD patients airway clearance was retarded and prolonged, 42+/-12 % followed the fast phase with a mean half time of 16.8+/-8.6 hours. The remaining fraction was cleared slowly with a half time of 121+/-25 days. In healthy NS 49+/-9 % of particles were cleared in the fast phase with a mean half time of 3.0+/-1.6 hours, characteristic of an intact mucociliary clearance. There was no difference in the slow clearance phase between PCD patients and healthy NS. Despite non-functioning cilia the effectiveness of airway clearance in PCD patients is comparable to healthy NS, with a prolonged kinetics of one week, which may primarily reflect the effectiveness of cough clearance. This prolonged airway clearance allows longer residence times of bacteria and viruses in the airways and may be one reason for increased frequency of infections in PCD patients