Characterizing gene functions with an overexpression ORF collection in Saccharomyces cerevisiae

My research project aims to discover new eukaryotic gene functions using yeast as a model organism. Yeast (Saccharomyces cerevisiae), are a prototypical eukaryotic model because of its high degree of genetic similarity to humans, fast generation time, and relatively low-cost maintenance. Identifying gene function in eukaryotes, such as humans, is an important, broad step in mapping gene and protein networks, predicting phenotypes, and understanding disease causation. This project uses a gain-of-function approach to characterizing new gene functions. We use a pool of transformed yeast with each yeast cell “over-expressing” a single gene carried by a plasmid, such that the quantity of the protein product encoded by the gene increases. The pool contains yeast transformed with plasmids representative of approximately 93% of all yeast genes. A plasmid is an exogenous piece of DNA that can be inserted into cells and engineered to carry specific genes of interest. Each plasmid in this pool has a galactose promoter that regulates the expression of a target gene, and only in the presence of galactose is that gene constitutively expressed. The yeast is treated with galactose to induce overexpression of a specific gene. This project aims to induce overexpression in a pool of yeast and monitor the change in abundance of each plasmid on a genome-wide scale using DNA microarrays. A DNA microarray measures the activity of thousands of yeast genes using the knowledge of complementary binding between nucleotides. By analyzing the patterns in which sets of plasmids are enriched and which drop out during overexpression, their functions may be inferred and characterized. Another aim of this project is to troubleshoot the 4 overexpression screen: from growing the yeast, to isolating the DNA from the yeast cells, and to amplifying the plasmid DNA. After several troubleshooting experiments, we found the PCR yield for yeast grown in galactose to be lower than that of yeast grown in glucose and yeast grown in raffinose. Additionally, PCR yield decreased the longer yeast grew in galactose, such that 12 hours of galactose induction yielded the lowest PCR. This could be due to the overexpression plasmids being lost from the yeast cells over time, the plasmids undergoing recombination with the genome, or plasmid DNA being lost or degraded during the DNA prep or PCR procedures. Our results also show changes in relative abundances of plasmid DNA as yeast change media from raffinose to galactose. Additionally, we found functional enrichment of distinct gene sets in yeast pools grown in raffinose and yeast pools grown in galactose. This illustrates the value this overexpression screen has in sorting genes into their functional networks, which in turn provides information in characterizing genes of unknown function.Biological Sciences, School o

Township-based bioenergy systems for distributed energy supply and efficient household waste re-utilisation: techno-economic and environmental feasibility

Sustainable waste management and climate change have been two of the major challenges worldwide. This study designed township-based bioenergy systems to treat solid waste in Glasgow based on anaerobic digestion and gasification technologies. The economic feasibility and environmental impacts (i.e. global warming potential, eutrophication potential, and acidification potential) were evaluated using Monte Carlo simulation-based cost-benefit analysis and life cycle assessment. It was found that township-based bioenergy systems could save over 300 kg of CO2 per tonne of municipal solid waste treated when biogenic carbon is excluded. It was shown that the proposed systems have profitability chances ranging from 68 to 98%, when the sale of by-products (digestate and biochar) is considered. This study also explored the effects of by-product selling and carbon tax on the economic feasibility of township-based bioenergy systems. The township-based bioenergy system can satisfy 20–23% of electricity demands and 4–5% of heat demands of each township served. The study can facilitate investors and policymakers to make informed decisions about planning distributed Waste-to-Energy (WtE) systems

Life cycle assessment and net present worth analysis of a community-based food waste treatment system

Food waste management has been a global challenge with significant economic and environmental impacts. A community-based food waste treatment scheme for Glasgow, UK is proposed. The food waste was treated by small-scale wet, mesophilic anaerobic digestion. Biogas was combusted in a combined heat and power plant to generate heat and electricity for each community. 201.39 kWh of electricity and 246.09 kWh of thermal energy could be provided to local communities per tonne of food waste treated. A total of 52,762 tonnes of food waste were produced each year in the city. Net-present worth analysis was employed to evaluate the scheme's economic feasibility. The scheme's environmental impacts were evaluated using life cycle assessment. The entire system saved 92.27 kg CO2-eq. per tonne of food waste treated and had a net-present worth of £ 3.187 million with a carbon tax of 50 £ tonne−1 and a biogas yield of 190 m3 tonne−1

Interpretable machine learning to model biomass and waste gasification

Machine learning has been regarded as a promising method to better model thermochemical processes such as gasification. However, their black box nature can limit how much one can trust and learn from the developed models. Here seven different machine learning methods have been adopted to model the gasification of biomass and waste across a wide range of operating conditions. Gradient boosting regression has been found to outperform the other model types with a coefficient of determination (R2) of 0.90 when averaged across ten key gasification outputs. Global and local model interpretability methods have been used to illuminate the developed black box models. The studied models were most strongly influenced by the feedstock’s particle size and the type of gasifying agent employed. By combining global and local interpretability methods, the understanding of black box models has been improved. This allows policy makers and investors to make more educated decisions about gasification process design

Tumour risks and genotype-phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD.

BACKGROUND: Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype-phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. METHODS: A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. RESULTS: Tumour risks analysis provided novel penetrance estimates and genotype-phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). CONCLUSIONS: Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype-tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers

A family of dual-activity glycosyltransferasesphosphorylases mediates mannogen turnover and virulence in Leishmania parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches

Financial Characteristics of Companies Audited by Large Audit Firms

Purpose “ The purpose of this paper is to examine how financial characteristics associated with the choice of a big audit firm with further investigation on the agency costs of free cash flows.Design/methodology/approach “ The sample used for this work includes industrial listed companies from Germany and France. To test our hypothesis, we used a number of logit models, extending the standard model selection audit firm, to include the variables of interest. Following previous work, our dependent dummy variable is Big4 or non-Big4.Findings “ We observed that most independent variables in the German companies show similar results to previous work, but we did not have the same results for the French industry. Moreover, our findings suggest that the total debt and dividends can be an important reason for determining the choice of a large audit firm, reducing agency costs of free cash flows.Research limitations/implications “ This study has some limitations on the measurements of the cost of the audit fees and also generates opportunities for additional searching.Originality/value “ The paper provides only one aspect to explain the relationship between the problems of agency costs of free cash flow and influence in choosing a large auditing firm, which stems from investors\u27 demand for higher quality audits

Effectiveness of smoking cessation therapies: a systematic review and meta-analysis

BACKGROUND: Smoking remains the leading preventable cause of premature deaths. Several pharmacological interventions now exist to aid smokers in cessation. These include Nicotine Replacement Therapy [NRT], bupropion, and varenicline. We aimed to assess their relative efficacy in smoking cessation by conducting a systematic review and meta-analysis. METHODS: We searched 10 electronic medical databases (inception to Sept. 2006) and bibliographies of published reviews. We selected randomized controlled trials [RCTs] evaluating interventions for smoking cessation at 1 year, through chemical confirmation. Our primary endpoint was smoking cessation at 1 year. Secondary endpoints included short-term smoking cessation (~3 months) and adverse events. We conducted random-effects meta-analysis and meta-regression. We compared treatment effects across interventions using head-to-head trials and when these did not exist, we calculated indirect comparisons. RESULTS: We identified 70 trials of NRT versus control at 1 year, Odds Ratio [OR] 1.71, 95% Confidence Interval [CI], 1.55–1.88, P =< 0.0001). This was consistent when examining all placebo-controlled trials (49 RCTs, OR 1.78, 95% CI, 1.60–1.99), NRT gum (OR 1.60, 95% CI, 1.37–1.86) or patch (OR 1.63, 95% CI, 1.41–1.89). NRT also reduced smoking at 3 months (OR 1.98, 95% CI, 1.77–2.21). Bupropion trials were superior to controls at 1 year (12 RCTs, OR1.56, 95% CI, 1.10–2.21, P = 0.01) and at 3 months (OR 2.13, 95% CI, 1.72–2.64). Two RCTs evaluated the superiority of bupropion versus NRT at 1 year (OR 1.14, 95% CI, 0.20–6.42). Varenicline was superior to placebo at 1 year (4 RCTs, OR 2.96, 95% CI, 2.12–4.12, P =< 0.0001) and also at approximately 3 months (OR 3.75, 95% CI, 2.65–5.30). Three RCTs evaluated the effectiveness of varenicline versus bupropion at 1 year (OR 1.58, 95% CI, 1.22–2.05) and at approximately 3 months (OR 1.61, 95% CI, 1.16–2.21). Using indirect comparisons, varenicline was superior to NRT when compared to placebo controls (OR 1.66, 95% CI 1.17–2.36, P = 0.004) or to all controls at 1 year (OR 1.73, 95% CI 1.22–2.45, P = 0.001). This was also the case for 3-month data. Adverse events were not systematically different across studies. CONCLUSION: NRT, bupropion and varenicline all provide therapeutic effects in assisting with smoking cessation. Direct and indirect comparisons identify a hierarchy of effectiveness