Characterisation of self-assembled engineered proteins on gold nanoparticles and their application to biosensing

PhD ThesisThe use of gold nanoparticles (AuNP) has a long and varied history, thought to cover several thousand years. More recently the unique properties of nanoscale materials have stimulated extensive work on nanoparticles and other nanomaterials leading to their use in novel technologies. AuNPs have been of particular interest for bioscience applications due to their biocompatibility and the ease with which biological molecules can be conjugated to their surface. In this study the assembly of engineered proteins, specifically the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM), onto the surface of AuNPs was investigated both in solution and with the particles attached to a SiO2 substrate. AuNPs were adhered to SiO2 surfaces using a novel silane treatment developed by the industrial sponsor and were characterised using spectroscopy, electron and atomic force microscopy. The addition of a single cysteine residue to the OmpATM structure was shown, by UV-Vis and fluorescence spectroscopy, to increase protein binding at equilibrium and form higher stability protein-AuNP complexes in solution. Following this, engineered OmpATM proteins containing tandem antibody-binding domains from Streptococcal protein G were assembled on the AuNP surface and their structure interrogated using neutron and light scattering. This revealed an oriented protein layer where the functional domains extend away from the AuNP surface and are available to bind antibodies. OmpATM-AuNP conjugates were used to develop biosensing assays using both well-established methods, such as lateral flow assays, and novel spectroscopic methods, which use the unique optical properties of AuNPs. Detection of influenza A nucleoprotein, an antigen used to clinically diagnose influenza, was achieved using a bespoke anti-nucleoprotein single-chain antibody domain fused to OmpATM and assembled on 20 nm diameter AuNPs. The results demonstrate that engineered OmpATM proteins conjugated to AuNPs can be used to develop novel diagnostics using a range of read out technologies

National Policies Affect Poverty Rates in Rich Nations

The labour market alone cannot move struggling families out of poverty. The social welfare programs in a country have a great impact on the level of poverty among minority and native population.York's Knowledge Mobilization Unit provides services and funding for faculty, graduate students, and community organizations seeking to maximize the impact of academic research and expertise on public policy, social programming, and professional practice. It is supported by SSHRC and CIHR grants, and by the Office of the Vice-President Research & Innovation. [email protected] www.researchimpact.c

Generic high-capacity protein capture and release by pH control

Techniques for immobilization and release of proteins are of general interest but challenging to develop. Here we show a new method for high-capacity (several \ub5g cm-2) immobilization of proteins in polyelectrolyte brushes by multivalent hydrogen bonds. Upon increasing pH, the proteins are fully released with preserved structure and activity

Evaluation of ‘Eyelander’: a video game designed to engage children and young people with homonymous visual field loss in compensatory training

Introduction: Rehabilitation can improve visual outcomes for adults with acquired homonymous visual field loss. However, it is unclear whether (re)habilitation improves visual outcomes for children because previous training schedules have been tiresome, uninteresting, and failed to keep them engaged. In this study we assessed whether children and young people with homonymous visual field loss would adhere to six weeks of unsupervised compensatory training using a specialised video game. Methods: Participants aged between 7 and 25 with homonymous visual field loss completed table-top assessments of visual search across four site visits. Two baseline assessments separated by four weeks evaluated spontaneous improvements before training began. Participants were then given a copy of the video game to use unsupervised at home for six weeks. Two follow-up assessments separated by four weeks were then conducted to evaluate immediate and acutely maintained effects of training. Results. 15 candidates met the inclusion-exclusion criteria, 9 participated, and 8 completed the study. Participants completed an average of 5.6 hours training unsupervised over the six weeks. Improvements on in-game metrics plateaued during week 3 of training. The time taken to find objects during table-top activities improved by an average of 24% (95% CI [2%, 46%]) after training. Discussion: The findings demonstrate that children and young people with homonymous visual field loss will engage with gamified compensatory training, and can improve visual outcomes with less time commitment than adults have required with non-gamified training in previous studies. Appropriately powered, randomised controlled trials are required to evaluate the validity and generalisability of observed training effects. Implications for practitioners: We conclude that (re)habilitation specialists can use specialist video games and gamification to engage children and young people with homonymous visual field loss in long-term unsupervised training schedules

Auditory, phonological and semantic factors in the recovery from Wernicke’s aphasia post stroke: predictive value and implications for rehabilitation

Background: Understanding the factors that influence language recovery in aphasia is important for improving prognosis and treatment. Chronic comprehension impairments Wernicke’s-type aphasia (WA) are associated with impairments in auditory and phonological processing, compounded by semantic and executive difficulties. This study investigated whether the recovery of auditory, phonological, semantic or executive factors underpins the recovery from WA comprehension impairments by charting changes in the neuropsychological profiles from the sub-acute to the chronic phase. Method: This study used a prospective, longitudinal, observational design. Twelve WA participants with superior temporal lobe lesions were recruited before 2 months post stroke onset (MPO). Language comprehension was measured alongside a neuropsychological profile of auditory, phonological and semantic processing alongside phonological short-term memory and nonverbal reasoning at three post stroke time points: 2.5, 5 and 9MPO. Results: Language comprehension displayed a strong and consistent recovery between 2.5 and 9MPO. Improvements were also seen for slow auditory temporal processing, phonological short-term memory, and semantic processing, but not for rapid auditory temporal, spectrotemporal and phonological processing. Despite their lack of improvement, rapid auditory temporal processing at 2.5MPO and phonological processing at 5MPO predicated comprehension outcomes at 9MPO. Conclusions: These results indicate that recovery of language comprehension in WA can be predicted from fixed auditory processing in the subacute stage. This suggests that speech comprehension recovery in WA results from reorganisation of the remaining language comprehension network to enable the residual speech signal to be processed more efficiently, rather than partial recovery of underlying auditory, phonological or semantic processing abilities

Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR

Endocytosis, an important macromolecule uptake process in cells, is known to be dysregulated in cancer. Clathrin and caveolin-1 proteins play a major role in receptor-mediated endocytosis. We have used a quantitative, unbiased and semi-automated method to measure in situ protein expression of clathrin and caveolin-1 in cancerous and paired normal (cancer adjacent, non-cancerous) human prostate tissue. There was a significant (p < 0.0001) increase in the expression of clathrin in prostate cancer samples (N = 29, n = 91) compared to normal tissue (N = 29, n = 67) (N = number of patients, n = number of cores in tissue arrays). Conversely, there was a significant (p < 0.0001) decrease in expression of caveolin-1 in prostate cancer tissue compared to normal prostate tissue. The opposite change in expression of the two proteins was highly correlated to increasing cancer aggressiveness. There was also a concurrent increase in the expression of epidermal growth factor receptor (EGFR), a key receptor in carcinogenesis, with clathrin in prostate cancer tissue, indicating recycling of EGFR through clathrin-mediated endocytosis (CME). These results indicate that in prostate cancer, caveolin-1-mediated endocytosis (CavME) may be acting as a brake and increase in CME may facilitate tumorigenicity and aggressiveness of prostate cancer through recycling of EGFR. Changes in the expression of these proteins can also potentially be used as a biomarker for prostate cancer to aid in diagnosis and prognosis and clinical decision-making

Structural and Functional Analysis of Cell Wall-Anchored PolypeptideAdhesin BspA in <i>Streptococcus agalactiae</i>

Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans. Complementary crystallographic and biophysical characterization of BspA reveal a novel β-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections