Conceptual design and development of a research tool: - the Vagina-on-chip (VOC)

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonBacterial Vaginosis is one of the most common vaginal infection that affect 50% of women globally between the ages of 14-49, yet its aetiology remains unknown. Antibiotics or vaginal creams are usually prescribed to women to treat the infection however, reoccurrence is common after a year of treatment. Therefore, this condition desperately needs a new approach to developing an effective treatment. The aim of this thesis was to develop a microfluidic platform that can realistically mimic the in vivo vaginal epithelium tissue as an in vitro system which can then be used by clinicians and researchers to gain a better understanding of bacterial vaginosis (BV). The Vagina-on-chip (VOC) was developed using multiple techniques that combined micro-engineering, 3D printing, electrospinning, and cell culture to mimic the mechanical, biochemical and physical aspects of vaginal tissue. VOC platform comprised of three layers, top and bottom microfluidic channels to provide nutrients to the central layer, the membrane held in suspension to support the growth of vaginal tissue. The membrane was fabricated using natural and synthetic polymers via electrospinning technique. Composite membrane made with gelatine (GE) and polycaprolactone (PCL), a mixture of natural and synthetic polymers was found to be the optimal membrane for cell culture. This membrane was chosen due to its fibre size (257.25 ±72.92nm) and wettability CA (29.66 °) which provided a large surface area and hydrophilic exterior to support growth and cell adhesion of vaginal cells. Mechanical testing revealed that composite membrane exhibited similar mechanical properties to vaginal epithelium from non-prolapsed women. The composite membrane had a stress failure at 1.6MPa with strain failure at 12%. Cell viability assays were also conducted on the membranes to test for biocompatibility, which confirmed the composite membrane to be the most appropriate membrane for the VOC platform. Scanning electron microscopy was performed to visualise cell attachment on all membranes which showed the vaginal cells merging to the fibres of PCL/GE, PCL/COL and composite membrane indicating that these as-spun scaffolds promoted cellular attachments and spreading. Together with these results, the first VOC platform prototype was constructed. Due to its early stages in development, further experimentation and optimisation is required to evaluate its performance, to support the growth of vaginal tissue and potentially becoming a realistic research tool to study BV.EPSR

The zinc cluster protein Sut1 contributes to filamentation in Saccharomyces cerevisiae

Copyright © 2013, American Society for Microbiology. All Rights ReservedSut1 is a transcriptional regulator of the Zn(II)(2)Cys(6) family in the budding yeast Saccharomyces cerevisiae. The only function that has been attributed to Sut1 is sterol uptake under anaerobic conditions. Here, we show that Sut1 is also expressed in the presence of oxygen, and we identify a novel function for Sut1. SUT1 overexpression blocks filamentous growth, a response to nutrient limitation, in both haploid and diploid cells. This inhibition by Sut1 is independent of its function in sterol uptake. Sut1 downregulates the expression of GAT2, HAP4, MGA1, MSN4, NCE102, PRR2, RHO3, and RHO5. Several of these Sut1 targets (GAT2, HAP4, MGA1, RHO3, and RHO5) are essential for filamentation in haploids and/or diploids. Furthermore, the expression of the Sut1 target genes, with the exception of MGA1, is induced during filamentous growth. We also show that SUT1 expression is autoregulated and inhibited by Ste12, a key transcriptional regulator of filamentation. We propose that Sut1 partially represses the expression of GAT2, HAP4, MGA1, MSN4, NCE102, PRR2, RHO3, and RHO5 when nutrients are plentiful. Filamentation-inducing conditions relieve this repression by Sut1, and the increased expression of Sut1 targets triggers filamentous growth.The project was supported by Deutsche Forschungsgemeinschaft grant HO 2098/

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/μL and 100ng/μL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×106 of sample DNA through thermophillic helicase dependant amplification and 1×105 copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests.This work is funded under the UKCRC Translational Infection Research (TIR) Initiative supported by the Medical Research Council (Grant number G0901608) with contributions to the grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, the Chief Scientist Office of the Scottish Government Health Directorates and the Wellcome Trust. Axxin are thanked for sup- plying the T16-ISO platform