General health screening is needed to decrease the risk of pandemic in high volume areas. Thermal characterization, via infrared imaging, is an effective technique for fever detection, however, strict use requirements in combination with highly controlled environmental conditions compromise the practicality of such a system. Combining advanced processing techniques to thermograms of individuals can remove some of these requirements allowing for more flexible classification algorithms. The purpose of this research was to identify individuals who had febrile status utilizing modern thermal imaging and machine learning techniques in a minimally controlled setting. Two methods were evaluated with data that contained environmental, and acclimation noise due to data gathering technique. The first was a pretrained VGG16 Convolutional Neural Network found to have F1 score of 0.77 (accuracy of 76%) on a balanced dataset. The second was a VGG16 Feature Extractor that gives inputs to a principle components analysis and utilizes a support vector machine for classification. This technique obtained a F1 score of 0.84 (accuracy of 85%) on balanced data sets. These results demonstrate that machine learning is an extremely viable technique to classify febrile status independent of noise affiliated.

Staphyloccocus aureus is an opportunisitic pathogen that causes soft skin and tissue infections (SSTI) such as endocarditis, osteomyelitis and meningitis. In recent years, the re-emergence of antibiotic-resistant S. aureus such as MRSA presents a formidable challenge for infection management worldwide. Amidst this global epidemic of antimicrobial resistance, several research efforts have turned their focus towards exploiting the cell-wall biosynthesis pathway for novel anti-bacterial targets. Recently, the lipoteichoic acid (LTA) biosynthesis pathway has emerged as a potential anti-bacterial target. LTA is an anionic polymer found on the cell envelope of Gram-positive bacteria. It comprises of repeating units of glycerol-phosphate (GroP) and is important for bacterial cell physiology and virulence. For example, it is critically involved in regulating ion homeostasis, cell division, host colonization and immune system invasion. Several reports showed that bacteria lacking LTA are unable to grow. At the same time, they suffer from severe cell division defects and also exhibit aberrant cell morphologies. The key protein involved in the LTA biosynthesis pathway is the Lipoteichoic acid synthase (LtaS). LtaS is located on the cell membrane of Gram-positive bacteria and can be divided into two parts: a transmembrane domain and an extra-cellular domain responsible for its enzymatic activity (annotated eLtaS). Given that LtaS is important for bacterial survival and there are no known eLtaS homologues in eukaryotic cells, this protein is an attractive antibacterial target. In 2013, a small molecule eLtaS inhibitor (termed 1771) was discovered. Although 1771 was able to deplete LTA production, the binding mechanism of 1771 to eLtaS remains unknown. Additionally, 1771 could only prolong the survival of infected mice temporarily because of its in vivo instability. Therefore, the need for finding more potent and metabolically stable inhibitors of eLtaS still remains. Computational-aided drug design (CADD) is a cost-effective and useful approach that has been widely integrated into the drug discovery process. The protein eLtaS lends itself to be a good target for CADD since its crystal structure and a known inhibitor (with limited structure-activity data) is available. In this work, I have targeted eLtaS using CADD methodology followed by prospective validation using various biophysical, biochemical and microbiological assays. My project can broadly be sub-divided into three phases: (a) identification of small molecule binding “hot spots”, (b) optimization of existing inhibitor and (c) discovery of new hits. Through a systematic use of different computational approaches, I modelled a plausible 1771-bound eLtaS complex and used the structural insights to generate new inhibitors against eLtaS. To this end, I discovered EN-19, which is a more potent inhibitor of eLtaS. Additionally, by targeting transient cryptic pockets predicted by Molecular Dynamic simulations, I have discovered a new inhibitor chemotype that seems to exhibit a different mode of action against eLtaS. Taken together, my work presents a computational platform for future drug design against eLtaS and reinforces the notion that targeting eLtaS is a viable strategy to combat Gram-positive infections.

Graviditetsdiabetes innebär diabetes som nyupptäcks under graviditeten. Graviditetsdiabetes innebär en förändring av det dagliga livet. Forskning har visat att förändringen medför både positiva och negativa upplevelser, dels ökad medicinsk uppmärksamhet beroende på sjukdomen, dels ett mer inrutat liv än tidigare eftersom kvinnan måste kontrollera sitt blodsocker upprepade gånger varje dag.. Därmed blir hon också hela tiden påmind om sin graviditetsdiabetes. Syftet med studien var att undersöka hur kvinnor som får diabetes under sin graviditet upplever sin situation. Intervjuer genomfördes med fem gravida kvinnor. En kvalitativ innehållsanalys utfördes, vilket innebär ställa frågor om kvinnornas upplevelser och att lyssna på dem för att förstå hur de upplever sin graviditet efter att de fått besked om att de fått graviditetsdiabetes. Resultatet tydliggörs i två huvudteman (trygghet och oro) och sju underteman (välmående, tillit till personalen, lättnad med extra kontroller, bekräftelse, förvirring, rädsla/chock, brist i självkänslan). Resultaten visar att kvinnorna är oroliga och rädda precis när de fått diagnosen graviditetsdiabetes, rädslan gäller främst barnets hälsa. Barnmorskan har en central roll i vårdmötet men kvinnorna känner en stor tillit till all personal som är inblandad. Kvinnorna upplever att de mår bra under graviditeten. De tänker inte så mycket på att de har graviditetsdiabetes. Kvinnorna lever i princip som de gjorde innan diagnosen sattes förutom att de beskriver att de har minskat sitt intag av socker.. Kvinnorna beskriver också att de känner en stor lättnad och trygghet i att extra kontroller på barnet som t.ex. ultraljud görs. / Program: Barnmorskeutbildning

Many droplet microfluidic assays have been described in the literature over the last decade of research, however, there has been little reported industrial use of droplet microfluidics in drug discovery compound screening, and in particular that of P450 enzyme inhibition assays for profiling drug-drug interactions. This is partly for Intellectual Property reasons, since Pharmaceutical companies do not wish to give away trade secrets in a competitive market, but also because the technology is not yet 'proven' and remains in the proof-of-concept stage. In droplet microfluidics, where at least two liquid phases are encountered, it is important that leakage of material between phases is addressed. This effect has been extensively reported in the literature using fluorescent dyes, however there is very little evidence of research using large compound sets of diverse chemistry. This is probably because few researchers have access to the large pharmaceutical libraries necessary for this work. This project assessed the feasibility of translating a widely used microtitre plate-based P450 enzyme inhibition assay to droplet format; determined the extent of partitioning from droplets using a large pharmaceutical library set and attempted to model this behaviour, and thirdly, considered the pharmacological impact the droplet format may have on the assay. The P450 cytochrome 1A2 enzyme type (isoform) was chosen for translation to the micro-droplet format. Assays of this type are often conducted using fluorogenic substrates, making them favourable for relatively easy fluorescent detection in droplet format using simple optical detection assemblies. Oil selection was investigated to determine which oil systems would be better suited in respect of droplet formation. The use of surfactants in the oil phase and its impact on droplet formation was studied and the synthesis, preparation and characterisation of a custom perfluoropolyether (PFPE) surfactant ('AZF') conducted. Droplet chips were designed and fabricated to produce droplets of 200-300 µm diameter using novel channel designs and sealing techniques. The droplets were analysed by fluorescence spectroscopy using bespoke detector apparatus. Partitioning from aqueous to oil phase was studied for a small range of compounds and oils (with and without surfactant for fluorous oils). Partitioning was lowest using fluorous oils alone, and increased substantially when surfactant was included. Results from the large pharmaceutical test set suggested the percentage of compounds that may partition readily to the oil phase is low even when using surfactant. However, attempts to correlate this to known physicochemical properties and to develop a predictive model for fluorous solubility proved largely unsuccessful. Partitioning in the droplet chip using a droplet collection pooling method was difficult to quantify as a consequence of the profound impact turbulence had on partitioning. Miniaturisation of the P450 cytochrome inhibition assay to the droplet format initially gave poorly reproducible low signals. Possible causes included detector insensitivity, partitioning of reagent and/or fluorescent metabolite over longer incubation times, and binding of the 1A2 P450 cytochrome enzyme-protein at the droplet interface. Protein interaction at the droplet-oil boundary was studied by fluorescence labelling a protein contained in 200µm droplets and observing the extent of fluorescence localisation at the interface by epifluorescent and confocal fluorescence microscopy. The data from this work indicates a pronounced localisation of protein at the droplet interface, possibly leading to enzyme deactivation and the loss of signal seen for the assay in the droplet chip. A number of protein titrations were co-added to the droplets as 'blocking proteins' which were found to improve the reaction output, however were also noted to affect the pharmacology of the assay, noted by an order of magnitude shift in the reported IC50 for the test inhibitor used (fluvoxamine). The effects of compound leakage from droplets, and the possible detrimental impact on biological reagents by interaction at the droplet-oil interface, is a challenge that may limit widespread adoption of droplet MF systems in drug screening operations. Appropriate control measures and/or a means to reduce these effects are essential to enable accurate quantification with industrial drug discovery environments. The findings in this work highlight the challenges that have to be addressed for droplet microfluidic technology to be successfully incorporated into key areas of assay screening within drug discovery. In terms of further research, there is a significant requirement for the research community to delve further into these challenges and work closely with the industry sector to understand the beneficial role microfluidics can have and how to develop effective robust strategies the industry can easily adopt to progress this area of science.

Taiwanese lesbians have been found to utilize screening tests for breast cancer at lower rates when compared to women in general in Taiwan. However, there is a lack of evidence regarding the factors which influence Taiwanese lesbians' breast healthcare behaviour and intentions. A two-phase sequential exploratory mixed-methods study was employed to explore the factors influencing Taiwanese lesbians' breast healthcare behaviour and intentions, including semi-structured interviews and an online survey. Taiwanese women aged 20 years or above and who self-identified themselves as lesbians or as partnered with the same gender were targeted and recruited, using purposive and snowball sampling. Thirty-seven semi-structured interviews were conducted initially. According to the interview findings and two existing health psychology models (the health belief model and the theory of reasoned action), a questionnaire was developed and an online questionnaire survey was undertaken with a larger population. A total of 284 women completed the online survey. The findings showed that the lesbians' breast healthcare behaviour and intentions were directly or indirectly affected by their gender identity, gender role expression, patient-provider interaction and partners' support. In addition, it was also found that the lesbians may share similar views about breast cancer and breast cancer screenings, self-efficacy and cues to action with women in general in Taiwan. Some of these factors had an important effect on the lesbians' breast healthcare behaviour and/or intentions, in particular the perceived barriers to performing and/or having breast cancer screenings, the perceived susceptibility to breast cancer, self-efficacy and cues to action. Based on the PhD findings and social-ecological model, four levels of recommendations were proposed in order to encourage Taiwanese lesbians' utilization of breast cancer screenings and to promote their breast health.

Background: Fear of birth (FOB) is becoming increasingly recognised as a clinical issue that can have profound effects on the mother and her experience of pregnancy and birth. Failure to identify women with FOB could potentially lead to them feeling isolated and unsupported, and impact on their psychological health and the health of their baby. Aim: The main aim of this study was to gain an understanding of FOB and the associated impact on health professional practice, clinical outcomes and women's experiences of birth. The objectives were to: identify the most effective way of measuring FOB in clinical practice; investigate the most appropriate antenatal intervention to support women who fear childbirth; inform the study design for an RCT to assess the effectiveness of the intervention; and assess the most meaningful outcomes to include in future work. Methods: An explanatory mixed-methods study design was used. The first phase was a two-part online survey sent to Heads of Midwifery at 202 maternity units in the UK via Survey Monkey. Respondents were asked to give details of their unit in part 1 and service provision and evaluation for women with FOB in part 2. The second phase was a prospective cohort study of 148 women who had not experienced childbirth who were consecutively attending the Elisabeth Garrett Anderson and Obstetric Hospital, London or St Mary's Hospital, Manchester. Demographic data and details of sources of information on pregnancy were collected from participants in the first trimester along with their score on the tool chosen to measure FOB, the Fear Of Birth Scale (FOBS), and a saliva sample to measure cortisol level. In the third trimester, a second FOBS score and saliva sample were collected, and the Personal Health Questionnaire-9 (PHQ-9) and General Anxiety Disorder-7 (GAD-7) were administered to measure depression and anxiety respectively. Birth outcomes for the participants were collected from clinical records. In the third phase, 15 women participating in the second phase were purposively selected to reflect a range of FOBS scores and interviewed by telephone using a semi-structured interview to find out their experiences of pregnancy, being part of the study and service provision. Results: Response rates for the online survey were 63% for part 1 and 54% for part 2. Consultant obstetricians 25% had the most involvement in the care of women with FOB, followed by consultant midwives 21% and 30% had a designated midwife for dealing with FOB and only 32% provided specialist midwifery-led services for women with FOB, with 16% referring to a consultant obstetrician and 47% providing no specialist provision. No unit provided an evaluation of their services, although 19% had undertaken local audits. In the cohort study, using a cut-off of 54 for the FOBS, 30/148 (20%) had a FOB in the first trimester while 21/80 (26%) had a FOB in the third trimester. Compared with the first trimester, 51/80 women showed an increase in FOBS score, with 14 gaining and 7 losing a FOB. FOBS scores were not correlated with salivary cortisol in either trimester but they were correlated with PQH-9 and GAD-7 scores in the third trimester. They were also associated with a previous history of depression but only in the first trimester (p=0.011). FOBS scores showed considerable variability and a high measurement error, indicating a need for further refinement and psychometric testing. The qualitative interviews identified three themes underlying FOB: fearing the worst (pain, fear for the baby and fear of the unknown and complications), pathways to fear (friend-induced fear, mother-induced fear or reassurance and media-induced fear) and igniting or reducing fear (sources of information, support and communication). Conclusions: The FOBS is a potentially effective way of measuring FOB in clinical practice and research, but it requires enhancement informed by the themes identified by this study and psychometric testing in all three trimesters. An enhanced version of the FOBS could be used as the primary outcome to measure FOB during pregnancy in an RCT assessing the effectiveness of a suitable intervention, with the PHQ-9 and GAD-7 as secondary outcomes to measure depression and anxiety during pregnancy. An intervention to support primiparous women with FOB should be developed informed by the findings of this study, including components such as psychological education, relaxation, social support, reliable information sources and continuity of carer.

The aim of the study is to examine the one-to-one interventions and methods used by special education teachers for pupils with reading disabilities. The study highlights how special education teachers plan, implement and evaluate the interventions, based on analysis of qualitative interviews. The survey consists of eleven special education teachers active in eleven different schools. The study shows that the special education teachers consider early, intense interventions and a combination of evidence-based methods and materials as a means for good results. A majority of the teachers emphasize high competence and use current research in planning, implementation and evaluation of reading interventions. The special education teachers are unanimous in that cooperation between different competences and with guardians is a necessity to best support pupils with reading difficulties. All special education teachers emphasize that a respectful relationship with the pupil must permeate the individual reading interventions. In summary, the special education teachers express the need of more time for early intense interventions regarding pupils with reading disabilities. A question that arises is why those responsible in the municipality, in spite of thorough screening and research, do not sufficiently prioritize early intense interventions.

Advances in combinatorial chemistry have facilitated the production of large chemical libraries that can be used as tools to discover biological probes and therapeutics. High-throughput screening (HTS) strategies have emerged as the standard method to assess the biological activity of small molecules. These screens involve the individual analysis of each small molecule in multi-well plates, often requiring expensive automated methods and development of robust assays that may not translate to physiologically relevant contexts. This problem of evaluating large numbers of reagents in physiologically relevant cell and animal models has been addressed for genetic reagents such as RNAi, CRISPR, and cDNA by creating barcoded retroviral libraries that can be used to infect target cells in culture or in animal models. Using these tools, effective reagents can be selected and decoded using a rapid and inexpensive procedure compared to testing of individual reagents one at a time in an arrayed fashion. In order to more efficiently analyze small molecules, a pooled approach would similarly be useful. This dissertation describes the studies towards developing a pooled screening strategy for small molecules in cellular contexts. Through an initial screen, we set to phenotypically profile small molecule biological activity in a pooled fashion, while simultaneously gain insight about an individual, active molecule’s mechanism of action. I first describe the design of the pooled screen and define the goals necessary for successful application. Next, I outline the steps taken and challenges encountered during the invention of each component of the technology. Finally, I discuss a computational, target-based approach to design small molecules appropriate for future applications of the new screening technology.

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan degradation. KMO is emerging as an increasingly important target for drug development. The enzyme is implicated in the development and progression of several neurodegenerative disorders, in the regulation of the immune response and in sterile systemic inflammation. Production of recombinant human enzyme is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Although several in vitro KMO assay techniques have been reported in the literature these methods are typically insensitive or require purified protein for use in high-throughput screening assays of human KMO enzyme. The first report of bacterial expression of soluble active human KMO enzyme is described here. Fusion protein tags were used to optimise soluble expression and enable characterisation and partial purification of the active protein constructs. Functional enzyme was used to develop several novel high-throughput drug screening techniques for the discovery of inhibitors specifically targeting human KMO. These screening techniques were fully characterised and validated using known KMO inhibitors from the patent literature. One of the novel KMO assay techniques was implemented for compound screening and several hit compounds were identified, validated and their in vitro DMPK characteristics determined. In addition to assay development, KMO was characterised at the cellular level when overexpressed in HEK293 cells. These experiments indicated that KMO overexpressing cells undergo bidirectional adaptation via alteration of kynurenine pathway homeostasis. As a result, these cells are protected from cytotoxicity mediated by 3-hydroxykynurenine (3-HK), the toxic product of KMO catalysis. The development of novel high throughput screening techniques targeting KMO has enabled screening of potential new inhibitors specifically targeting the human enzyme. Implementation of these screening assays will allow accelerated and improved discovery and development of novel KMO inhibitors for the potential treatment of numerous disease states.

A major area of interest is the identification of proteins that play a role in hormone dependent cancers and in collaboration with the MRC Centre for Reproductive Health we studied the gonadotropin releasing hormone receptor (GnRH-R). Other targets described in the thesis are the SH3 domain of PSD-95 and the protein BLyS. In order to identify potential inhibitory small molecules we have used a variety of computational data base mining approaches as well as using and developing experimental binding assays. It has become increasingly challenging to evaluate the most representative drug like small molecule compounds when using traditional high throughput screening methods. This thesis assesses the use of in silico tools to probe key protein-protein and protein-peptide interactions. These tools provide a means to identify enriched compound datasets which can be purchased and tested in vitro in a time and cost efficient way. The transmembrane protein GnRH-R provides an interesting opportunity to identify small molecules that could inhibit the binding of its peptide ligand GnRH. This is a challenging project as there are few examples in the literature of drug-like molecules that bind to such protein-peptide interfaces. The first step involved receptor modelling using solved crystal structures of homologous proteins. The model was then validated by developing structure activity relationships for established high affinity ligands. We also performed crystallographic and biophysical studies on the native GnRH decapeptide. Two other protein-protein systems were also examined using the same virtual screening and experimental ligand binding methodology. SH3 domains play an important role in cell signalling and we used the PSD-95 protein as our target for study as a crystal structure has been published. As well as identifying potential ligands we characterised structural properties of PSD-95 fusion proteins and also developed the basis for compound assay. The third system studied was B Lymphocyte Stimulator (BLyS) which is a target for treatment of a number of autoimmune diseases. This presented an interesting target for study as the protein binds to multiple receptors depending on its multimeric state. BLyS protein was characterised using electron microscopy and other biophysical techniques.