The retina of the human eye has the potential to reveal crucial information about several diseases such as diabetes. Several signs such as microaneurysms (MA) manifest themselves as early indicators of Diabetic Retinopathy (DR). Detection of these early signs is important from a clinical perspective in order to suggest appropriate treatment for DR patients. This work aims to improve the detection accuracy of MAs in colour fundus images. While it is expected that multiple images per eye are available in a clinical setup, proposed segmentation algorithms in the literature do not make use of these multiple images. This work introduces a novel MA detection algorithm and a framework for combining spatial and temporal images. A new MA detection method has been proposed which uses a Gaussian matched filter and an ensemble classifier with 70 features for the detection of candidates. The proposed method was evaluated on three public datasets (171 images in total) and has shown improvement in performance for two of the sets when compared to a state-of-the-art method. For lesion-based performance, the proposed method has achieved Retinopathy Online Challenge (ROC) scores of 0.3923, 2109 and 0.1523 in the MESSIDOR, DIARETDB1 and ROC datasets respectively. Based on the ensemble algorithm, a framework for the information combination is developed and consists of image alignment, detecting candidates with likelihood scores, matching candidates from aligned images, and finally fusing the scores from the aligned image pairs. This framework is used to combine information both spatially and temporally. A dataset of 320 images that consists of both spatial and temporal pairs was used for the evaluation. An improvement of performance by 2% is shown after combining spatial information. The framework is applied to temporal image pairs and the results of combining temporal information are analyzed and discussed.

The study of cell signalling in schistosomes is crucial in deepening our knowledge of the biology of these blood flukes, which affect hundreds of millions of people worldwide. Here, Akt/protein kinase B (PKB) signalling has been functionally characterised and mapped in Schistosoma mansoni; an Akt variant of approximately 52 kDa has been characterised and RNA interference of the S. mansoni Akt gene, resulted in an 84% reduction in Akt expression. The phosphorylation (activation) status of the characterised Akt protein was increased by host molecules, including insulin and L-arginine in somules and adult worms, and L-arginine and linoleic acid in cercariae. Akt phosphorylation (activation) was also attenuated by Akt Inhibitor X and herbimycin A treatment. Immunohistochemistry/confocal laser scanning microscopy revealed phosphorylated Akt in all S. mansoni human infective/resident life stages. Somules and adult worms displayed activated Akt primarily in the tegument, particularly the tubercles and gynaecophoric canal of adult males. Cercariae exhibited activated Akt in the nervous system and punctate regions along the length of the tail prompting investigation into the role of Akt in cercarial motility. Behavioural studies demonstrated a significant increase in cercarial swimming in response to host factors, which was attenuated following exposure to Akt inhibitor X. The striking activation of Akt observed in the tegument of adult worms stimulated research into its possible role in glucose uptake in this host-interactive layer. RNAi of Akt resulted in a 59% and 47% reduction in SGTP4 glucose transporter expression in male and female adult worms respectively with a concomitant reduction in glucose uptake by the parasite. In somules, the expression of SGTP4 and its evolution at the apical tegument membrane during transformation were significantly attenuated by Akt Inhibitor X; a 74% reduction in glucose uptake was also demonstrated following Akt inhibition. Bioinformatic analysis of S. mansoni Akt interacting proteins uncovered a putative connection between Akt and Rab vesicle trafficking proteins and a mechanistic model illuminating the possible role of Akt in the translocation of SGTP4 to the parasite surface was proposed. Collectively, this research highlights the significance of Akt in schistosome homeostasis and host-parasite interactions and thus demonstrates that Akt may be a suitable target for anti-schistosome drug development strategies.

In February 2017, the World Health organization published a list of 12 antibiotic-resistant "priority pathogens" that pose the greatest threat to human health. Amongst these, in the high priority group was 'Neisseria gonorrhoea'. When considering 'N. meningitdis' the highest rates of incidence are seen in infants with peak seen in adolescents and the elderly in some countries. CREE have been indentified near virulence and metabolic genes as well being present in the pathogenic 'Neisseria' species. It was therefore seen as an interesting area to undertake gene expression research. To look at the transcriptome and effective method of RNA extraction needed to be considered. Experiments found that the Qiagen RNeasy kit was the most successful kit to extract RNA from cultures of 'N. gonorrhoeae' and 'N. meningitidis'. This research, in the assessment of the transcriptome, has demonstrated that the presence of CREE is associated with the presence on ncRNAs in the genome. CREE locations in the neisserial genomes are frequently found near or overlapping SIPHT predicted ncRNAs. The transcriptome has also shown that CREE can invert at its location with the potential to impact gene expression. Research set out in this thesis has set the ground work for some interesting areas to be taken up for further investigation.

A number of innovative techniques were developed for the extra-vascular delivery of insulin, of which oral delivery of insulin being one of the most active fields of study in pharmaceutics. Interest in this domain is due to two factors: the therapeutic potential of the approach and lack of delivery systems which demonstrate promising results for clinical implementation. Oral delivery of insulin is of a particular challenge due to highly evolved and complex barriers presented by the gastrointestinal tract (GIT). Long-term s.c. injections are invasive and are associated with major drawbacks such as pain, weight gain, hypoglycaemia, hyperinsulinemia, leading to low patient compliance and adherence. The present work aims to develop novel insulin-loaded chitosan (CS) and pegylated chitosan (CS-O-mPEG) based nanoparticles (NPs) and investigate them for potential colonic delivery. PEG-Chitosan was chemically conjugated using low molecular weight chitosan and mPEG-2000. Insulin loaded pegylated chitosan (CS-O-mPEG) NPs were prepared via the iontropic gelation technique by cross-linking with tripolyphosphate (TPP). The characteristics of the NPs i.e. particle morphology, particle size and zeta potential was evaluated. The effect of pH and the polymer:TPP wieght ratios on NP characteristics was also evaluated. An HPLC analytical method to quantify insulin was developed and validated. In vitro release and entrapment studies of the nanoparticles were conducted using the Franz diffusion cells. Prepared nanoparticle formulations were assessed for biocompatibility using the MTT (Tetrazolim dye) assay and permeability studies on the Caco-2 and MDCK monolayers. Successful CS-O-mPEG conjugation was confirmed by FTIR and 1H NMR spectroscopy. SEM revealed the spherical nature of CS and CS-O-mPEG NPs. A mean diameter ranging from 50 - 250 nm was recorded for the NPs. Characterisation of NPs for zeta potential was carried out for both the CS and CS-O-mPEG formulations. Particle size measurements for the NPs revealed size ranges between 110-250 nm, in accordance to the hydradynamic diameter measured by DLS. RP- HPLC analytical method was developed and validated according to ICH guidelines to quantify Insulin. The data showed presence of well-defined single insulin peak, being successfully recovered at retention time between 7.5 - 9 minutes. CS-O-mPEG NPs demonstrated maximum release in simulated intestinal fluids (SIF). There was some encouraging data obtained in regard to the biocompatibility studies for the prepared Insulin loaded NP formulations using MTT assay. Permeability studies were also conducted for the prepared NP formulations on Caco-2 and MDCK monolayers, revealing better permeation of insulin through the CS-O-mPEG NPs. Insulin-loaded NPs of CS and CS-O-mPEG were successfully formulated. CS-O-mPEG NPs demonstrated superior in vitro characteristics over the conventional CS NPs in terms of aqueous solubility, particle size, entrapment efficiency and drug release profile, In addition, permeation studies revealed that CS-O-mPEG NPs enabled a significantly higher insulin transfer across Caco-2 and MDCK cell monolayer models compared to the CS NPs making the former a promising candidate for oral delivery of insulin.

For the first time, an in vitro cystic fibrosis (CF) artificial sputum model (ASM) was found to support the growth and survival of a clinical epidemic strain of meticillin-resistant Staphylococcus aureus (EMRSAl6-252). Specific components, which included mucin. DNA and others, were removed from ASM and the physiological impact of this was fully explored using viable counts and light microscopy. As CF patients are known to develop cystic fibrosis-related diabetes or CFRD, glucose was added to ASM (GASM), to explore the physiological impact of glucose on the growth and survival MRSA252. Total RNA was extracted from the corresponding log phases of MRSA252 grown in brain heart infusion (BHI) as a laboratory control, as well as ASM and GASM. RNA was extracted in order to conduct microarray analysis. MRSA252 DNA was used as a control. RNA (from the samples) was labelled with Cy5 and control DNA was labelled with Cy3. Once labelled and amplified, the Cy5/Cy3 mixture was then purified and hybridised onto an array containing seven sequenced S. aureus : genomes (N315, Mu50, MW2, MRSA252, MSSA476, COL and NCTC8325).

The Fallopian tube provides physiological support to both gametes and the embryo. Experimental approaches to improve the outcome of infertility treatment have included co-culture systems, whereby sperm, ova and/or embryos are cultured in a monolayer of Fallopian tube epithelial cells. This approach has been shown to be superior to other methods based on co-culture with other cell types. In addition, the role of gap junction intercellular communication (GJIC) in the regulation of cell proliferation and differentiation is becoming increasingly recognised as one of the major cellular functions. GJIC plays an important role in fertilisation as well as in the normal development of both the embryo and foetus. It is also involved in the sexual maturation of the adult and in the maintenance of health throughout life. As such, GJIC may be the key element in the understanding of cellular functions especially during the reproductive process. Therefore, the aims of this thesis were to examine some of the cellular and molecular aspects of the human Fallopian tube epithelium in relation to infertility. In the first part of this study, the gap junction proteins, connexins were examined by immunohistochemical and Western blotting techniques. Fallopian tubes were removed from women at different stages of the ovarian cycle. The results demonstrated the presence of gap junction proteins, connexins (cx) in the human Fallopian tube throughout the ovarian cycle. Fimbrial and ampullary regions were separated and subsequently processed for Western blotting analysis using a range of monoclonal or polyclonal antibodies directed against cx26, cx32 and cx43.The intensity of staining varied depending upon the hormonal status of the patients examined and appeared to be upregulated during the secretory stage as opposed to the proliferative phase of the ovarian cycle. Human Fallopian tube did not express cx32, regardless of the anatomical site examined. In all cases though, the expression of both cx26 and 43 appeared to be more prevalent in the ampullary region. The second part of this investigation focussed on transforming Fallopian tube epithelial cells into an immortal cell line. Normal cells cannot be sustained indefinitely, as cell degeneration occurs with continuous passaging. In most IVF treatments, sperm and ova are allowed to fertilise in a conventional medium but there is the potential that the Fallopian tube epithelial cells may be employed in IVF treatments in the future. Therefore, current techniques used to isolate and culture epithelial cells from the Fallopian tube were evaluated, in order to assess the best method that provides an optimal yield of Fallopian tube epithelial cells, both qualitatively and quantitatively. The cells were isolated using (i) a mechanical technique, whereby the mucosal layer of the tube were minced finely using a pair of scissors and (ii) an enzymatic method which consisted of the enzymes, trypsin and pancreatin. The results showed that enzymatic isolation provided a large number of cells, but there was a significant detrimental effect on cell survival and their secretory status. In contrast, cells isolated by the mechanical method were fewer in number than those obtained by the enzymatic method but cell survival and secretory status were relatively better. Regardless, of the isolation technique employed, the Fallopian tube epithelial cells had a limited lifespan in culture. Bearing this in mind and coupled with the realisation that the interactions between the tubal epithelial cells and gametes/embryos cannot adequately be studied in vivo, the next part of the study investigated ways of establishing an immortalised human Fallopian tube cell line. Such a cell line would be able to survive in culture indefinitely and therefore could be a potential model, which could mimic the in vivo milieu more closely. In order to obtain a 'bank' of Fallopian tube epithelial cells, cells isolated by either the mechanical or enzymatic method were cultured in growth medium for 24-48 hours prior to being transfected with a transforming gene, the SV40 Large T-antigen. Various techniques were used to transfect the epithelial cells in culture; these included well-established methods such as DNA co-precipitation as well as more advanced methods using cationic liposomes. The results from this study demonstrated that human Fallopian tube epithelial cells can be transiently transfected and could survive for over 102 days in culture, whilst still retaining many of the morphological characteristics of the original epithelial cell type. By examining both the cellular and molecular aspects of the tube as described above, it was anticipated that further insight into the complex nature of this structure would be achieved. Furthermore, it was hoped to improve the current understanding and knowledge of the Fallopian tube's functions, thereby facilitating a greater awareness of the issues important in establishing and maintaining an in vitro model of the Fallopian tube.

Neonates born to mothers with an active gonorrhoea infection can develop serious sight threatening eye infections. The causative agent, Neisseria gonorrhoeae, is passed to the neonate during birth, and infects the eyes of the neonate. The condition, ophthalmia neonatorum, develops 0-14 days after birth and initially presents as a painful inflammation of the eye with yellowish purulent discharge from one or both eyes. One preventative action used by some countries is the use of an ophthalmic prophylaxis which is usually an ophthalmic ointment which contains an antibiotic. This its self can cause chemical conjunctivitis. This study evaluates the potential to use a fatty acid or fatty acid derivative as the active antimicrobial agent in an ophthalmic prophylaxis. A panel of thirty-seven initial candidates were screened for anti-gonococcal properties. Seven of this panel were selected and tested against for ocular irritation potential using in vitro models and anti-gonococcal properties tested further in simulated tear fluid. Finally a single candidate, monocaprin, was selected as the main drug candidate. Ophthalmic formulations of liquid and semi-solid dosage forms were made and evaluated. Liquid dosage forms performed the best in in vitro tested and were further evaluated in cell culture and explanted models. The cell culture model suggested that monocaprin could be used to prevent infection 90 minutes after the cell were inoculated with the bacteria. An explanted corneal infection model was used to assess the potential formulations. It was shown that the anti-gonococcal properties of the drug candidate were inhibited on the ocular surface but this this could be countered by increasing the amount of monocaprin in the formulation. The formulations containing 0.188 % and 0.25 % (w/w) monocaprin were in some cases able to totally clear inoculations of higher cell numbers on the surface of the eye. Passage on agar plates containing monocaprin showed that increasing resistance due to genomic mutation is not likely and that existing mechanisms of fatty acid resistance did not give cross-resistance to monocaprin. However, duplicate samples passaged on monocaprin both acquired identical mutations in the dksA gene which may confer a small decrease in susceptibility. Also, work done on the processing of natural sources of fatty acids showed that treatment of coconut oil by use of a purified lipase or a lipase secreting yeast produced powerful anti-gonococcal substances. This could has the potential to be used in developing nations treat gonococcal and other bacterial infection. Overall, the work in thesis demonstrates that there is potential in the use a fatty acid or fatty acid derivative, most likely monocaprin, to be used as the active antimicrobial agent in an ophthalmic prophylaxis but more evaluation in terms of in vivo testing is required to demonstrate that the higher levels of monocaprin do not cause irritation to the eye.

Adoptive Cell Therapy of cancer using T cells is entering mainstream practice after years as a research method. Central to the efficacy of this “living therapy” is the function of the T cells transferred. T-cells, like other primary tissue cells, undergo differentiation and death. Clinical and preclinical data shows that lesser differentiated, less glycolytic, and more proliferative-capable cells used for the adoptive transfer yield superior tumor responses. This introductory section will describe discoveries which elucidate and control T-cell differentiation and function for the improvement of adoptive cell therapy. Namely by use of inhibition of the PI3k/AKT pathways, and through the discovery of a dual function of death and differentiation by the canonical death receptor Fas, which can be parsed apart with a mutation from valine to cysteine at the 194 position (C194V), differentiation can be withheld while leaving cell proliferation unhindered during T-cell stimulation and expansion. The data also reveals that the differentiation signal caused by extracellular Fas ligation passes through AKT, revealing both Fas and AKT as points of intervention for targeting differentiation along the same pathway. From further investigation, this introduction will describe the effect of AKT inhibition on T-cell differentiation on a transciptional and metabolic level. The data reveals AKT inhibition promoted FOXO1 intranuclear organization, which creates a more naive-like phenotype to the cells, and lower glycolytic status, another phenotype associated with persistent and long-lived cells. Furthermore, this control of AKT and Fas in T-cells yields benefits in several modalities of pre-clinical models of adoptive T-cell immunotherapy of cancer, in both use of a chimeric antigen receptor (CAR) and with use of Tumor Infiltrating Lymphocytes (TIL). Finally, the real-world applicability of the finding including the use of AKT inhibition in current approved Adoptive T-cell immunotherapies will be discussed.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disorder that affects 1 in 3600 male births. It is caused by genetic mutations in the dystrophin gene. This study investigated several aspects of the neuromuscular disorder within a population of Brazilian DMD boys and their families. This study's framework was laid out within the prism of an interacting cycle of genetic factors, cognitive functioning, and psychosocial aspects that underlie the neuromuscular disorder. It focuses on DMD's aetiology, history and previous research on genetic, cognitive and psychosocial aspects. Mixed methods were adopted to allow for a more encompassing view of the neuromuscular disorder: cognitive tests, an emotion recognition battery, genetic analyses, well-being questionnaires, and interviews were applied. Correspondent, quantitative and qualitative data analysis was carried out. The findings of 32 DMD patients (mean age 10.4 years, SD= 2 years) and 31 control subjects (mean age 9.4 years, SD= 3 years) revealed severe cognitive dysfunctioning in all assessed cognitive domains in the DMD population, as well as in the ability of emotion recognition. In the DMD group, it could be shown that poor executive functioning stood in a positive correlation with a poor ability of emotion recognition. The DMD patients' cognitive phenotypes were correlated with the genetic mutations in their dystrophin gene, but no relationship between the patients' genotype and cognitive phenotype could be confirmed. These results were contrary to previous research, which suggested that specific mutations in the dystrophin gene cause cognitive impairment. The DMD group scored poorly on the emotion recognition task, which is also a characteristic of autism spectrum disorder. However, when diagnosing for autistic characteristics through means of an interview, only a few similarities between the two disorders could be found. In order to assess the psychosocial components that come along with the disorder, well-being questionnaires were supplied. Interestingly, DMD boys scored higher on well-being than the boys in the control group. Moreover, 30 of the DMD caregivers (mean age app. 31 years) also revealed high levels of well-being, which correlated positively with the well-being of their sons, suggesting high levels of resilience. Given the participants' socio-economic hardship and the lack of governmental help, it was concluded that participants showed an incredible level of resilience that most likely resided within their faith, which nearly all of them stated to be the reason for their strength to strive. The relevant and new information about cognitive, genetic and social aspects of DMD uncovered in this study will pave the way for further (and much needed) studies into psychosocial aspects of the disorder.

Erythroid Kruppel-like factor (EKLF) is an erythroid specific transcription factor that binds to the proximal [beta]-globin gene promoter and is essential for high level expression. In addition, EKLF binds to the far upstream enhancer commonly referred to as the Locus Control Region (LCR). The nature of these two events, their relationship to other events at the multigene [beta]-globin locus and the precise and required interaction of these cis-acting sequences remains unclear. Equally, the mode of action ofEKLF at other erythroid-specific gene loci that are not regulated by an LCR has not yet been reported. These targets include chaperones, membrane-bound proteins, and enzymes of the heme biosynthesis pathway that are essential for red blood cell function. To elucidate the role of EKLF at the multigene locus, and at other erythroid genes, we monitored the temporal EKLF-directed events across the [beta]-globin locus, at the AHSP and Dematin as well as the ALAD promoter using a unique 4-0H-Tamoxifen EKLF-inducible erythroid cell line (JH31), developed in the laboratory, chromatin immunoprecipitation (ChIP) studies, and DNasel hypersensitivity assays. We demonstrate here that EKLF is not required for priming of the [beta]-globin locus for expression. However, EKLF is essential for maximal erythroid factor occupancy, recruitment of chromatin-modifying enzymes, and effective recruitment of the RNA Polymerase II complex. We show that EKLF recruits these complexes first to the LCR prior to the [beta]-globin promoter, suggesting that the LCR serves as a docking element. Finally we provide evidence that the LCR/promoter interacting factor, Ldb-l, is recruited to the [beta]-promoter in an EKLF-dependent manner. We extend our observations to several EKLF-regulated genes outside the [beta]-globin cluster, demonstrating not only the kinetics of transcriptional activation, but also a previously unknown mechanism of chromatin remodeling that implicates histone eviction. Finally, we report a discrepancy between the roles of the histone acetyltransferases CBP and p300 at EKLF target gene promoters, challenging conservative notions of basic transcriptional events. Together, our observations deepen our understanding of the mechanisms of action of EKLF, and provide a platform for additional studies.