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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Human papillomavirus genome status in cervical samples

Marongiu, L. January 2014 (has links)
Infection with oncogenic Papillomavirus genotypes is considered a major risk factor for the development of cervical cancer; nonetheless only a small proportion of the infected patients actually develop malignancies. The specific identification of high grade lesions is pivotal to increase the effectiveness of the cervical cancer screening. In the present study promising candidate molecular biomarkers based on the Human Papillomavirus (HPV) genomes were assessed in a cross-sectional cohort formed by samples singly infected with the most prevalent oncogenic genotypes 16, 18, 31 and 45. The candidate markers under investigation were the DNA viral load (VL), viral CpG methylation and viral integration. For the HPV16 samples, sequence variation within the regulatory region was also assessed. The viral integration was evaluated in a smaller longitudinal set. The results obtained showed that the DNA VL was lowest in subclinical lesions. The viral methylation was highest in severe dysplastic samples and differences in methylation profiles were observed between HPV species. The viral integration displayed a significant depletion of HPV16 episomal forms in cancerous lesions and the presence of viral integrants in all cytology grades. The analysis of the HPV16 variants identified eight novel polymorphisms and mutations profiles were specifically recovered in high grade cervical lesions. It was concluded that the viral genome modifications allowed the prediction of the cervical lesions. The combination of the molecular markers might allow a higher clinical specificity in the identification of cervical cancer precursor lesions.
22

Contribution of Gag and protease to variation in susceptibility to protease inhibitors between different strains of HIV-1

Sutherland, K. January 2014 (has links)
Recent reports have shown that HIV-1 Gag can directly affect susceptibility to protease inhibitors (PIs) in the absence of known resistance mutations in protease. Inclusion of co-evolved Gag alongside protease in phenotypic drug susceptibility assays can alter PI susceptibility in comparison to protease with a wild-type Gag. Using a single replication-cycle assay encompassing full-length Gag together with protease, we demonstrate significant variation in PI susceptibility between a number of PI-naïve subtype B viruses. Six publicly available subtype B molecular clones, namely HXB2, NL4-3, SF2, YU2, JRFL and 89.6, displayed up to 9-fold reduction in PI susceptibility. For two molecular clones, YU2 and JRFL, Gag contributed solely to the observed reduction in susceptibility. Gag and protease from treatment-naïve, patient-derived viruses also demonstrated significant variation in susceptibility, with up to a 17-fold reduction to atazanavir. In contrast to the molecular clones, protease was the main determinant of the reduced susceptibility. Common polymorphisms in protease including I13V, L63P and A71T were shown to contribute to this reduction in PI susceptibility, in the absence of major resistance mutations. The role of variation in PI susceptibility on LPV/r monotherapy treatment failure was investigated. The contribution of suboptimal adherence to treatment failure was shown and the development of reduced PI susceptibility during treatment observed. In addition, reduced PI susceptibility and single-round infectivity were associated with subsequent treatment failure. This study demonstrates significant variation in PI susceptibility of treatment-naïve patient viruses and provides further evidence of the independent role of Gag, the protease substrate, and in particular the amino terminus of Gag in PI susceptibility. It also highlights the importance of considering co-evolved Gag and protease when assessing PI susceptibility. These data indicate that reduced PI susceptibility at baseline may contribute to treatment failure on PI monotherapy.
23

Exploring the impact of antiretroviral drugs on the cell-to-cell spread of HIV-1

Titanji, B. K. January 2015 (has links)
Recent observations on the reduced susceptibility of HIV-1 cell-to-cell infection to inhibition by Reverse Transcriptase Inhibitors (RTIs) have raised questions on the bearing this mode of spread may have on the successful treatment of HIV-1, the maintenance of viral reservoirs and viral pathogenesis. This thesis presents a detailed assessment of the individual drug classes, which constitute first-line and second-line antiretroviral therapy, with regard to their ability to inhibit HIV-1 cell-to-cell infection in comparison to cell-free infection. Special emphases is given to the study of Protease Inhibitors (PIs), which have a mechanism of action different from RTIs, present a higher barrier to the selection of drug-resistant viruses, are highly potent and very important in both first-line and second-line treatment of HIV-1 infection. Also, PIs have not been studied before in the context of cell-to-cell spread of HIV-1. The results obtained show that different classes of antiretroviral drugs have different potencies against cell-to-cell spread of HIV-1. While PIs are equally effective at inhibiting cell-to-cell and cell-free spread of HIV-1, RTIs especially those of the Nucleoside Reverse Transcriptase Inhibitor (NRTI) class are ineffective inhibitors of cell-to-cell spread of the virus. This thesis also assesses the impact of combination antiretroviral therapy on these two modes of viral infection, using drug synergy analysis by the median effect principle. We show that combination antiretroviral therapy is effective against both cell-to-cell and cell-free HIV-1 infection. However in the context of antiretroviral drug resistance, cell-to-cell spread may contribute to a reduced efficiency of combination antiretroviral therapy in blocking the spread of infection. Overall, the study provides a better understanding of the impact of antiretroviral therapy on cell-to-cell spread of HIV-1 and within reason, bearing in mind the limitations of in vitro models, gives some insight on the possible clinical implications of these observations for current HIV-1 therapy.
24

Characterisation of keratinocyte-derived epidermal signals in the initiation of contact hypersensitivity to chemicals

Summerfield, V. L. January 2015 (has links)
Contact hypersensitivity reactions result from an adaptive immune response following topical exposure to chemical allergen. The initiation of this response requires the production of an inflammatory micro-environment, however the contribution of epidermal keratinocytes in creating this environment is not well-characterised. In particular, the molecular processes that regulate inflammatory mediator release in response to chemical allergen, and the function of keratinocyte-derived mediators in dendritic cell migration and maturation, have not been fully explored. Keratinocytes (HaCaT cell line) and three-dimensional reconstructed human epidermis were evaluated as possible models for investigating these questions. We have found that, in response to chemical allergen, keratinocytes transcribe and release inflammatory mediators (interleukin-6 (IL-6) and IL-8), and a cytokine known to induce dendritic cell migration (IL-1). Furthermore, the production of these mediators was greater than that induced by a simple chemical irritant, and different to lipopolysaccharide/interferon- stimulation. Transcriptomic analysis was performed to investigate the molecular mechanisms behind the inflammatory mediator release. Principal component analysis of the data showed distinct clustering of gene expression profiles associated with different doses of 2,4-dinitrochlorobenzene (DNCB, allergen) and sodium dodecyl sulfate (SDS, irritant) exposed keratinocytes. Many of the differentially expressed genes that were found in response to DNCB map to components of stress-induced pathways such as nuclear factor -light-chain-enhancer of activated B cells (NF-B) and nuclear factor erythroid 2-related factor 2 (Nrf2). Investigation of the upstream signal transduction cascades revealed activation of mitogen-activated protein kinase (MAPK) pathways, specifically phosphorylation of p38MAPK and c-Jun N-terminal kinase (JNK), with a simultaneous decrease in the phosphorylation level of extracellular-signal-regulated kinase (ERK). There was also evidence of partial activation of the NF-B pathway. The mechanism by which chemical allergens initiate these responses is unknown, however we have identified two possible triggers: production of reactive oxygen species, and activation of a receptor tyrosine kinase.
25

Lentivector-based cancer immunotherapy silencing PD-L1 and modulating cytokine priming : development of ex vivo myeloid-derived suppressor cells to assess therapeutic efficacy

Liechtenstein, T. M. January 2015 (has links)
Cancer immunotherapy strategies based on lentiviral vector (lentivector) transduction have recently been shown to be safe and effective in the clinic. The objective of cancer immunotherapy is to induce anti-cancer immune responses. Cytotoxic T cell (CTL) responses are particularly effective at recognizing and eradicating cancer cells and their activation is the main objective of most cancer immunotherapy treatments. Effective CTL responses depend on activation by antigen presenting cells (APCs), presenting tumour-associated antigens (TAAs) in the context of appropriate co-stimulatory and cytokine signals. In general, cancer immunotherapy has largely been ineffective due to tumour-induced immune suppression. One immunosuppressive mechanism employed by tumours relies on the accumulation of tumour-infiltrating myeloid-derived suppressor cells (MDSCs). Effective cancer immunotherapy treatments therefore need to stimulate tumour-specific CTLs as well as counteract the activity of tumour-infiltrating immunosuppressive cells. The first part of this thesis is based on the construction and evaluation of lentivector vaccines. The lentivector vaccines simultaneously expressed TAAs and cytokines, combined with silencing of the co-inhibitory molecule programmed death 1 ligand 1 (PD-L1). A collection of lentivector vaccines expressing an array of different T cell-polarising cytokines was generated and their T cell stimulatory and anti-tumour efficacies were assessed in vitro and in vivo. In the second part of this thesis a highly efficient and rapid method to produce large numbers of melanoma-infiltrating MDSCs ex vivo was developed, without inducing tumours in mice. Ex vivo MDSC phenotype, differentiation, and immunosuppressive activities were extensively studied. The novel ex vivo melanoma MDSCs were further used to evaluate the lentivector vaccines generated in the first part of this thesis. Simultaneous delivery of IL12 and a PD-L1-silencing microRNA was the only combination that could counteract ex vivo MDSC suppressive activities, correlating with therapeutically relevant anti-melanoma activities in a syngeneic B16 melanoma mouse model.
26

The role of Akt1 in skin barrier formation

Naeem, A. S. January 2014 (has links)
Atopic Dermatitis (AD) is a chronic inflammatory disease characterised by pruritus, hyperkeratosis, parakeratosis and dry skin. Association of filaggrin mutations with AD has been reported, however not all AD patients have filaggrin mutations suggesting other mechanisms give rise to the barrier defect present in AD. Akt1 activity is essential for cornified envelope formation and correct profilaggrin processing, although the functional role of Akt1 in these processes remains unclear. The aim of this study was to investigate the role of downstream targets of Akt1 signaling in profilaggrin processing and cornified envelope formation. Using shRNA to knock down Akt1 activity in rat epidermal keratinocyte cell lines, an in vitro organotypic model was created that phenocopies AD displaying hyperkeratosis, parakeratosis and impaired filaggrin processing. Results show that reduced Akt activity led to decreased lamin A/C degradation disrupting nuclear disintegration process giving rise to parakeratosis. HspB1 is reported to interact with filaggrin and also involved in filaggrin processing. Inhibition of Akt activity demonstrated a switch between HspB1-filaggrin to HspB1- actin interaction in the upper epidermis, which may interfere with filaggrin processing. Cathepsin H (Ctsh) was down regulated 4-fold in our Akt1 knockdown cultures and its expression co-localized with filaggrin in the granular layer of human epidermis. Filaggrin processing was impaired in Ctsh shRNA knockdown cell lines, and both Ctsh+/− and Ctsh−/− mice displayed hyperkeratosis and reduced filaggrin expression. Inhibition of RAPTOR (a component of mTORC1) has previously been reported to increase Akt1 activity. Raptor overexpression in REKs showed a decrease in Akt phosphorylation, Ctsh and filaggrin processing, and treatment with Rapamycin, an mTORC1 inhibitor, reverses these effects. In uninvolved AD skin, increase in RAPTOR correlated with decrease in both Akt phosphorylation and filaggrin expression. This thesis presents a novel mechanism where increase in RAPTOR can lead to a reduction in epidermal granular Akt1 phosphorylation leading to impaired filaggrin processing and barrier defects in AD.
27

The effects of Neisseria meningitidis infection on endothelial E-selectin, and consequences for neutrophil adhesion and transmigration

Dusart, P. J. January 2014 (has links)
Severe septicaemia due to the bacterium Neisseria meningitidis is characterised by substantial recruitment and activation of neutrophils on inflamed endothelium, and is associated with vascular damage. N.meningitidis associates with endothelial cells, E-selectin, and neutrophils in patient biopsies, and E-selectin can co-localise beneath adherent bacteria in vitro. This thesis aims to investigate whether N.meningitidis affects E-selectin distribution on the endothelial surface and whether meningococcal-E-selectin interactions have functional consequences on neutrophil rolling, adhesion and transmigration across vascular endothelium. Lentiviral constructs were created containing full-length E-selectin (ES) or a cytoplasmic domain deficient mutant (ΔC). Transfection of primary HUVEC demonstrated that ES E-selectin forms spontaneous clusters, while ΔC is expressed in a diffuse membrane pattern. Capsulated N.meningitidis was unreliable at inducing E-selectin clustering and co-localisation. In contrast, an unencapsulated SiaD- strain could co-localise with endogenous IL-1β induced E-selectin, although not with transfected ES or ΔC. Thus the E-selectin cytoplasmic domain affects both the molecule’s distribution on the endothelial membrane, and also N.meningitidis mediated redistribution, which additionally requires some factor present on IL-1β stimulated, but not ES or ΔC transfected, endothelium. Functional aspects of N.meningitidis interactions with E-selectin on neutrophil behaviour were also investigated. Brief co-culture of N.meningitidis with ES transfected HUVEC induced increased neutrophil adhesion under static conditions and greater transmigration under physiological flow. Neutrophil adhesion to N.meningitidis stimulated ΔC transfected HUVEC however, was no greater than untransfected cells. ΔC HUVEC also only saw a small increase in transmigration despite having comparable adhesion and rolling velocity to ES cells under flow. Taken together these results show that the E-selectin cytoplasmic domain has an important yet unstudied role in neutrophil adhesion and transmigration. These data should lead to a better understanding of the underlying processes surrounding neutrophil mediated endothelial damage caused by N.meningitidis, and could have broader relevance for other inflammatory responses, particularly in sepsis.
28

Overcoming T cell tolerance to tumour antigens : an evaluation of the role of helper responses

Ghorashian, S. January 2014 (has links)
Tumour associated antigens (TAAs) are up-regulated on malignant cells but may be expressed on normal tissues. Transfer of highly-avid cytotoxic T cells specific for TAAs may either cause auto-immune damage to normal tissues or transferred cells may lose efficacy through deletion or development of tolerance. CD8+ T cells recognising a Major Histocompatibility Complex class 1 (MHC-I) restricted epitope of the Murine Double Minute (MDM2) TAA were generated by T cell receptor (TCR) gene transduction. I then designed a model of tolerance in which MDM2 specific CD8+ T cells were transferred into lymphodepleted antigen-bearing hosts. These cells persisted but showed defective specific in vivo cytotoxicity where antigen was ubiquitous, or confined to non-haematopoietic tissues. In antigen-free hosts they remained efficient killers. Provision of CD4 help by OT-II T cells led to increased frequency and cytotoxicity of MDM2-specific CD8+ T cells. I then tested whether MDM2 TCR-transduced CD4+ cells could act as a helper population. Co-transfer of MDM2 TCR-transduced CD4+ and CD8+ cells improved antigen-specific killing of the CD8+ T cells. This was not due to specific killing activity of the MDM2 specific CD4+ T cells, and was not provided by a mock-transduced CD4+ population. Thus, transduction with an MHC-I restricted TCR generated TAA specific CD8+ T cells which became dysfunctional on antigen exposure, but could also generate a helper population capable of rescuing them from tolerance in vivo. Since the MDM2 TCR is CD8 dependent, CD4 T cells bearing this TCR have a lower avidity than their CD8+ counterparts. They therefore fell below the activation threshold for development of tolerance in antigen-bearing hosts, and could act as helpers in vivo. This strategy may be a novel approach to overcoming CD8+ T cell tolerance to TAAs.
29

The immune response in Influenza A-S. pneumoniae coinfection

Ellis, G. T. January 2014 (has links)
Streptococcus pneumoniae coinfection is a major cause of influenza-associated mortality. In this thesis the underlying disease mechanisms and the role of the immune response are investigated in a mouse model. Coinfection with otherwise mild influenza and S. pneumoniae strains is shown to synergistically cause mortality and severe disease. Loss of bacterial but not viral control, and subsequent outgrowth, is identified as the main driver of mortality. Influenza-mediated immune impairment and lung damage have been proposed as mechanisms of coinfection. Here the aspects of the immune response profiled are not impaired; in contrast, coinfection induces a strong proinflammatory cytokine response and an influx of functional neutrophils. Depletion of neutrophils or TNF-α blockade exacerbates disease and bacterial outgrowth, showing these aspects of the immune response are protective. In addition to profiling the downstream response to bacterial outgrowth, the upstream causes of bacterial colonization are investigated. CCR2-/- mice are shown to be more resistant to coinfection. Influenza-infected CCR2-/- lungs lack inflammatory monocytes and exhibit reduced damage prior to coinfection. How inflammatory monocyte derived damage is mediated is investigated. Blockade of TRAIL - a cell-death inducing ligand - during the viral phase prior to coinfection ameliorates disease. Inflammatory monocytes are shown to comprise the majority of TRAIL-expressing cells during influenza infection, and TRAIL expression is largely absent in CCR2-/- mice. Therefore a mechanism is proposed for coinfection where influenza-induced TRAIL-expressing inflammatory monocytes cause lung damage, allowing bacterial colonization, while neutrophils and TNF-α counter subsequent bacterial outgrowth. Other aspects of coinfection, such as bacterial spread to the brain and other facets of the immune response, are also investigated.
30

The parts are greater than the sum of the whole : exploring the process of change in a pain management programme using single case study design

Pike, A. R. January 2014 (has links)
This three-part thesis reviews the effectiveness of psychological interventions for chronic non-cancer pain on healthcare use and sick leave from work, and explores the process of change in a pain management programme using single case methods. Part 1 is a meta-analysis of 16 randomised controlled trials of psychological interventions in a chronic pain population. Small to moderate effect sizes were found for reduced healthcare use but no significant benefit for sick leave. Part 2 is a study using single case design methodology to explore trajectories of change in 8 patients attending a CBT-based chronic pain management programme. Baseline, intervention and bi-weekly follow-up self-report of catastrophic thinking, mood, self-efficacy, and goal attainment, and of process variables of working alliance and adherence, were supplemented by a post treatment change telephone interview which was qualitatively analysed. Detailed examination of change for each participant provided rich data: three participants improved significantly over the course of the programme, three deteriorated, and all improved in at least one goal. Therapeutic alliance was high and participants rated central elements of the programme, explanations of their pain, and peer support/group membership as important. Part 3 is a critical appraisal of the study and the review, contrasting the approaches, and concluding with a personal reflection on the process.

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