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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.

Determining the action spectrum of UVR-induced mitochondrial DNA damage, and related UVR-induced cellular effects in human skin

Latimer, Jennifer Anne January 2013 (has links)
Biological responses of human skin to ultraviolet radiation (UVR) are largely wavelength-dependent as shown by the action spectrum of UVR-induced erythema and nuclear DNA (nDNA) damage. As mitochondrial DNA (mtDNA) damage has been shown to be a reliable and sensitive biomarker of UVR exposure in human skin, the aim of this study was to determine the action spectrum for UVR-induced mtDNA damage in human skin cells. This involved the irradiation of both HaCaT (immortalised keratinocytes) and HDFn (human fibroblasts) cell lines and primary human keratinocyte cells with increasing doses of UVR. A wide variety of UVR sources were chosen for their unique overlapping UVR profiles, covering the entire UVA and UVB spectrum. Overall UVR induced mtDNA damage was assessed using a quantitative long real time PCR to amplify an 11 kb segment of the mitochondrial genome. Specific UVR-induced mtDNA deletions (3895 bp and 4977 bp) were investigated, as was the effect on cellular respiration using the novel Seahorse extracellular flux analyzer. Furthermore investigations sought to determine UVR-induced nDNA damage: This included the induction of p53 mutations, generation of the oxidative stress marker 8-OHdG, photoproduct production as well as differential gene expression. For investigations of general UVR-induced mtDNA damage, dose curves were produced for each of the UVR sources and cell types. From this an action spectrum was determined by a method known as de-convolution. A higher sensitivity was seen in the fibroblast cell type. The overall trend of the action spectrum of the UVR-induced mtDNA damage followed that of various previously determined action spectra with the most detrimental effects occurring over the shorter wavelengths of UVR. This was also shown for the production of photoproducts and with the differential gene expression analysis. UVA and UVB both also affected cellular respiration and gene expression. The UVR exposures used in the study were not effective in inducing detectable levels of either of the mtDNA specific deletions, mutations within the p53 gene or levels of 8-OHdG and this may have been affected by limitations of the analytical techniques. Further experiments saw that the addition of antioxidant type entities provided protection and the possible reversal of some of the UVR damaging effects.

Systematic development of a behavioural intervention to promote sun-protection behaviours amongst holidaymakers

Rodrigues, Angela Margarete Martins January 2014 (has links)
Intermittent UV-exposure is a risk factor for melanoma. Recreational sun-exposure (e.g. holiday) is associated with melanoma incidence. Effective and affordable interventions to promote sun-protection behaviours (SPB) are needed. This PhD thesis describes the development of a behavioural change intervention to promote SPB amongst holidaymakers and a pilot of acceptability, feasibility, and fidelity of the intervention. A systematic review was conducted to appraise efficacy of behavioural interventions to change SPB and experience of sunburn. Twenty-three randomised-controlled trials (RCT) were included and no evidence was found for the efficacy of interventions in reducing tanning, promoting protective clothing and seeking shade. Larger effects were observed for self-reported sun-exposure and number of sunburn experienced. Moderator analyses showed that effective interventions were more likely to stimulate social norms and provide appearance-based information about photoaging. A qualitative study based on the theory domain framework was conducted to investigate perceptions of sun-related experiences and determinants of SPB. In a semi-structured interview, 17 holidaymakers showed a desire to tan attributing a high value to it during holidays. Most respondents knew how to perform SPB and identified key barriers to SPB. Findings from systematic review and qualitative work informed the development and design of an evidence-based intervention. The prototype of the mobile phone based (app) intervention was initially tested using a user-centred design: 17 participants were satisfied with the prototype and expressed willingness to use it, with minor changes being introduced to optimise acceptability. Novel outcome measures to assess sun protection behaviours were also explored. The two newly developed methods of outcome assessment (sunscreen use events classifier and mDNA damage caused by UV exposure) show robust evidence for the assessment of sun protection behaviours and skin damage during holidays. This work contributed to the development of a full protocol for the outcome assessment in a definitive trial. Another systematic review was conducted to synthesize evidence on the question-behaviour effect (QBE) for health-related behaviours. Forty-one studies were included assessing a range of health behaviours. Findings showed a small QBE. Studies showed moderate heterogeneity, variable methodological quality and evidence for publication bias. No dose-response relationship was found. Risk of bias within studies and publication bias indicate that the observed small effect size may be an over-estimate. Based on these findings, no changes would be introduced to the protocol of the definitive trial to tackle QBE. A pilot study assessing the acceptability, feasibility and fidelity of the app use showed that the intervention was feasible and highly acceptable. Findings from the pilot study will inform a definitive RCT.

From therapy to theory : a cognitive perspective of the psychosocial impact of dermatological disease

Walker, Carl January 2003 (has links)
No description available.

A molecular genetic analysis of Crohn's disease susceptibility loci in psoriasis

Quaranta, Maria January 2012 (has links)
Psoriasis is an immune-mediated skin disorder that is inherited as a complex trait. Genome-wide linkage and association studies have identified a major disease susceptibility locus (PSORS1) and several genetic determinants of smaller effect. At least two of these (the IL12B and IL23R genes) have independently been associated with Crohn’’s disease (CD). Thus, the aim of this project was to investigate the genetic overlap between psoriasis and CD, with a view to identifying shared pathogenic pathways. In the first phase of the study, 26 CD variants were genotyped in 1,256 psoriatic patients and 2,938 unaffected individuals. Significant associations (FDR < 0.01) were observed for three markers, mapping to chromosomes 6p22, 21q21 and 21q22. The analysis of an independently ascertained dataset (1,348 cases vs. 1,368 controls) validated the chromosome 6p22 association, with the critical SNP (rs6908425) yielding a combined P value of 4 x 10-6. This marker lies within the CDKAL1 gene, which also harbours type 2 diabetes (T2D) associated alleles. Since the mechanisms mediating the pathogenic effect of CDKAL1 are poorly understood, an investigation into gene function was undertaken in the second part of the study. Real-time PCR analyses of multiple cDNA panels showed that CDKAL1 is abundantly expressed in immune cells, especially in CD4+ and CD19+ lymphocytes. Stable CDKAL1 knock-down cell lines generated by sh-RNA lentiviral transduction were also analysed. This showed that CDKAL1 silencing results in reduced cell proliferation and altered cell cycle progression. Transcription profiling of the knock- down cells identified a number of differentially expressed genes, mostly involved in housekeeping functions and inflammatory responses. Taken together, these findings indicate that CDKAL1 is a pleiotropic gene conferring susceptibility to psoriasis, CD and T2D. The results of functional studies suggest that this pathogenic role may be mediated by an effect on inflammatory responses and cell cycle progression.

Molecular mechanisms controlling the expression of the HLA-Cw*06 psoriasis susceptibility allele

Bertoni, Anna January 2013 (has links)
Psoriasis is an inflammatory skin disorder that is inherited as a common and complex trait. Linkage studies have repeatedly identified a primary disease susceptibility locus (PSORS1) spanning 250 kb within the Major Histocompatibility Complex. Deep sequencing experiments and genome-wide association scans have highlighted HLA-C as the most likely PSORS1 gene and unambiguously demonstrated that the HLA-Cw*0602 allele confers increased disease risk in a wide range of populations. In this context, the aim of this project was to investigate the impact of PSORS1 genetic variation on HLA-C transcription, with a particular focus on HLA-Cw*0602 expression. In the first part of the study, genetic variants mapping to the regulatory elements of different HLA-C alleles were characterized, by measuring the basal and cytokineinduced reporter activity driven by the HLA-Cw*0602, -Cw*0702 and -Cw*0304 promoters. These experiments identified two regulatory polymorphisms that virtually abolished the response to TNF-α (rs2524094) and IFN-γ (rs10657191) in HLA-Cw*0602 constructs. The existence of a differential cytokine response was also confirmed by the real-time PCR analysis of primary keratinocytes treated with TNF-α and IFN-γ. Since little is known about the role of epigenetic factors in the regulation of HLA-C expression, the presence of HLA-Cw*0602 specific modification patterns was examined in the second part of the project. The methylation status of the five PSORS1 CpG islands (CGIs) was initially investigated. This targeted approach revealed lack of methylation at four CGIs, two of which mapped within the HLA-C gene region. Partial methylation was detected at a fifth CGI, lying upstream of POU5F1. To complement these data, a methylation profile of the entire PSORS1 locus was generated in two HLA Cw*0602,-Cw*0702 heterozygous individuals. This showed that the majority of methylated cytosines mapped between the POU5F1 and C6orf15 transcripts, and upstream of HLA-C. Neither the targeted CGI analysis nor the systematic profiling of the entire PSORS1 region revealed any substantial differences in the methylation status of haplotypes bearing different HLA-C alleles. Taken together, the results of this study indicate that HLA-C expression is tightly controlled by promoter variants and immunological stimuli. Further studies will be required to fully explore the impact of genetic variation on DNA methylation and to determine whether PSORS1 epigenetic modifications play a role in psoriasis susceptibility.

Functional analysis of MICA polymorphism : with an emphasis on Behcet's disease

Shafi, Seema January 2013 (has links)
Behcet’s disease (BD) is a multi-organ inflammatory pathology. A main factor of genetic predisposition locates to HLA-B*51, but MIC A* 009 is also reported to be strongly associated with BD. The aim of the thesis is to undertake a functional analysis of MICA polymorphism, and determine the functional effects of the HLA-B*51-MICA* 009 combination. Isogenic stable cell lines expressing MICA*009,*008,*009v and ULBP2 were separately created. These were used in killing assays to compare the cell biology of the different MICA isoforms and to assess whether that encoded by MICA*009 varies from other isoforms in its capacity to promote killing by NKG2D positive cells from healthy controls and BD patients. Similarly, double transfectants: HLA-B*51-MICA*009 and HLA-B52*MICA*009 (control) were created and used in similar assays to determine the inhibitory effect, if any, of HLA-B*51 on the targeting of MICA+ cells by patients’ lymphocytes. Data from killing assays show a most unanticipated, donor-to-donor variation in the hierarchy with which different transfectants are targeted by lymphocytes from healthy controls and from patients. These hierarchies seem stable longitudinally and are validated by the CD107a assay. Differential killing cannot easily be explained by an affinity hierarchy because people’s cells kill different targets with different hierarchies. Variation among patients is even greater: Approximately 25% of patients preferentially target cells expressing MIC A* 009 versus cells expressing other MICA isoforms. HLA-B*51 and HLA-B*52 comparably inhibit the targeting of MICA*009+ cells by lymphocytes from healthy controls but for 45% of patients, HLA-B*51 shows distinctively stronger inhibition of the targeting of MICA*009+ cells. The variation is killing is not explained by either the number of NKG2D+ or KIR3DL1* cytotoxic cells nor the expression level of these molecules. In conclusion, this thesis advances the current understanding of human MICA biology by presenting novel data detailing the expression and function of MICA proteins. The data in this study provide insight into the "tuning" of the lymphocyte stress surveillance response and the impact upon this of HLA-B*51, with implications for the pathogenesis of Behcet’s disease.

Cellular responses in cutaneous sensitisation

Rose, Marlene January 1975 (has links)
No description available.

UVA-induced oxidative stress and DNA damage in human skin cells and photoprotection by antioxidant compounds

Delinassios, George January 2012 (has links)
Solar ultraviolet radiation (UVR), especially UVA (320-400nm), is a potent inducer of oxidative DNA damage in human skin cells. Such damage has mutagenic and carcinogenic potential. It is therefore important to understand the mechanisms that control its extent, and develop means of protecting against such damage. The main purpose of this work was to develop reliable quantitative methods to measure UVR-induced oxidative stress, assess its biological consequences and its inhibition, in human skin cells in vitro. Most of this work studied the effects of UVA radiation, as this is the major (&gt;95%) component of solar UVR and readily generates reactive oxygen species (ROS). Particular focuses of this work were the measurement of UVA-induced ROS, as well as the formation of two of the most intensively studied DNA photolesions, the guanine-derived lesion 8-oxo-7,8-dihydroguanine (8-oxoGua), and the cyclobutane pyrimidine dimer (CPD). These were assessed by an enzyme specific comet assay. Vitamin E was used as a model antioxidant and treatment of HaCaT keratinocytes pre-but also post-irradiation was found to reduce ROS levels, as well as UVA-induced 8-oxoGua and CPD formation. The post-UVR protection offered by vitamin E eliminated any possible sunscreen effects. Real-time PCR data of UVA-induced genes showed that vitamin E inhibited expression of heme oxygenase 1 (HOI) but not matrix metalloproteinase 12 (MMP12). Of three other antioxidants tested, only one (lanosterol) had an inhibitory effect on ROS and 8-oxoGua, but not on CPD formation. Studies with agents that modified ROS levels showed a clear link between ROS and 8-oxoGua but not CPD. Overall, the data showed that antioxidants have the potential to protect against potentially mutagenic epidermal DNA photodamage, an endpoint which, along with gene expression, can be readily applied to the in vivo human situation.

Towards hES cell-based therapy of epidermolysis bullosa

Petrova, Anastasia January 2012 (has links)
Regenerative medicine offers great hope for therapeutic innovation in many diseases, including those with defective skin, such as the group of inherited blistering skin diseases known as epidermolysis bullosa. The work in this thesis addresses some of the important pre-clinical approaches to generating keratinocytes (the main cell of the outer skin layer, epidermis) from pluripotent stem cells (human embryonic stem cells, hESCs). Undifferentiated hESCs were exposed to a complex microenvironment in organotypic cultures (to mimic conditions for epidermal stem cell development and maintenance); this yielded epidermis-like structures which stained positively for basal keratin 14 and several extracellular matrix molecules, although a stratified epidermis was not produced. To refine the protocol, hESCs were differentiated to an epidermal cell fate in monolayer cultures in vitro prior to subjecting them to organotypic culture. The most efficient technique involved culture of hESCs on native de-cellularized extracellular matrix produced by normal human dermal fibroblasts as a substrate for differentiation. Large epitheliaHike sheets positive for keratin 14 and p63 expression were observed. On subculture, these cells retained their morphological and immunocytochemical characteristics for up to 5 passages and could be successfully cryo-preserved and recovered. Overall, this study explored the potential of pluripotent stem cells as a source of epidermal progenitors, with results that provide proof-of-concept for future cell therapy innovations in defective skin diseases such as epidermolysis bullosa.

Characterisation of rare genetic variants conferring susceptibility to psoriasis

Onoufriadis, Alexandros January 2013 (has links)
Psoriasis is an immune-mediated skin disorder that is inherited as a complex trait. Although genome-wide association studies (GWAS) have identified a number of common disease susceptibility alleles, a substantial fraction of psoriasis heritability remains unexplained, suggesting the possibility that rare variants may also be pathogenic. The aim of this project was to further investigate this hypothesis and explore different approaches to the identification of rare susceptibility alleles. A candidate gene approach was initially undertaken, through the re-sequencing of RNF114, a gene that had been associated with psoriasis in various GWAS. This led to the identification of four novel promoter variants (c.-66C>A, C.-64C>A, C.-41C>T and c.-9A>C) which collectively demonstrate significant association with the disease (P &lt; 0.01). The functional characterisation of two representative substitutions showed that they both affected Spl binding as well as RNF114 promoter activity. In the second part of the study, a positional cloning approach was applied to the analysis of a multigenerational psoriasis pedigree, where the disease appeared to segregate as an autosomal dominant trait. Tentative evidence for linkage (LOD &gt; -2) was obtained for two regions on chromosomes 2p21 and 15q25. However, next-generation sequencing of an affected family member failed to identify any pathogenic mutation within the above intervals. In the final part of the study, five unrelated patients affected by a rare and severe variant of psoriasis (generalised pustular psoriasis) were analysed by whole-exome sequencing. This led to the identification of two recessive mutations (p.Ser113Leu and p.Arg48Trp) of the IL36RN gene, which encodes a protein antagonizing the activity of IL-36 cytokines. An ex-vivo analysis of patient cells demonstrated a marked increase in cytokine reduction upon IL-36A stimulation, suggesting that GPP mutations may affect the anti-iflammatory function of IL36RN. While this work suggests that rare variants are likely to contribute to the pathogenesis of psoriasis, it also highlights a number of methodological issues that will need to be considered in the design of future studies.

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