Despite many studies on the action of yellow light in acne, it is still unclear whether it improves inflammatory acne or has photoimmunologic activity against pro-inflammatory pathways like toll-like receptor 2 (TLR2) and its down-stream cytokines e.g. TNF-α. This work sought to determine whether 530 nm IPL could cause a clinical improvement in acne and if its photo-mechanism of action involved modifications of the expressions of TLR2, TNF-α, IL-10 and IL-8. Twenty-eight adult patients with mild to moderate acne vulgaris involving their backs received four 530-950 nm IPL treatments at two-week intervals. Assessments performed at baseline and one week after the final IPL session included inflamed and non-inflamed lesion counts, Leeds assessments and sebum excretion rate (SER). Biopsies within the treatment area were taken at these two time points and two days after the first irradiation. TLR2 expression was examined by immunohistochemistry and TaqMan® Low Density Arrays were used to measure changes in expression of TNF-α, TNFR, IL-8 and IL-10. The data from 21 patients was included in the final clinical analyses. Inflamed lesions fell significantly by 28.0% (p = 0.002), but was not associated with significant changes in the Leeds score, SER or non-inflamed lesions. TNF-α expression fell by 17.6% (p = 0.031) at the end of therapy, and appeared to correlate with the percentage change in lesion counts in the subjects evaluated. TLR2 expression fell by 2.6% (p < 0.001) a week after the final irradiation, but bore no relationship to lesion counts. Neither IL-10 nor IL-8 was significantly affected. Though 530nm IPL significantly reduces inflammatory lesions, treatment efficacy will have to be improved to make it a viable treatment option. Its mechanism seems to include an anti-TNF-α effect, independent of IL-10 up-regulation. This is a novel mechanism, not been previously described for 530nm IPL. Updated hypotheses are suggested in order to explore this phenomenon further.

Background: Necatoriasis is a neglected tropical disease caused by the insidious parasite Necator americanus. This hookworm infects and reinfects approximately 500 million individuals worldwide, with a further 5.1 billion at high risk for acquiring the infection. Despite the high level of reinfection, no lasting immunological memory develops in the host. Albeit the profound health implications, chronicity and public health burden in developing countries, many aspects of human Necator americanus infection, particularly early events at the interface with the host immune system, are under researched. These figures and facts highlight the need for new research elucidating the molecular interactions between Necator americanus and the innate immune system. This will aid in the rational design of innovative and more efficient intervention strategies against hookworm infection, which is an essential measure for disease prevention. Objectives: In the context of Necatoriasis, this thesis studied the physical interaction between infective Necator americanus larvae (L3) with human dendritic cells (DCs) and epidermal keratinocytes, investigating the biological consequences. In addition, the development of a platform consisting of human keratinocytes, fibroblast and DCs on a 3D scaffold was constructed as an in vitro model of human skin. Results: The present thesis provides new insights into early immunological events at the interface of DCs and Necator americanus larvae and could explain how L3 affect immunity upon initial interaction with antigen presenting cells. For the first time, the data presented illustrates the sequestration of human DCs onto the sheath of L3 infective Necator americanus larvae, triggering the hookworm to exsheath. Intriguingly, the exposed cuticle of the larvae had negligible interaction with the free DCs. The findings also illustrate that the interaction between DCs and the larvae is mediated via a mandatory interaction with C-type lectin receptors, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor (MR). Blocking of either receptors with antibodies resulted in an inhibition of DC sequestration and aggregate formation. This demonstrates the biological relevance of previously identified lectin binding molecules on the Necator americanus larvae (L3) sheath in the context of interacting with DCs. These findings allude to a disparity between the surface chemistry of the sheath and larvae that could explain their differential ability to interact with DCs. While the exact nature of differences in the surface properties of the larvae and sheath are yet to be characterised, this data clearly indicates the presence of distinct chemical signatures on the cuticle sheath that attract DCs. However, this not only induces exsheathing but also enables larvae migration without being recognised or challenged by antigen presenting cells. A potential escape mechanism through which the larvae could bypass the immune cells, creating a possible site of ‘temporary immune privilege’. DCs incubated with viable axenic larvae exhibited an immature phenotype as evidenced by the low expression of the maturation markers CD80, CD83, CD86, CD40, and HLA-DR. Subsequently, the ability of DCs to acquire a mature phenotype in response to co-stimulation with lipopolysaccharide (LPS) in the presence of Necator americanus was assessed. These data show that DCs treated with the larvae will remain responsive to LPS stimulation. Additionally while the axenised larvae do not induce any cytokine production by DCs, they seem to suppress LPS induced cytokine expression, however these changes were not statistically significant (p value ≤0.3). Furthermore, the cell-free culture media from DCs, matured in the presence of LPS, had no visible effects on the larvae. Intriguingly, matured DCs in LPS-free culture media render the larvae non-viable through a lysing mechanism, alluding to a modified paracrine signalling response by mature immune cells in culture with the parasite. Interestingly, in the presence of epidermal keratinocytes, ex-sheathing was not mandatory to enable larval burrowing. In fact, only a small number of the larvae sheaths were recoverable from the apical surface of the keratinocyte layer; indicating preferential ensheathed larval burrowing. The data also illustrated the novel behavioural strategies promoting host invasion by Necator americanus larvae, in the presence of epidermal keratinocytes. Larvae were notably slower to exsheath in culture with keratinocytes and exhibited no vigorous movements as observed in DC cultures. This was thought to prevent early exsheathing, as the advantage of larvae maintaining their sheath during the initial stages of infection is in theory highly beneficial. Finally an immunocompetent tri-culture was developed on 3D layered PET scaffolds, encompassing epidermal keratinocytes and dermal fibroblasts, interspersed with DCs cultured at air liquid interface. A functional barrier was optimised, following which immune cell migration within the tri-culture system was observed successfully. Conclusion: Collectively, the sequestration of DCs onto the larvae sheath, suppression of maturation and cytokine expression, provides a possible explanation for the lack of a lasting immune response. These data provide novel insights into early immunological events at the interface of DCs, epidermal keratinocytes and Necator americanus larvae, which could explain how L3 evade immunity upon initial interaction with antigen presenting cells.

Light-based therapies, including photodynamic therapy (PDT), for acne are gaining popularity in dermatology. Based largely upon in vitro data, their beneficial outcome in acne is thought to be related to their bactericidal effects on Propionibacterium acnes. This randomised controlled study sought to determine the efficacy and tolerability of 610-950 nm IPL (administered as IPL-Placebo) and IPL-assisted methyl aminolaevulinate PDT (IPL-MAL) vs. adapalene 0.1% gel in the treatment of acne and to identify their mode of action, looking specifically at the effect on surface density of P. acnes. Thirty seven patients (31% of target due to slow recruitment) with mild to moderate facial acne were randomly allocated to IPL-MAL treatment, IPL-Placebo or adapalene. Both IPL groups received four treatments to the whole face, 2 weeks apart, while the third group was given adapalene nightly for 12 weeks. Assessments performed at baseline and weeks 8, 11, and 16 included inflamed, noninflamed and total lesion counts, Leeds grading, follicular porphyrin fluorescence, the Family Dermatology Life Quality Index and Dermatology Life Quality Index scores, and patient’s perspective of clinical improvement by the visual analogue scale (VAS). Cutaneous microflora was collected from all patients at similar intervals. Of the 37 patients randomised, only 30 completed the trial (10 in each group) and were included in the final analyses. Adapalene was found to be significantly superior to IPL-MAL and IPL-Placebo in reducing the noninflamed (adapalene 37.6% vs. IPL-MAL 3.4% vs. IPL-Placebo −9.7%) and total lesion counts (adapalene 35.7% vs. IPL-MAL 4.3% vs. IPL-Placebo −8.4%) at week 16. This was accompanied by a significant decrease (52.9%) in the DLQI score in this group (p = 0.031). The maximum improvement in inflamed lesion counts from baseline was seen at week 11 in the IPL-MAL (20.7%) and IPL-Placebo (13.4%) groups but occurred at week 16 in the adapalene group (26.5%). Statistical significance, however, was not reached in any group. There was no significant difference within or between the groups in the VAS, Leeds, FDLQI and porphyrin fluorescence results pre- and post- treatment. A significant increase in the density of propionibacteria (p = 0.021) and xxi coagulase-negative staphylococci (p = 0.039) was seen in the IPL-Placebo and IPL- MAL groups at week 16 and week 8, respectively; however, there was no significant difference between the groups. All the treatments were well tolerated. Adapalene remains an effective first line treatment in mild to moderate facial acne. However, the present study has remained indecisive (due to being underpowered) in drawing any firm conclusions regarding the efficacy of IPL and IPL-MAL on inflamed acne lesions. Further research is therefore warranted before their use can be advocated for acne treatment. An alternative mode of action for IPL and IPL-assisted MAL-PDT other than photodynamic destruction of P. acnes is suggested from the results of this study.

This thesis discusses some aspects of the use of ultraviolet rays to treat skin disease. The early history of dermatological phototherapy in Scotland is summarised as an introduction to the more recent developments described in a chapter on the Scottish phototherapy and photochemotherapy audit of 1996/1997 (funded by the Clinical Resource and Audit Group, the Scottish Office). This audit revealed some aspects of the phototherapy service that could be improved, and identified areas of particular importance for future research. Firstly, what type of UVB phototherapy lamp is most effective: the narrow-band (311-313nm) TL-01 lamp, or the broad-band UVB lamp? A meta-analysis conducted as part of this thesis gave a clear answer. TL-01 UVB is much more effective than broad-band UVB. The second included study was a randomised, controlled study that has contributed to deciding the optimal treatment frequency for TL-01 UVB phototherapy of psoriasis. Although 5x weekly treatment cleared psoriasis slightly more quickly than 3x weekly treatment, the difference in speed of clearance was too small to warrant the significantly greater frequency of acute erythema during treatment, and the greater number of exposures and dose required. The final question, answered by a randomised, controlled trial, in conjunction with a systematic review of the previous literature, was: for chronic plaque psoriasis, is TL-01 UVB or psoralen-UVA photochemotherapy to be preferred? The study conducted for this thesis showed TL-01 UVB to be more effective. Heterogeneity in findings of the studies addressing this question highlighted the importance of the particular treatment regimens selected for comparison, but the overall conclusion was that TL-01 UVB is the first choice of these two therapies.

This thesis contributes to the understanding of the psychosocial factors associated with skin conditions and the lived experience of visible difference. Chapter one is a systematic literature review that identifies the psychosocial factors associated with the onset and living with alopecia for children and young people. A systematic review of the literature indicated relational factors and frequency of negative life events were associated with the onset of alopecia for young people. Although the findings into the psychosocial factors associated with living with alopecia are mixed, anxiety was the most frequently reported factor for children and young people. The quality of the papers included in the review are mixed, with varying population samples, measures and methodological limitations. Clinical and research implications are discussed. Chapter two is a qualitative research study that explored the lived experience of six women with rosacea using interpretative phenomenological analysis (IPA). Participants’ experiences of rosacea were characterised by an internal struggle to feel in control of their skin condition whilst externally, learning to navigate complex social interactions. Clinical and research implications are discussed. Chapter three is a reflective account of the researcher’s experiences during the research process. The researcher’s reflections have been structured around the Acceptance and Commitment Therapy (ACT) Hexaflex model. Particular attention has been paid to the experiences that reflect the researcher’s values, cognitive defusion, acceptance, contact with the present moment, self as context and committed action.

MC1R pathways) would provide an up-to-date, superior mouse model able to mimic molecular aetiology of human melanoma to investigate the functions and mechanisms of other genes such as MITF, B-Raf, MC1R etc involved in the development of human melanoma.

The mechanism of action of IPL in acne treatment is not clearly understood, but an immunomodulatory role has been suggested. Furthermore, inflammatory cytokines and matrix degrading enzymes play a key role in acne pathogenesis. Therefore, curbing the production of these mediators may assist acne resolution. In photorejuvenation studies, IPL has been shown to induce the expression of a key immunomodulatory cytokine, TGF-β. Interestingly, TGF-β has been demonstrated to mediate immunosuppression, inhibition of keratinocyte proliferation and MMP-1 repression through a Smad3-mediated signalling pathway. Therefore, we sought to investigate the in vivo effects of IPL used for acne treatment. Biopsies obtained from 20 patients with inflammatory acne vulgaris at baseline and post-IPL treatment (48 hrs after the first treatment and 1 week after the final treatment) were immunohistochemically analysed to investigate the expression of TGF-β1, TGF-β2, TGF-β3, Smad3, MMP-1 and IL-8. Digital images were semi-qualitatively assessed using image analysis software. In addition, quantitative PCR analysis of TGF-β1, Smad3 and IL-8 was performed on biopsies from seven cases. Immunohistochemical analysis demonstrated that IPL elicited a statistically significant increase in epidermal TGF-β1 expression. However, no statistically significant difference was observed in the expression of TGF-β2/β3. Increased nuclear immunolocalisation of Smad3 was demonstrated in the post-IPL biopsies, which was statistically significant. Although not statistically significant, both IL-8 and MMP-1 expression showed a downward trend in the majority of cases. No statistically significant change was detected in the gene expression of TGF-β1, Smad3 and IL-8, which may be attributed to the small sample in which PCR was carried out. The data from this study suggests that Smad3-mediated TGF-β1 signalling may play a role in IPL-induced resolution of acne vulgaris. The therapeutic effect of TGF-β1 in inflammatory acne vulgaris could be attributed to its immunosuppressive effect and its ability to inhibit matrix degradation and keratinocyte proliferation.

Thyroid dysfunction is frequently associated with skin and hair diseases; however, the underlying pathogenic mechanisms are poorly understood. Pathological activation of the thyroid stimulating hormone receptor (TSHR) is the key feature of both hyper- and hypo-thyrodism. Expression of the (TSHR) has been reported in several extra-thyroidal locations including adipose tissue, bone and skin fibroblasts. TSHR expression may explain the association between the thyroid and skin disease. The TSHR can also be activated by a newly discovered glycoprotein hormone, known as thyrostimulin. This hormone is composed of a dimer of unique α 2 and β 5 subunits. Although thyrostimulin has not been detected in the circulation. However, both subunits have been shown to be expressed in different tissues including the skin. The aim of this study is to examine the expression of the TSHR and thyrostimulin in the skin. In addition, to investigate the expression of a variant form of the TSHR in human and mouse skin and, other mouse tissues. RT-PCR using primers specific for the full length receptor and the truncated variant revealed that although the variant was widely expressed in mouse tissues including skin, it was not found in human skin. The full length receptor and thyrostimulin were found to be co-expressed in eye, testis and skin. Immunohistochemistry of frozen skin and thyroid sections using commercially available antibodies against the extracellular (A9) and transmembrane domains (A7) of TSHR demonstrates that TSHR is not expressed in the epidermis but expressed in dermal fibroblasts and in myoepithelium around sweat glands. A new β5 antibody was characterised by western blotting and immunohistochemistry for future investigation of β5 expression in the skin. These data suggest a functional role for TSHR signalling possibly via thyrostimulin in the skin and that the variant form,although potentially present in some tissues, is unlikely to be important in human skin.

This thesis sets out to demonstrate the significant contribution to the field of podiatric dermatology, through the use of various forms of published documentary evidence, made by the author. In addition to the published papers submitted, a content analysis of British podiatric literature over a period of 21 years has mapped the emergence and development of the specialism of podiatric dermatology within the United Kingdom. This work demonstrates a significant increase in professional interest within this area during this period. This is evidenced through increased reporting of dermatological topics within podiatry journals in terms of related news items, advertisements and editorials. This is accompanied by an increasing number of case studies, peerreviewed papers and continuing professional development articles evident within the literature. The author has presented within this thesis a suite of fifty published articles along with verifiable evidence of professional activities related to the promotion and development of podiatric dermatology. Collectively this evidence represents a significant contribution to the development and evolution of dermatology as a specialist area within podiatry in the United Kingdom over the last fifteen years.

The purpose of this study was to investigate ROCK2 activation in squamous cell carcinogenesis and assess its co-operation with rasHa and fos oncogene activation together with loss of PTEN mediated AKT regulation. The analysis of ROCK deregulation with these genes in the MAP Kinase and PI3K pathways, two of the most commonly deregulated signalling systems, employed a well-characterised, transgenic mouse skin model of multi-stage carcinogenesis. A major goal was to study co-operation between these genes in the conversion of benign tumours to malignancy and investigate subsequent progression to aggressive carcinomas, given these are the most significant clinical stages of carcinogenesis from a patient’s viewpoint; and also investigated effects of ROCK2 deregulation on the processes of normal epidermal differentiation. ROCK2 is an effector protein of RhoA, which is a member of the ras superfamily and ROCK2 activation has been associated with tumour progression via an increase in tissue stiffness mediated by changes in actomyosin cytoskeleton leading to increased cellular motility. Thus, ROCK2 expression is commonly associated with the later events in cancer. Furthermore, there are many studies investigating ROCK2 in cancer given that it may be a useful therapeutic target in being downstream of oncogenic ras signalling. Yet, relatively few studies have explored the possibilities of a definite link that confirms the co-operation status of ROCK2 activation with ras/MAPK/fos and /or PTEN/PI3K/AKT pathways in SCC aetiology. Thus, questions exist as to exactly when does ROCK2 activation become causal; and what are the collaborating genes involved in the mechanism that drive the early or late stage events in carcinogenesis. To begin to answer these questions, inducible ROCK2 activation has been introduced into a well-characterised transgenic mouse skin carcinogenesis model that expressed a combination of ras and fos activation, driven by a modified human keratin 1 vector (HK1). Thus, exclusive epidermal expression of activated rasHa and fos oncogenes, in proliferative basal layers, gave hyperplasia and papillomatogenesis; but with no evidence of spontaneous malignant conversion. This stability of phenotype is thus ideal to assess roles for multiple transgene co-operations in the development of benign tumours and their conversion to malignancy. Hence, ROCK transgenic mice that expressed a conditionally active, 4-hydroxytamoxifen (4-HT)-regulated of human ROCK2 transgene were crossed with mice expressing activated rasHa and /or fos exclusively in epidermal transit amplifying keratinocytes (HK1.ras, HK1.fos). Inducible PTEN tumour suppressor gene mutation via exon 5 ablation (K14.Cre/D5PTENflx) and thus loss of AKT regulation was also incorporated into this model. This was achieved via deletion of exon 5 employing the RU486-mediated cre/loxP system, driven by keratin K14 promoter expression in basal layer keratinocytes. Therefore, to facilitate this investigation, a new and unpublished inducible ROCK2 system was employed in order to target the identical keratinocytes as PTEN loss. This new transgenic line of lsl.ROCKer transgenic mice employed the same 4-HT inducible ROCKer transgene but now driven by a generic CAG promoter following cre mediated ablation of the stop cassette once treated with RU486. In bi-genic K14.ROCKer/HK1.ras1205 mice, synergism between ROCK2 activation and (wound-promotion) sensitive HK1.ras1205 line showed direct co-operation and achieved malignant conversion of benign papillomas to well-differentiated squamous cell carcinoma (wdSCCs) histotypes (12 weeks of 4-HT treatment). This placed ROCK2 activity as the causal event driving malignant conversion, but in the absence of a wound promotion stimulus (loss of ear tag), papillomas did not convert. The correct papilloma context was required for ROCK to become causal proved to be the case on employing the wound insensitive HK1.ras1276 line. Here, K14.ROCKer/HK1.ras1276 mice failed to exhibit any papillomas and required the constitutive promotion stimulus from additional fos activation. Interestingly, following cessation of 4-HT, two intriguing observations were recorded. Firstly, that once bi-genic ROCK/ras1205 achieved malignancy, exogenous ROCKer expression appeared to show no involvement once squamous cell carcinomas (SCCs) progressed to poorly differentiated squamous cell carcinomas (pdSCCs), given the elevated expression of endogenous ROCK upon malignant progression. Secondly, the rapid growth of papilloma appeared upon cessation of 4-HT with highly intense p21 expression indicated the requirement of exogenous ROCK2 for malignant conversion and the possibility of a papilloma inhibition by 4-HT anti-cancer activity. Another major novel finding demonstrated ROCK2 activation could act as an initiator in co-operation with fos activation. Direct co-operation between ROCK2 and fos activation produced benign squamous papillomas yet, whereas ROCK2 activation alone induced hyperplasia as did fos activation alone at this time point, given papilloma formation in HK1.fos mice occur over 4-5 months. However, unlike deregulation of MAP Kinase signalling in bi-genic ROCK/ras1205 mice, in bi-genic ROCK/fos mice, no malignant conversion was observed due to high levels of compensatory p53/p21 expression. Thus, this bi-genic K14.cre/lsl.ROCKer/HK1.fos model suggests the requirement of additional mutation event for malignant conversion. An unexpected result appeared in bi-genic ROCK/Δ5PTENflx co-operation experiments where K14.cre/lsl.ROCKer/Δ5PTENflx cohorts exhibited epidermal hyperplasia with folded papillomatous appearance to the epidermis, but without papillomatogenesis even after seven month of period. This either indicates a weak co-operation between ROCK2 and Δ5PTENflx which may be due to the unexpected low levels of p-AKT from a compensatory increased p21 expression; and additional events needed to fill in the oncogenic gap in this bi-genic ROCK/Δ5PTENflx model, or may possibly highlight redundancy in the oncogenic hits provided by ROCK and PTEN. This latter suggest similar links exist between their normal roles in the epidermis which may be accountable for the alterations observed in keratinocyte differentiation. In both in vitro and in vivo experiments, K14.cre/lsl.ROCKer/Δ5PTENflx cohorts showed alterations in epidermal differentiation via anomalous K1 and low levels of K6 expression. Interestingly, activated ROCK2 appeared to induce or accelerate differentiation activity in K14.cre/lsl.ROCKer keratinocytes via increased K1 (early differentiation marker) and reduced K6 (proliferation marker) expression profiles. These results were consistent with in vivo data where K6 was expressed in low levels in K14.cre/lsl.ROCKer hyperplasia histotypes. In contrast, in K14.cre/lsl.ROCKer/Δ5PTENflx keratinocytes, inactivation of PTEN-mediated AKT activity may be accountable for restored keratin K6 and anomalous keratin K1 expression; as keratin K1 was expressed in a similar fashion of normal keratinocytes in K14.cre/lsl.ROCKer/fos keratinocytes. Interestingly, all tri-genic cohorts: K14.cre/lsl.ROCKer/ras1276/fos, K14.cre/lsl.ROCKer/fos/Δ5PTENflx and K14.cre/lsl.ROCKer/ras1276/Δ5PTENflx synergisms exhibited malignant conversion and /or malignant progression in all animals highlighting a novel role of ROCK2 activation. Further, the stage-specific consequences in each model in this study were shown to be influenced by p53/p21 status, where typically p53 expression disappeared in late stage papillomas yet, p21 expression persisted. This demonstrated the importance of compensatory p53/p21 expression in modulating tumour pathogenesis in all these models. Given that this study incorporated PTEN mutation, the influence of AKT activity was investigated in the SCC progression of tri-genic ROCK/ras1276/PTENflx and ROCK/fos/PTENflx cohorts; revealing a crucial antagonism between p21 and AKT. However, this study revealed that the malignancy in tri-genic ROCK/ras1276/fos cohorts was not influenced by p-AKT expression, and as this tri-genic model achieved wdSCCs only. This suggests that as the roles of ROCK in altering cytoskeletal organisation leading to increase in tissue stiffness are overlaid onto both MAP Kinase and AKT deregulation, the outcome is a very aggressive pdSCC. Thus, suggesting ROCK signalling to be a potential therapeutic target for ras/MAPK/fos pathway in carcinogenesis. Overall, this study showed the involvement of ROCK2 activation in the initiation stage for papillomatogenesis with fos oncogene, and demonstrated ROCK2 as a converter again and also in malignant progression with ras/fos/Δ5PTENflx mutations. This indicates the link of ROCK2 signalling with both MAPK and PI3K pathways, thus targeting ROCK2 would aid in development of cancer therapy.