Cutaneous malignancies represent a quarter of all new cancer diagnoses in the UK. The key to reducing the tumours’ associated mortality and morbidity is early diagnosis and treatment. Prompt diagnosis remains predominately a clinical skill, but relatively little investigation of the cognitive psychology underpinning expertise in this domain has been undertaken. This thesis aims to improve understanding of these processes and investigate how lesional diagnostic expertise might be enhanced. A large database of diagnostically tagged images was captured specifically for this project. A series of separate studies were undertaken to give insight into how lesional diagnosis occurs and how it can be improved. The studies highlighted that non-analytical pattern recognition (NAPR) is likely to predominate in distinguishing malignant and non-malignant skin lesions and that the widely-promoted rules advocating analytical pattern recognition (APR) are not effective for discriminating melanoma from benign pigmented lesions. The keystone to promoting the development of NAPR and thus diagnostic expertise would seem to be increasing a novice’s personal library of examples with relevant feedback. Studies demonstrated that current undergraduate exposure was variable but universally sparse, so simulation by way of diagnostically tagged images was developed which showed accuracy could be improved by increased exposure. This improvement occurred in both a content specific and dose responsive manner. These studies also highlighted that the learning curves for skin lesions are not uniform. Further studies demonstrated that the choice of images had implications on the development of diagnostic expertise; suggesting it was important that these images represent clinical practice rather than “classic” examples traditionally advocated for teaching purposes. In addition, studies highlighted the potential benefit of the 3D models developed during this project. Building on the idea that a personal catalogue of relevant referent images was crucial to enhanced diagnostic accuracy, prototype software was developed to exteriorise the experts’ library of examples; in the tests described novices utilising the software delivered superior accuracy than medical students on the completion of their undergraduate teaching. In summation, the work described shows that by utilising dermato-informatic approaches lesional diagnostic competence can be improved significantly.

Die Anpassung des Aktinzytoskeletts an extrazelluläre Gewebsstrukturen ist Voraussetzung für die Interaktion mit der extrazellulären Matrix und für die Zellbewegung, einschließlich der Invasion und Metastasierung von Tumorzellen. Wir untersuchten bei invasiven B16/F1 GFP-Aktin Mausmelanomzellen, ob und wie sich Zellform, Art und Effizienz der Bewegung an physikalisch unterschiedlich beschaffene kollagenöse Umgebungen anpassen: 1) mit Kollagen-Monomeren beschichtete 2D Objektträger, 2) 2D Oberfläche einer fibrillären Kollagenmatrix und 3) Zellen, die in einer 3D Kollagenmatrix eingebettet waren. Zur Darstellung des Aktinzytoskeletts wurden Zellen eingesetzt, die GFP-Aktin Fusionsprotein exprimierten, und mittels Zeitraffer-Videomikroskopie und Konfokalmikroskopie untersucht. Im direkten Vergleich waren Struktur und Dynamik des Aktinzytoskelett wie auch Zellform und Art der Migration unterschiedlich in den verschiedenen Umgebungen. Auf 2D planer Oberfläche erfolgte eine rasche Adhäsion und Abflachung der Zellen (Spreading) mit nachfolgender Migration mit Bildung fokaler Adhäsionszonen, in die kabelartige Aktinstrukturen (Stress fibers) einstrahlten. Dagegen entwickelte sich in 3D Kollagenmatrices eine spindelförmige, fibroblastenähnliche Zellform (mesenchymal) mit zylindrischen fingerförmigen vorderen Pseudopodien, die Zug der Zelle nach vorne bewirken und hochdynamisches polymeres Aktin, nicht jedoch Stress Fibers enthielten. Eine ähnliche Zellform und Struktur des Zytoskeletts entwickelte sich in Zellen auf 2D fibrillärem Kollagen. Die Kontaktfindung und Migrationseffizienz auf oder in fibrillären Matrices war im Vergleich zu 2D kollagenbeschichteter Oberfläche erschwert, die Migrationseffizienz verringert. In Kontrollversuchen wurden Migration und polarisierte Bildung von Aktindynamik durch Inhibitoren des Aktinzytoskeletts (Cytochalasin D, Latrunculin B, Jasplakinolide) stark gehemmt. Diese Befunde zeigen , dass die Struktur und Dynamik des Aktinzytoskeletts sowie die Art der Migration in Tumorzellen stärker als bisher angenommen durch die umgebende Kollagenstruktur bestimmt wird. Während 3D Kollagenmatrices in vivo ähnliche bipolare Zytoskelettstruktur fördern, müssen Abflachung der Zellen mit Bildung von Stress Fibers als spezifische Charakteristika von 2D Modellen angesehen werden. / The dynamics and the adaptation of the actin cytoskeleton in response to extracellular matrix structures is the prerequisite for cell polarisation, shape change, and migration, including the invasion and metastasis of tumor cells. In invasive B16-mouse melanoma cells expressing GFP-actin fusion protein we directly imaged cytoskeletal dynamics, adaptation and movement in response to physically different collagen substrata using time-lapse videomicroscopy and confocal microscopy: 1) cells on 2D surfaces coated with monomeric collagen, 2) 2D surfaces composed of fibrilliar collagen, and 3) cells which were embedded in 3D collagen matrices. In directly comparision the structure and dynamic of the actin cytoskeleton, cell shape and migration efficiency were different between the different collagen substrata. On 2D monomeric collagen quick cell adhesion, spreading, and cell flattening were followed by migration driven by focal contacts in which cable like actin structures (stress fibres) inserted. In 3D collagen matrices however, cells developed a spindle like (mesenchymal) shape with cylindrical finger-like pseudopods which generated the forward-driving force towards collagen fibres. These pseudopods contained dynamic polymerized actin yet lacked stress fibres. A similar mesenchymal cell shape and structure of the actin cytosceleton that lacked stringent focal contacts and stress fibres developed on 2D fibrilliar collagen matrices. The migration efficiency in 3D collagen was significantly lower, compared to 2D substrata, suggesting an impact of matrix barriers on the migration velocity. Both, actin polymerization and migration were severely impaired by inhibitors of the actin cytoskeleton (Cytochalasin D, Latrunculin B, Jasplakinolide), causing cell rounding and oscillatory “running on the spot”. These findings show the dynamics of the actin cytoskeleton in living melanoma cells critically dependent on and respond to the physical structure of the ECM. 3D collagen matrices hence favour in vivo-like cell shape and cytoskeletal organization while flat cell spreading and formation of stress fibres are specific cell characteristics of cells on 2D.

Skin, more specifically the outermost skin layer, the stratum corneum (SC), forms an extremely effective barrier, preventing both the loss of heat and water, and the ingress of micro-organisms and chemicals. Assessing the rate and extent of drug permeation into or through the skin is important both to evaluate the usefulness of a drug for topical or transdermal delivery, and to compare different formulations to assess their bioequivalence. Prediction of drug permeation is logistically, ethically and economically preferable to in vivo measurements. The recent progress that has been made with empirical and mechanistic mathematical models, along with in vitro diffusion cell techniques has been reviewed. However, currently, in vivo measurements, in man, are still required. For new chemical entities, the need for clinical trials is clear. In the case of generic products, however, there is considerable effort currently being expended to replace expensive, subjective clinical trials with objective, validated measurements of drug permeation, in vivo, in particular to assess bioequivalence. The tape stripping technique has emerged as a promising technique to objectively measure drug permeation through skin, and is the focus of this thesis. After formulation application and removal, layers of SC are sequentially removed by adhesive tapes. As the SC performs the main barrier function of the skin, measuring the rate and extent of drug permeation through this layer is assumed to be related to overall topical bioavailability. The work in this thesis concentrates on performing tape stripping studies such that all tapes are analysed individually, and drug concentration as a function of SC depth is measured. The concentration depth profiles across the SC may be fitted to an appropriate solution of Fick's second law of diffusion to obtain estimates of the vehicle-SC partition coefficient and the drug's diffusivity in the membrane. These dermato-pharmacokinetic parameters may be compared for different formulations.