Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...

Machine learning methods provide an opportunity to improve the classification of skin lesions and the early diagnosis of melanoma by providing decision support for general practitioners. So far most studies have been looking at the creation of features that best indicate melanoma. Representation learning methods such as neural networks have outperformed hand-crafted features in many areas. This work aims to evaluate the performance of convolutional neural networks in relation to earlier machine learning algorithms and expert diagnosis. In this work, convolutional neural networks were trained on datasets of dermoscopy images using weights initialized from a random distribution, a network trained on the ImageNet dataset and a network trained on Dermnet, a skin disease atlas.  The ensemble sum prediction of the networks achieved an accuracy of 89.3% with a sensitivity of 77.1% and a specificity of 93.0% when based on the weights learned from the ImageNet dataset and the Dermnet skin disease atlas and trained on non-polarized light dermoscopy images.  The results from the different networks trained on little or no prior data confirms the idea that certain features are transferable between different data. Similar classification accuracies to that of the highest scoring network are achieved by expert dermatologists and slightly higher results are achieved by referenced hand-crafted classifiers.  The trained networks are found to be comparable to practicing dermatologists and state-of-the-art machine learning methods in binary classification accuracy, benign – melanoma, with only little pre-processing and tuning.

La voie de signalisation Ras/MAPK (Ras/mitogen-activated protein kinase) régule une variété de protéines intracellulaires qui jouent un rôle important dans la croissance et la prolifération cellulaire. La régulation inappropriée de cette voie de signalisation conduit au développement de nombreux cancers comme le mélanome, qui est caractérisé par des mutations activatrices au niveau des gènes NRAS et BRAF. La protéine kinase RSK (p90 ribosomal S6 kinase) est un composant central de la voie Ras/MAPK, mais son rôle dans la croissance et la prolifération cellulaire n’est pas bien compris. RSK a été montrée pour participer à la résistance des mélanomes aux chimiothérapies, mais le mécanisme moléculaire reste encore à élucider. Nous montrons à l’aide d’un anticorps phospho-spécifique que MDM2 est phosphorylée en réponse à des agonistes et des mutations oncogéniques activant spécifiquement la voie Ras/MAPK. En utilisant des méthodes in vitro et in vivo, nous avons constaté que RSK phosphoryle directement MDM2 sur les Sérines 166 et 186, ce qui suggère que MDM2 est un substrat de RSK. La mutagénèse dirigée envers ces sites nous indique que ces résidus régulent l’ubiquitination de MDM2, suggérant que RSK régule la stabilité de MDM2 et de p53. De plus, nous avons observé que l’inhibition de RSK conduit à une augmentation du niveau protéique de p53 après un dommage à l’ADN dans les cellules de mélanomes. En conclusion, nos travaux suggèrent un rôle important de la protéine kinase RSK dans la régulation de MDM2 et de sa cible, p53. L’étude de ces mécanismes moléculaires aidera à mieux définir le rôle de RSK dans la croissance tumorale, mais également dans la résistance aux agents chimiothérapeutiques. / The Ras/mitogen-activated protein kinase (Ras/MAPK) signaling cascade regulates various intracellular targets involved in growth and proliferation. Inappropriate regulation of this pathway leads to many types of cancer, including melanomas, which are characterized by activating mutations in NRAS and BRAF. The protein kinase RSK (p90 ribosomal S6 kinase) is a central component of the Ras/MAPK pathway, but its role in cell growth and proliferation is not well understood. RSK has also been shown to participate in the resistance of melanoma cells to chemotherapy, but the mechanisms involved remain elusive. We show that MDM2 becomes phosphorylated in response to agonists and oncogenes of the Ras/MAPK pathway. Using in vitro and in vivo approaches, we found that RSK directly phosphorylates MDM2 at Ser166 and Ser186, suggesting that MDM2 is a bona fide RSK substrate. Site-directed mutagenesis indicated that these residues regulate MDM2 ubiquitination, suggesting that RSK regulates p53 function in an MDM2-dependent manner. Overexpression of active and inactive mutants of RSK revealed that this kinase regulates p53 stability, suggesting a role for RSK in the DNA damage response. Taken together, our results suggest an important role for RSK in the regulation of MDM2 and its target p53. In view of the role of p53 in the response to DNA-damaging agents, our results provide a potential mechanism involved in melanoma chemoresistance.

52 7.2 Souhrn v anglickém jazyce Malignant melanoma is the most lethal skin cancer whose incidence has been worldwide increasing. The prognosis of the disease is dependent on many factors - the effected locality, age, form of the tumor and the growth stage at which it was surgically removed. There has been no reliable adjuvant treatment available so far; although cytokins have been recently administered to high-risk patients. The accent is therefore put mainly on the timely detection and the relevant extent of surgical intervention whose degree depends on the stage of the disease. The result of the above mentioned facts is the necessity of an early diagnosis of new cases and permanent follow-up of the patients in whom melanoma has been already confirmed. The patients in the follow-up care are regularly checked, for the rest of their lives, at by the so called melanoma commissions, usually at dermatological clinics. The permanent contact with fact of the possible presence of the disease (information about the diagnosis, repeated follow-ups, waiting for the results of the examination) is stressful for these patients, although they become, to a certain extent, gradually adapted to stress. A good adaptation is however dependent also on the extent of social support, depression, the severity of the disease and...

No description available.

The reduction of intentional exposure to ultraviolet (UV) radiation is an important area of skin cancer prevention. Hillhouse et al. (Cancer 113:3257-3266, 2008) have developed an appearance-focused intervention with evidence of efficacy in lowering indoor tanning UV exposure in young women. In the current study, a subgroup approach was used to determine moderators of intervention efficacy. Undergraduate females in two regions of the United States (n = 362) were randomized into an intervention or control condition. Latent profile analysis was used to identify subgroups of indoor tanners based on patterns of indoor tanning motives. Intervention efficacy was examined within each subgroup. We found evidence for 4 subgroups of tanners: knowledgeable-appearance tanners, low-knowledge tanners, low-knowledge, relaxation tanners, and knowledgeable, low-appearance and lowrelaxation tanners. The intervention significantly reduced indoor tanning for the low-knowledge subgroup (34% of the sample). The utility of the subgroup approach in developing targeted behavioral skin cancer interventions is discussed.

Four known small molecule uracil-DNA glycosylase (UNG) inhibitors were synthesized and tested against human melanoma cells, IgR3 and MM200. They were found to be effective against cell proliferation at micromolar concentrations and to operate through a nonapoptotic mechanism. Thus, small molecules that target UNG may be useful as potential chemotherapeutic agents against human melanoma.

Given the prevalence and risk associated with indoor tanning among college students, university campuses constitute a prime target for skin cancer prevention. This report identifies the successes and challenges faced in promoting a campus-wide tan-free policy through the National Council on Skin Cancer Prevention (NCSCP) Indoor Tan-Free Skin Smart Campus Initiative. Beginning in February 2016, we communicated with university faculty or staff members who have participated in skin cancer prevention via education, clinical care, or research at 20 universities regarding the steps to adopt the tan-free policy. One campus, East Tennessee State University (ETSU), successfully fulfilled all criteria and implemented the policy change to become the first US Indoor Tan-Free Skin Smart Campus. The greatest challenge faced in recruiting campuses was gaining administrative support. Reported reasons for not adopting the policy change included wanting to wait for other schools to join first and not seeing it as a top priority. Despite the importance of improving skin cancer awareness and decreasing tanning among university students, we faced several challenges in promoting campus-wide policy change. We identify a need for research on effective ways to disseminate university health policies and increased involvement of healthcare providers in policy-related work.

Indiana University-Purdue University Indianapolis (IUPUI) / In the age of big data, better use of large, real-world datasets is needed, especially ultra-large databases that leverage health information exchange (HIE) systems to gather data from multiple sources. Promising as this process is, there have been challenges analyzing big data in healthcare due to big data attributes, mainly regarding volume, variety, and velocity. Thus, these health data require not only computational approaches but also context-based controls.In this research, we systematically examined associations among select neural and dermal conditions in an ultra-large healthcare database derived from an HIE, in which computational approaches with epidemiological measures were used. After a systematic cleaning, a binary logistic model-based methodology was used to search for associations, controlling for race and gender. Age groups were chosen using an algorithm to find the highest incidence rates for each condition pair. A binomial test was conducted to check for significant temporal direction among conditions to infer cause and effect. Gene-disease association data were used to evaluate the association among the conditions and assess the shared genetic background. The results were adjusted for multiple testing, and network graphs of significant associations were created. Findings among methodologies were compared to each other and with prior studies in the literature. In the results, seemingly distant neural and dermal conditions had an extensive number of associations. Controlling for race and gender tightened these associations, especially for racial factors affecting dermal conditions, like melanoma, and gender differences on conditions like migraine. Temporal and gene associations helped explain some of the results, but not all. Network visualizations summarized results, highlighting central conditions and stronger associations. Healthcare data are confounded by many factors that hide associations of interest. Triangulating associations with separate analyses helped with the interpretation of results. There are still numerous confounders in these data that bias associations. Aside from what is known, our approach with limited variables may inform hypothesis generation. Using additional variables with controlled-computational methods that require minimal external input may provide results that can guide healthcare, health policy, and further research.

During regeneration, cells must coordinate proliferation and differentiation to rebuild tissues that are lost. Understanding how source cells execute the regeneration process has been a longstanding goal in regenerative biology with implications in wound healing and cell replacement therapies. Melanocytes are pigment-producing cells in the skin of vertebrates that can be lost during hair graying, injury and disease-related depigmentation. Melanoma is an aggressive skin cancer that develops from melanocytes, and it is hypothesized that melanoma cells have properties that are similar to melanocyte stem cells. To gain insight into melanocyte regeneration we set out to identify the source of regeneration melanocytes in adult zebrafish and the path through which progenitor cells reconstitute the pigment pattern. Using targeted cell ablation and single cell lineage-tracing analyses we identified that a majority of regeneration melanocytes arise through direct differentiation of mitfa-expressing progenitor cells. Concurrently, other mitfa-expressing cells divide symmetrically to generate additional mitfa-positive progenitors, thus maintaining regeneration capability. Using reporter assays and drug studies, we found that Wnt signaling gets turned on in progenitor cells during regeneration and Wnt inhibition after melanocyte ablation blocks regeneration. Based on our finding that Wnt signaling is active in differentiated melanocytes but not in the progenitor cells, we explored the role of Wnt signaling in tumor initiation. We found that approximately half of the melanomas are Wnt silent, and overexpression of dkk1b, a negative regulator of canonical Wnt signaling, accelerates melanoma onset. This work defines an unappreciated contribution by direct differentiation in melanocyte regeneration and suggests a broader role for this process in the maintenance of epithelial sheets. This study also identifies a shared pathway between melanocyte progenitors and melanoma cells, which could be applicable to other cancers.