One key to understanding how cells integrate and how they respond to diverse stimuli in order to direct a transcriptional response is knowing how a transcription factor may be directed to an appropriate subset of its target genes. One mechanism with which this may be achieved is by modulation of the transcription factor’s post-translational modification status. The microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage, and it is also a lineage addiction gene in melanoma. Low or high levels of MITF expression induce a reversible cell cycle arrest. Invasive behaviour is characteristic of low MITF expression; differentiation a product of high MITF activity; and moderate levels of MITF expression promote proliferation. A major, unaddressed problem is how DNA binding by MITF may be differentially directed such that it regulates either a proliferation-associated or a differentiation-associated gene expression programme appropriate to the cellular microenvironment. This thesis explores the function and regulation of the signalling pathways controlling novel post-translational modifications of MITF. One such modification, in the DNA binding domain of MITF, defines a key switch that controls MITF’s DNA binding affinity and specificity. Moreover, a novel set of MITF target genes are revealed that extend its control beyond pigmentation and cell cycle regulation to implicate MITF as an overall regulator of cell behaviour in the melanocyte lineage.

Bc.Veronika Hrabcová BRAF mutations in metastatic malignant melanoma. Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Healthcare bioanalytics - Specialist in Laboratory Methods Backround: Melanoma is malignant disease with increasing incidency. Treatment of advanced stage of melanoma is still limited. With a progress of knowledge in genetics and tumorigenesis, the incidence of mutated BRAF protein was observed at 50 % of melanomas. In 80-90 % mutated melanomas contain BRAF V600E mutation. The aim of study was to establish a suitable molecular biological method for the diagnosis of mutations in codon V600 BRAF. Methods: Cobas 4800 BRAF V600 mutation test and BRAF StripAssay test were used to analyze DNA. Cobas 4800 BRAF V600 mutation test is based on PCR using TaqMan probes designed for the wild-type and mutant BRAF V600E sequence. BRAF StripAssay test is based on PCR amplification with biotinylated primers and subsequent hybridization of the stripped with allele-specific oligonucleotide probes. Examined DNA samples were derived from 35 patients with advanced malignant melanoma or from archive of laboratory. Results: BRAF V600 mutation was detected in approximately half of the tumors, consistent with the results of other studies. In comparison methods Cobas test...

The study of regulatory T cells (Tregs) is a relatively new field. Within the past few decades, research on Tregs has greatly deepened scientists’ understanding of the link between the immune system and cancer. The study of melanoma is one such cancer that has benefited greatly from this area of study. Tregs are a subset of CD4+ T cells (TCs) that are either generated in the thymus or in the periphery. The main role of Tregs in normal immune physiology is to suppress immune cells. This is an essential component in the prevention of autoimmunity. In melanoma, however, Tregs prevent components of the immune system from mounting a robust response to cancerous lesions and tumors. Tregs have been observed to infiltrate melanoma tumors due to chemokines and other soluble signaling molecules such as CCL1 and CCL22. Once Tregs accumulate inside melanoma tumors, they generate an immunosuppressive microenvironment in a contact-dependent and contact-independent manner. IL-10 secretion and use of the CTLA-4 pathway were observed to be the most well characterized modes of suppression but other mechanisms are still being discovered. Clinicians can take advantage of new therapeutics to modulate the activity of Tregs. Exogenous administration of antibodies that bind to CTLA-4, PD-1, CCR4 and other receptors and molecules can prevent Treg development and action. Preventing Tregs from carrying out their suppressive function may allow other elements of the immune system, such as CD8+ TCs, to target and destroy melanoma cells. Clinicians can also measure the relative abundance of Tregs or use the ratio of effector TCs (Teff) and Tregs to predict patient outcomes and survival. More research is needed to determine that precise mechanisms of Treg infiltration and accumulation within the tumor and the mode of Treg suppression. This paper finds that there is no standard Treg identification marker. This can lead to aberrant results and failures such as the inability to distinguish Tregs from melanoma cells that also express Treg-like markers or a failure to identify other Treg subtypes. Lack of consensus also extends to the prognostic value of Tregs due, in part, to small sample sizes and the inability to accurately identify Tregs in vivo. Future research must focus on Treg identification, action, and the elucidation of therapeutic mechanisms. These future studies will ensure that clinicians have the correct information to choose the proper melanoma treatment that will target the specific Treg populations found within patients’ melanoma tumors.

Doctor en Bioquímica / El cáncer, una de las causas más frecuentes de muerte en el mundo, es un gran desafío para el desarrollo de terapias específicas anti-tumores o de sus metástasis. Ya sea la inmunoterapia anti-tumoral ó la terapia anti-angiogénica, son estrategias con un muy buen potencial en la lucha contra el cáncer. La primera busca activar la respuesta de linfocitos T tumor-específicos, mientras que la segunda bloquea o retarda el crecimiento del tumor al interferir con el abastecimiento adecuado de nutrientes y oxígeno y con la remoción de productos catabólicos tóxicos. La inmunoterapia requiere de la identificación de Antígenos Asociados Tumores (TAA) que se expresen preferencialmente en tejidos con alto nivel proliferativo, como los tumores. Un TAA adecuado para este tipo de terapia debe ser esencial para la mantención de la viabilidad del tumor y ser blanco de linfocitos T citotóxicos. Survivina (Surv), cumple con estos requisitos. Por otra parte, en vertebrados, Calreticulina (CRT), proteína altamente pleiotrópica, además de su participación en procesos de presentación antigénica en células presentadoras de antígeno (APCs), puede translocarse a la membrana externa e interactuar con C1 del complemento. A pesar de la enorme distancia evolutiva entre el Trypanosoma cruzi, agente causal de la enfermedad de Chagas, y los mamíferos, CRT del parásito (TcCRT) comparte una gran cantidad de características estructurales y funcionales con sus homólogos vertebrados. Recientemente, hemos propuesto que el efecto anti-neoplásico asociado a la infección por T. cruzi, se debería, al menos parcialmente, a la capacidad anti-angiogénica de TcCRT, al interactuar con células endoteliales, interfiriendo con su multiplicación, movilidad y capacidad morfogénica capilar. En estas actividades, la proteína parasitaria es más efectiva que su contraparte humana. Por estas razones, nuestra Hipótesis de Trabajo propuso que, la inmunización genética con Surv y la administración concomitante de TcCRT, genera una actividad anti-tumoral mayor que con solo uno de estos productos. Los principales resultados obtenidos señalan que: i) De acuerdo a lo esperado, rTcCRT no afecta la proliferación in vitro de células de un melanoma murino B16-F10, ii) la inmunización genética con pSurv, en conjunto con rTcCRT (ó su dominio R), retrasa el crecimiento del tumor, iii) aumentando la sobrevida de los animales tratados. iv) El tratamiento combinado pSurv-rTcCRT inhibe la angiogénesis tumoral. v) rTcCRT se une a los melanocitos, promueve la incorporación de C1q humano y la fagocitosis macrofágica y, vi) La combinación de la inmunización con pSurv y pSec-Surv, más la inoculación i.v. con rTcCRT, inhibe sinérgicamente la metástasis pulmonar. Es probable que una secuencia de eventos se desencadene primariamente por la actividad anti-angiogénica de TcCRT, en el tumor, generando una respuesta al estrés derivado, siendo translocada CRT de la célula tumoral a la membrana externa, donde captura C1, evento que promueve la fagocitosis de la célula tumoral, por parte de las APCs. La presentación de péptidos derivados de antígenos TAA debiera ocurrir (Surv incluida), con la consiguiente inducción de actividad inmune anti-tumor. Independientemente, Surv podría también mediar actividad inmune anti-células endoteliales. En consecuencia, el retardo en el crecimiento tumoral y aumento en la sobrevida de los animales tratados con pSurv-rTcCRT se debería a la combinación de efectos sobre distintos blancos moleculares, colaborando y potenciando el efecto final. / Cancer, the most frequent cause of death worldwide, is a great challenge for the development of specific anti-tumor and metastasis treatment. Either immune anti-tumor or anti-angiogenic therapies, are potentially promising in the struggle against cancer. Immune therapy seeks to activate the response of antigen-specific T lymphocytes, thus allowing selective elimination of tumor cells, while anti-angiogenesis blocks or delays tumor growth by interfering with nutrient or oxygen supply and with the removal of catabolic toxic products. Immune therapy for cancer treatment requires the identification of Tumor Associated Antigens (TAA), specifically expressed in tissues with high proliferative levels, such as tumors. A TAA useful for this therapy must be essential for tumor liability and also serve as a target for cytotoxic T lymphocytes. Survivin (Surv), fulfills these requirements. On the other hand, in vertebrates, Calreticulin (CRT), a highly pleiotropic protein, besides its participation in antigen presenting cells (APCs), can translocate to the external membrane and interact with complement C1. In spite of the great evolutionary distance between Trypanosoma cruzi, the agent of Chagas´ disease, and mammals, parasite CRT (TcCRT) shares many structural and functional properties with its vertebrate counterparts. Recently, we have proposed that the anti-tumor effect associated to T. cruzi infection is at least partially due to the TcCRT anti-angiogenic capacity. Thus, upon interacting with endothelial cells, TcCRT interferes with their multiplication, mobility and capacity to generate capillaries. In all these activities, the parasite protein is more effective than its human counterpart. For these reasons, the Working Hypothesis proposes that genetic immunization with Surv and concomitant TcCRT administration, generates a greater anti-tumor activity than each of these products, administered separately. The principal results obtained indicate that: i) As expected, while rTcCRT does not affect the in vitro proliferation of B16-F10 tumor cells, ii) genetic immunization with pSurv, together with rTcCRT (or its R domain), slows the growth of the B16-F10 murine melanoma iii) increasing survival of treated animal. iv) pSurv-rTcCRT treatment inhibits tumor angiogenesis. v) rTcCRT binds to B16-F10 melanocytes, promotes the incorporation of human C1q and consequent macrophage phagocytosis and, vi) The combination of immunization with pSurv and pSecSurv plus rTcCRT i.v. inoculation, synergistically inhibits lung metastasis. Perhaps, a sequence of events is primarily unleashed by the TcCRT anti-angiogenic activity in the tumor. As a response to derived stress, tumor cell CRT is translocated to the external membrane, where it captures C1, an event that promotes tumor phagocytosis by APCs. TAA-derived antigen presentation (Surv included) should follow, with the induction of anti-tumor immune activity. Independently, Surv could also mediate anti-endothelial cell immune activity. Consequently, the slower tumor growth and increased survival of the animals treated with pSurv-rTcCRT is due to the combined effects on different molecular targets, collaborating and enhancing the overall effect. / Fondap

Non-melanoma skin (NMSC) cancer is the most common human malignancy. Cutaneous squamous cell carcinoma (cSCC) and its precursor, actinic keratosis (AK) affect tens of thousands of people each year in the UK. Merkel cell carcinoma is a rare, yet aggressive type of NMSC recently linked with Merkel Cell Polyomavirus (MCPyV). In spite of the clinical burden of NMSC, key molecular regulatory patterns remain largely unknown. The aims of this thesis were to investigate genome-wide genetic, epigenetic and transcriptional changes in AK and cSCC, and assess the prevalence of MCPyV and its effect on methylation in NMSC. Copy-number analysis revealed that AK harbours significantly more genomic aberrations compared to skin, the majority of which occurs on chromosomes 8 and 9. Transcriptional profiling has found 292 and 308 genes as differentially expressed in AK compared to non-sunexposed and sun-exposed skin, respectively, and gene-set enrichment analysis (GSEA) revealed dysregulation of PPAR pathway in this lesion. Expression profiling of cSCC and AK has revealed 346 differentially expressed genes, and GSEA detected dysregulation in several canonical pathways including TGF-β and MAPK pathway. Aberrant methylation in cSCC cell lines occurs in the promoters of many developmental genes. A total of 1085 hyper- and 833 hypomethylated genes were detected in cSCCs, and GSEA revealed dysregulation of critical signalling pathways (WNT, MAPK signalling pathways). Methylation analysis of AK revealed a total of 4194 differentially methylated genes, and implicated FOXF2, PITX2, RUNX1 and SMAD3 transcription factors in this lesions. MiRNA profiling of cSCC and normal skin revealed significant dysregulation of 38 miRNAs including several of viral origin. MCPyV was shown to be common in NMSC, yet MCPyV nor human papillomavirus does not affect cSCC methylation. Taken together, this work provides novel insight into molecular regulation of cSCC oncogenesis, and identifies potential epigenetic targets for functional evaluation in this malignancy.

Melanoma is one of the most common cancers in the world. For primary melanoma, early diagnosis and surgical excision are effective treatments but, despite the new targeted therapies and immunotherapies, there is still a need for more effective treatment options to improve overall survival for patients with metastatic melanoma. Chemotherapy with genotoxic agents remains the main approach for most cancers, but DNA repair pathways in cancer cells reduce their effectiveness. So disruption of key DNA repair pathways, such as nucleotide excision repair (NER), could be an effective option to combine with chemotherapy for melanoma. The structure-specific endonuclease ERCC1-XPF, which heterodimerises through the C-terminal helix-hairpin-helix (HhH)2 domains of both proteins, is essential for NER. The aim of my project was to determine the mechanism involved in regulating the level of the ERCC1-XPF heterodimer with a view to disrupting NER activity. The project started by determining the ERCC1 and XPF response in six melanoma cell lines to the chemotherapeutic cisplatin at the mRNA and protein levels. Although the mRNA and protein levels of both ERCC1 and XPF increased, there was variable consistency between the cell lines, raising the possibility that post translational modification may play an important role in the regulation of ERCC1- XPF activity. We chose to focus on ubiquitination, because it can affect a protein’s activity at both expression and activation levels and several examples of ubiquitinated DNA repair proteins were known. In the pilot study we found that ERCC1 was accumulated after proteasome inhibitor treatment and decreased by treatment with a translation inhibitor in two melanoma cell lines, suggesting that ERCC1 may be ubiquitinated. By cotransfecting His-tagged ubiquitin and non-tagged ERCC1 constructs into melanoma cells and performing an ubiquitin assay, we found that ERCC1 was degraded by the proteasome system through polyubiquitination or multiple monoubiquitination. To determine the nature of the ubiquitination type, we mutated each of the seven Lys residues on ubiquitin and carried out additional assays with ubiquitin single and combination mutants, and discovered that Lys33 was most likely involved in the proteasome dependent degradation of ERCC1. By immunoprecipitation with an antibody to linear ubiquitin from melanoma cell extracts containing a ubiquitin construct with all seven Lys residues mutated to Arg, we found that the N-Met of ubiquitin was also most likely involved in ERCC1 ubiquitination. To determine which Lys of ERCC1 is used by ubiquitin, we did another series of in vivo ubiquitin assays with full length and truncated ERCC1 constructs and found that the key amino acid is most likely within the C-terminal XPF binding domain of ERCC1. By cotransfecting the full length ERCC1 and ERCC1 truncation constructs together with full length XPF, we showed that the ubiquitination of ERCC1 was not an artefact resulting from overexpression of ERCC1 alone and that the stability of XPF was dependent on the overexpression and stability of ERCC1. We then made single lysine and lysine combination mutants in the XPF binding domain of ERCC1 and found that none of the lysines were essential for ubiquitination of ERCC1, indicating that a non- lysine amino acid might be used for ubiquitination. However, using a transfection-based NER assay in ERCC1-deficient cells, we found that ubiquitination of Lys 295 could be involved in regulating the DNA repair activity of ERCC1-XPF. The in vivo ubiquitin assay result after cotransfection of ERCC1 and XPF, which showed that XPF was dependent on the presence of ERCC1 for stability, but not vice versa, was inconsistent with previous published data suggesting that heterodimerization was essential for the stability of both proteins. Instead we hypothesised that homodimerization of ERCC1 might be another mechanism to keep ERCC1 stable and obtained evidence for this at the overexpression level by immunoprecipitation following cotransfection of Myc-tagged ERCC1 and Flag-tagged ERCC1 or ERCC1 truncations, which was supported at the endogenous expression level by size exclusion chromatography on melanoma cell extracts to identify ERCC1 in different molecular weight fractions. In the previous in vivo ubiquitin assay, we found that levels of transfected full length ERCC1 and XPF were dramatically reduced by cotransfection with the Flag-tagged ERCC1 (220-297) construct that just contains the XPF binding domain of ERCC1. This led to another hypothesis, that the ERCC1 (220-297) peptide can decrease endogenous levels of ERCC1 and XPF and so be a potential drug in combination with cisplatin chemotherapy. This hypothesis was verified in stable transgenic cell lines expressing ERCC1 (220-297) which showed reduced levels of ERCC1 and XPF and of NER and increased sensitivity to cisplatin and UV irradiation. Based on the above results and supporting bioinformatics analysis we have made the following conclusions: ERCC1 is regulated by the ubiquitin-proteasome degradation pathway through linkages most likely involving Lys33 and N-Met; the XPF binding domain is most likely the key domain for ERCC1 ubiquitination; XPF stability is dependent on the presence of ERCC1 and seems affected by ERCC1 ubiquitination; ERCC1 seems to be ubiquitinated in a non-conventional lysine-independent manner and ubiquitination of Lys 295 might be involved in the regulation of the DNA repair activity of ERCC1- XPF; homodimerization is most likely a novel mechanism to keep ERCC1 stable; the ERCC1 (220-297) peptide can destabilise both ERCC1 and XPF and could be a potential drug in combination with genotoxic therapies.

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS (tede2@pucrs.br) on 2015-08-27T11:35:28Z No. of bitstreams: 1 474248 - Texto Completo.pdf: 1288519 bytes, checksum: 76a0f632ccb7c577dfc183ba0e91a44b (MD5) / Made available in DSpace on 2015-08-27T11:35:28Z (GMT). No. of bitstreams: 1 474248 - Texto Completo.pdf: 1288519 bytes, checksum: 76a0f632ccb7c577dfc183ba0e91a44b (MD5) Previous issue date: 2015-07-10 / Objectives: To evaluate the characteristics of cell nuclei of melanocytes using digital nuclear morphometric analysis. To evaluate the nuclear morphometric differences between nevi excised in winter or summer; demonstrating the presence of characteristics and irregularities in nuclear morphology caused by solar radiation on nevi. To consider whether there are morphological differences between common acquired nevi, dysplastic nevi and melanomas. To access the potential use of nuclear morphometry to detect cell senescence, apoptosis and other nuclear irregularities of nevi and melanomas in HE stained slides of paraffin embedded specimens. Methodology: Histopathological specimens were subjected to digital image analysis. Results: Through this pilot study, glass slides obtained from biopsies of 30 patients were analyzed; 10 nevi excised in winter, 5 nevi excised in summer, 9 dysplastic nevi and 6 melanomas. Between winter and summer nevi; secondary variables able to perform such a separation were 'variability Area' and % of nuclei with NII higher than 1SD. Conclusion: There are differences of nuclear morphometric characteristics of excised nevi in winter and summer and can be caused by cumulative effects of solar radiation on nevi. There are morphometric differences between common acquired nevi, dysplastic nevi and melanomas. The dysplastic nevi and melanomas were separated by the average area and percentage of nuclei with area above one standard deviation of the mean area. / Objetivos: Avaliar as caracter?sticas dos n?cleos celulares de melan?citos atrav?s de an?lise morfom?trica nuclear digital e as diferen?as morfom?tricas nucleares entre nevos excisados no inverno e nevos excisados ap?s o ver?o; para detectar poss?veis caracter?sticas e irregularidades na morfometria nuclear provocadas pela radia??o solar sobre nevos. Averiguar a possibilidade de identifica??o de diferen?as morfom?tricas entre nevos adquiridos comuns, nevos displ?sicos e melanomas e avaliar a possibilidade de detec??o de senesc?ncia, apoptose e outras irregularidades nucleares em nevos e melanomas por morfometria digital em cortes histol?gicos corado por HE a partir de blocos de parafina. Metodologia: Os esp?cimes histopatol?gicos foram submetidos ? an?lise de imagem digital realizada atrav?s de ferramentas de avalia??o nuclear digital. Resultados: Neste estudo piloto foram analisados os esp?cimes de bi?psia de 30 pacientes, compostos por: 10 nevos biopsiados no inverno, 5 nevos no ver?o, 9 nevos displ?sicos e 6 melanomas malignos. Nos nevos de inverno e nevos de ver?o; as vari?veis capazes de realizar tal separa??o foram ?Variabilidade da ?rea? e ?porcentagem de n?cleos com NII acima do valor da m?dia+1DP. Conclus?o: Foram detectadas diferen?as morfom?tricas nucleares entre nevos excisados no inverno e nevos excisados ap?s o ver?o, que podem ser provocadas pelos efeitos cumulativos da radia??o solar sobre nevos. Foram catacterizadas diferen?as morfom?tricas entre nevos adquiridos comuns, nevos displ?sicos e melanomas. Os nevos displ?sicos e melanomas foram separados pelas caracter?sticas de ?rea m?dia e porcentagem de n?cleos com ?rea acima de um desvio padr?o da m?dia.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2015. / A tese de doutorado intitulada “Avaliação dos efeitos citotóxicos e antiproliferativos da secreção cutânea e de peptídeos bioativos do anuro Physalaemus nattereri (steindachner, 1863)” desenvolvida por Andréa Cruz e Carvalho sob a orientação da profa. Dra. Mariana S. Castro teve o apoio financeiro do CNPq (processos n°. 563972/2010-6, Edital MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/PRÓ-CENTROOESTE nº 31/2010, n°. 302925/2012-0, Produtividade em Pesquisa - PQ – 2012 e n°. 407801-2013, Rede Centro-Oeste De Pós-Graduação , Pesquisa E Inovação - REDE PRÓ-CENTRO-OESTE), da FAPDF (processo n°. 193. 000.461/2011, Edital MCT/CNPQ/FNDCT/FAPS/MEC/CAPES/PRÓ-CENTRO-OESTE nº 31/2010), do CNPq por meio da concessão de bolsa de estudos (nível doutorado), da FINEP (CT-INFRA) E DA FUB-UNB. / As secreções cutâneas de anuros são fontes naturais de peptídeos antimicrobianos. A realização de novas pesquisas nessa área tem revelado que alguns peptídeos com atividade antimicrobiana também apresentam efeitos tóxicos sobre células tumorais. O objetivo do presente estudo foi investigar o potencial terapêutico da secreção cutânea de Physalaemus nattereri sobre células tumorais e determinar os mecanismos da ação citotóxica sobre céluluas de melanoma murino da linhagem B16F10. Nossos resultados demonstraram que a secreção bruta reduz a viabilidade de células B16F10, causando alterações na morfologia celular, (p.ex., arredondamento e encolhimento), ruptura da membrana plasmática, redução do potencial de membrana mitocondrial e parada do ciclo celular na fase S; tais mudanças em conjunto sugerem que as células tumorais morreram por apoptose. A secreção cutânea também foi submetida ao fracionamento cromatográfico empregando RP-HPLC e as frações eluídas foram avaliadas quanto à suas atividades antiproliferativas e antimicrobiana. Três frações ativas exibiram componentes de massa molecular compatíveis com peptídeos. Os três componentes majoritários presentes em tais frações foram purificados por RP-HPLC e analisados por MALDI-TOF/MS. Tais peptídeos mostraram-se ativos sobre bactérias e células tumorasi e foram identificados como pertencentes ao grupo das nattererinas, peptídeos antibacterianos previamente isolados desse animal por nosso grupo. Embora, o mecanismo específico envovlido na redução da viabilidade celular e citotoxicidade após o tratamento com essa secreção bruta ainda não seja conhecido, podemos supor que a atividade de moléculas, como peptídeos, presentes nessa secreção seja efetivos contra as células de melanoma murino (B16F10). Considerando-se a necessidade crescente por novas drogas anticâncer, o presente estudo fortemente reforça a validade do uso da secreção cutânea de P. nattereri como uma rica fonte de novas moléculas anticâncer, principalmente peptídeos. / Anuran secretions are rich natural sources of antimicrobial peptides. The emergence of new research in this area has shown that some peptides with antimicrobial activity also exert toxic effects against cancer cells. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against cancer cells, and to verify the mechanisms of its cytotoxic action on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduces the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), rupture of the plasma membrane, reduction in mitochondrial membrane potential, fragmentation of DNA, and cell cycle arrest in Sphase; together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also submitted to chromatographic fractionation using RP-HPLC and the eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular masses components with a molecular range compatible to peptides. Thes major petidic components present on these fractions were purified by RP-HPLC and analysed by MALDI-TOF/MS. These peptides were active against bacteria and cancel cells, and were identified as nattererins, antibacterial peptides previously isolated from this animal by our group. Although, the specific mechanism causing the reduction in cell viability and cytotoxicity after treatment with the crude secretion is still unknown, we can consider that the activity of molecules, such as peptides, within the secretion is effective against murine melanoma (B16F10) tumor cells. Considering the growing need for new anticancer drugs, the present data strongly reinforce the validity of crude secretion of P. nattereri as a rich source of new anticancer molecules, mainly peptides.

Le mélanome est un cancer qui représente la grande majorité de la morbidité et de la mortalité causées par tout cancer de la peau en raison de son implication dans les métastases. Parmi plusieurs biomarqueurs rapportés, les récepteurs du domaine de la discoïdine (DDR) et la métalloprotéinase Matrix (MMP) sont également considérés comme des acteurs potentiels du mélanome. Des études récentes démontrent que les DDR et les MMP sont surexprimés dans les mélanomes de mauvais pronostic, ce qui nécessite le recours à des sondes théranostiques à biomarqueurs spécifiques. De nombreux progrès ont été réalisés pour réduire le risque associé au mélanome métastatique à l'aide d'anticorps monoclonaux traditionnels, mais les inconvénients tels que la complexité de la production, la variabilité d'un lot à l'autre, la courte durée de conservation, l'immunogénicité, la réactivité croisée et le coût élevé ne peuvent être négligés. Par conséquent, au cours des dernières années, les aptamères ont acquis un grand intérêt comme substitut d’anticorps pouvant être synthétisés chimiquement avec un faible coût de production et dépassant les limitations associées aux anticorps. Les aptamères sont de courts oligonucléotides à haute affinité et spécificité vis-à-vis de la molécule cible, développés par une méthode combinatoire appelée SELEX (Evolution systématique de ligands par enrichissement exponentiel). Au cours de ma thèse, nous avons réalisé une étude SELEX contre les biomarqueurs DDR1, DDR2 et HMMP14 sur la base de supports alternatifs pour le tri des candidats et le séquençage à haut débit. Un groupe d'aptamères a été sélectionné contre DDR1 et le meilleur candidat a ensuite été dopé pour effectuer davantage de tours de sélection contre DDR1. Finalement, un aptamère contre DDR1 a été sélectionné avec un Kd dans la gamme nanomolaire basse. Cet aptamère peut être utilisé comme outil théranostique dans les études sur le mélanome associé à DDR1. Comme les aptamères présentent un avantage en termes de conjugaison et de modifications aux positions désirées, nous visons en outre à conjuguer cet aptamère avec des nanoparticules pour des études in vivo. / Melanoma is a cancer that accounts for the vast majority of morbidity and mortality caused by any skin cancer due to its involvement in metastasis. Among several reported biomarkers, Discoidin Domain Receptors (DDRs) and Matrix metalloproteinase (MMPs) are also considered as potential actors in melanoma. Recent studies demonstrate that DDRs and MMPs are overexpressed in melanoma with poor prognosis which demands the need of biomarkers specific theranostics probes. Though many advances has been made in order to reduce the risk associated with metastatic melanoma with the help of traditional monoclonal antibodies, but the drawbacks like production complexity, batch to batch variability, short shelf life, immunogenicity, cross reactivity and high cost cannot be neglected. Therefore in recent years, aptamers have gained high interests as a substitute of antibodies that can be synthesized chemically with low production cost and overcomes the limitations associated to antibodies. Aptamers are short oligonucleotides with high affinity and specificity against its target molecule, raised by a combinatorial method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). During my thesis, we performed a SELEX against DDR1, DDR2 and HMMP14 biomarkers based on alternative supports for candidates sorting and high throughput sequencing. A group of aptamers were selected against DDR1 and the best candidate was further doped to perform more rounds of selection against DDR1. Finally an aptamer against DDR1 was selected with a Kd in low nano-molar range. This aptamer can be used as a theranostics tool in DDR1 associated melanoma studies. As aptamers have an advantage of conjugation and modifications at desired positions so we further aim to conjugate this aptamer with nanoparticles for In-vivo studies.

During development, neural crest (NC) cells arise from the neural plate border and are further differentiated into multiple cell types, including melanocytes. Each step of this process is controlled by a gene regulatory network (GRN), and disruption of the GRN governing melanocyte differentiation contributes to the pathogenesis of pigmentation disorders and melanoma. While many of the factors within this network have been well studied, the role of Transcription Factor Activating Enhancer-Binding Protein 2 (TFAP2) paralogs has been unclear. TFAP2A and TFAP2C are required for NC induction. Later, TFAP2A is also expressed in melanocytes, and TFAP2A mutations cause pigmentation phenotypes in humans, mice, and zebrafish. Other paralogs with high homology to TFAP2A, particularly TFAP2B in mouse and Tfap2e in zebrafish, also function redundantly with TFAP2A in the melanocyte lineage. Here, we have used ChIP-seq and expression profiling to identify direct transcriptional targets of TFAP2A in melanocytes, which include genes involved in melanin synthesis and melanosome biology. Furthermore, we show that TFAP2A directly regulates many of the same genes as Microphthalmia-associated Transcription Factor (MITF), the “master regulator” of the melanocyte lineage. MITF activity has been described as a rheostat in melanoma, with high levels promoting differentiation and lower levels promoting invasiveness. The overlap between TFAP2A and MITF transcriptional targets in melanocytes suggests that TFAP2A may influence the MITF rheostat, driving it toward the differentiated state. To study the role of other TFAP2 paralogs in NC and melanocytes, we generated zebrafish lines that are double and triple mutant for tfap2a, tfap2c, and tfap2e and confirm genetic compensation among these paralogs. We also demonstrate that melanocyte-specific inhibition of Tfap2 activity by Kctd15 affects differentiation and that Kctd15 may participate in a negative feedback loop regulating Tfap2 expression. In support of a pro-differentiation role for TFAP2A in melanocytes, we show that overexpression of tfap2a in a zebrafish melanoma model significantly delays tumor formation. Together these results indicate that, in addition to its earlier roles in the NC, TFAP2A acts within the melanocyte GRN to directly regulate differentiation genes in parallel with MITF. This, combined with the tumor-suppressor function of TFAP2A in melanoma, implicates TFAP2A and the factors that regulate it as potential targets for melanoma therapies.