Ulcerative colitis and Crohn’s disease are chronic inflammatory bowel diseases (IBDs) which burden health systems worldwide; available pharmacological therapies are limited and cost-intensive. Use of peroxisome proliferator activated-receptor γ (PPARγ) ligands for IBD treatment, while promising, lacks solid evidences to ensure its efficacy. Annexin A1 (AnxA1), a glucocorticoid-modulated anti-inflammatory protein, plays a key role on IBD control and is a potential biomarker of IBD progression. We here investigated whether effects of pioglitazone, a PPARγ ligand, rely on AnxA1 actions to modulate IBD inflammation. Experimental colitis was evoked by 2% dextran sodium sulfate (DSS) in AnxA1 knockout (AnxA1−/−) or wild type (WT) C57BL/6 mice. Clinical and histological parameters were more severe for AnxA−/− than WT mice, and 10 mg/kg pioglitazone treatment attenuated disease parameters in WT mice only. AnxA1 expression was increased in tissue sections of diseased WT mice, correlating positively with presence of CD68+ macrophages. Metalloproteinase-9 (MMP-9) and inactive 33 kDa AnxA1 levels were increased in the colon of diseased WT mice, which were reduced by pioglitazone treatment. Cytokine secretion, reactive oxygen species generation and MMP-9 expression caused by lipopolysaccharide (LPS) treatment in AnxA1-expressing RAW 264.7 macrophages were reduced by pioglitazone treatment, effects not detected in AnxA1 knockdown macrophages. LPS-mediated increase of AnxA1 cleaving in RAW 264.7 macrophages was also attenuated by pioglitazone treatment. Finally, pioglitazone treatment increased extracellular signal-regulated kinase (ERK) phosphorylation in AnxA1-expressing RAW 264.7 macrophages, but not in AnxA1-knockdown macrophages. Thus, our data highlight AnxA1 as a crucial factor for the therapeutic actions of pioglitazone on IBDs.

The development of products for topical applications requires analyses of their skin effects before they are destined for the market. At present, the ban on animal use in several tests makes the search for in vitro models (such as artificial skin) necessary to characterize the risks involved. In this work, tissue engineering concepts were used to manufacture collagen-free three-dimensional scaffolds for cell growth and proliferation. Two different human skin models—reconstructed human epidermis and full-thickness skin—were developed from electrospun scaffolds using synthetic polymers such as polyethylene terephthalate, polybutylene terephthalate, and nylon 6/6. After the construction of these models, their histology was analyzed by H&E staining and immunohistochemistry. The results revealed a reconstructed epidermal tissue, duly stratified, obtained from the nylon scaffold. In this model, the presence of proteins involved in the epidermis stratification process (cytokeratin 14, cytokeratin 10, involucrin, and loricrin) was confirmed by immunohistochemistry and Western blot analysis. The nylon reconstructed human epidermis model’s applicability was evaluated as a platform to perform irritation and corrosion tests. Our results demonstrated that this model is a promising platform to assess the potential of dermal irritation/corrosion of chemical products.

Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large-scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence-profiled 524 American Joint Committee on Cancer Stage I-III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co-mutated, and an absence of co-occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole-genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 (IRF4) as a melanoma-associated dependency.

I would like to dedicate this thesis to my parents, Robilson and Celia, my brother Yuri, and my husband Bruno, for being my inspiration and always encouraging me to do my best at all times.First, I would like to thank my supervisor Silvya, for giving me the opportunity of concluding my PhD, also for your guidance and support throughout the years. I would also like to thank my co-supervisor Patricia, for all advice and patience when answering my endless questions. I began the CRISPR journey because of you.To everyone in Skin Biology group, for your welcome and project discussions. Special thanks to Silvia Berlanga and Silvia Romano for advice on experimental work. Denisse, my IC Juliana and Daniele, who unfortunately already left the lab, for the laughter and friendship.I would also like to say that I was lucky to have Dave as a mentor/co-supervisor during my sandwich PhD at Sanger. This thesis would not have been possible without Dave´s generosity and I truly appreciate him giving me the opportunity of staying is his laboratory for over a year, despite crazy pandemic situation. Thank you for putting your trust in me. Your kindness and dedication for science is really inspiring.To everybody in Adam´s team for the amazing hospitality at Sanger. I was very fortunate to be surrounded by lovely people such as you all. Thanks to Alastair, Victoria, Manik, and Roy for all bioinformatic analyses and kindness answering my doubts. Thanks to Saskia, Jen, Andrea, and Vicky for making my stay in Cambridge so joyful. Thanks for all encouragement, also for sharing your knowledge and experience with me, especially Vicky for all discussions and many hours spent in cell culture. Also, many thanks to Sofia Chen, who allowed me to be part of her work and for her supervision during experiments. I am also grateful to all my collaborators. Thanks to Mariana Boroni and Natasha Jorge for analyses on TCGA data and David Fraser from Gene Editing team at Sanger, for sequencing analyses. Finally, I would like to thank my big family, my grandparents, aunts, uncles, and cousins, for all celebrated achievements. Especially my mom, dad and my brother for constant love and support. Also, my husband Bruno, for sharing all frustrations, highs, and lows and for always being my inspiration. I wouldn't be the person I am today without you. I respect and admire you all. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001, CAPES-PRINT #88887.372178/2019-00, FAPESP #2017/04926-6, CNPq, and the Wellcome Trust Sanger Institute. "Nossas mentes possuem por natureza um insaciável desejo de saber a verdade" Padre Cícero RESUMO MATSUYAMA, L. S. A. S. Application of CRISPR-Cas9 to interrogate novel gene functions in cutaneous melanoma. 2021. 155 p. Tese (Doutorado em Ciências) -Faculdade