Orientador: Ana Marisa Fusco Almeida / Coorientador: Paulo Cesar Gomes / Banca: Mario Sergio Palma / Banca: Claudia Vianna Maurer Morelli / Banca: Tais Maria Bauab / Banca: Rosemeire Cristina Linhari Rodrigues Pietro / Resumo: Devido ao aumento das doenças fúngicas, do uso indiscriminado de antimicrobianos, associados ao aparecimento de cepas resistentes e formadoras de biofilmes, é essencial a busca pelo desenvolvimento de novos antifúngicos mais eficientes. As espécies de Candida são conhecidas pela capacidade de infectar humanos, sendo a formação de biofilmes um dos fatores de virulência mais importantes. No estudo interação patógeno-hospedeiro, bem como para eficácia terapêutica e segurança, algumas vantagens no uso de modelos animais alternativos são a redução de tempo de resposta em experimentos, eficiência e baixo custo quando comparado ao uso de camundongos. Desta forma, moléculas antifúngicas estão sendo testadas nestes animais alternativos, como Danio rerio (Zebrafish), um vertebrado que apresenta rápida reprodução e homologia genética e funcional com os mamíferos. Em relação às moléculas em testes, há um crescente estudo de peptídeos oriundos de venenos e da hemolinfa de insetos, como novos compostos antifúngicos, os quais possam apresentar além de boa atividade, baixa toxicidade e não-teratogenicidade. Neste trabalho, foram estudados peptídeos análogos de Mastoparanos de vespas (MKs) cuja atividade antimicrobiana já é conhecida, previamente sintetizados com modificações nas suas características estruturais, como número de resíduos de Lisina e carga líquida. Levando em consideração as características estruturais dos MKs, outros quatro peptídeos foram sintetizados e estudados, dos quais d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the increase of fungal diseases, indiscriminate use of antimicrobial linked to occurrence of resistance and biofilm - forming strains, the search for the development of new efficient antifungals is essential. Candida species are known for their ability to infect humans, and biofilm formation is one of the most important virulence factors. In respect pathogen - host interaction, as well as for therapeutic efficacy and safety, some advantages in the use of alternative animal models are the reduction of response time in experiments, efficiency and low cost when compared to the use of mice. Thus, antifungal molecules are being tested in these alternative animals, such as Danio rerio (Zebrafish), a vertebrate that exhibits rapid reproduction and genetic and functional homology with mammals. Regarding molecules under test, there is a growing study of peptides from poisons and insect hemolymph, as new antifungal compounds, which may present aside from to good activity, low toxicity and non-teratogenicity. In this work, it was studied was mastoparan analog peptides (MKs), whose antimicrobial activity is already known, previously synthesized with modifications in their structural characteristics, such as Lysine residues number and net charge. Taking into account the structural characteristics of MKs, four other peptides were synthesized and studied, of which two peptides (PepM1; PepM2) are analogues of the antimicrobial Moricin C3, derived from Galleria mellonella hemolymph, an... (Complete abstract click electronic access below) / Doutor

Low water pH is an ionoregulatory challenge to freshwater teleosts. Larval zebrafish (Danio rerio) exposed to pH 4 water experience increased loss of Na⁺ and respond with increases in ionocyte abundance and whole-body concentrations of cortisol. Because cortisol plays a role in regulating early development, particularly of the stress axis, the present study asked whether the increase in cortisol in embryos exposed to pH 4 water causes dysregulation of the stress axis in later life. Baseline whole-body cortisol levels measured at 4, 6, and 15 days post-fertilization (dpf) did not differ between pH 4-exposed and control fish. At 6 dpf, pH 4-exposed fish had higher concentrations of cortisol compared to control fish following a stressor, but no difference was detected at 15 dpf. In addition, transcript abundances for key genes of the stress axis did not differ between control and pH 4-exposed fish. Based on these results, exposure to pH 4 water in early life does not influence the stress axis or cortisol responses later in life. Increases in ionocyte abundance in response to low pH have the potential to alter gill morphology, thereby impairing gas transfer, a trade-off known as the osmorespiratory compromise. The present study tested the hypothesis that zebrafish reared in pH 4 water have reduced gas transfer capacity in accordance with the osmorespiratory compromise. Indicators of gas transfer and ionoregulation were measured at 6, 15, 30 and 90 dpf. Across all ages examined, fish reared in pH 4 water had significantly higher whole-body concentrations of Na⁺, higher ionocyte abundances and thicker gills than control fish. These differences were accompanied by higher ventilation frequencies and higher critical PO₂ (Pcrit) values. Additionally, adult fish raised in low pH had a significantly higher rate of oxygen consumption compared to control fish. These results support the hypothesis that development in water of low pH impairs gas transfer, as predicted by the osmorespiratory compromise.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, featuring motor signs such as tremors, bradykinesia, and impaired gait that are often preceded by nonmotor symptoms such as anxiety/depression and olfactory dysfunction. Interestingly, significant olfactory loss was found to be manifested in the majority of PD patients and may precede motor symptoms by years, and thus can be used for the risk assessment of developing PD in asymptomatic individuals. The main pathological feature of PD is the progressive and irreversible loss of dopaminergic (DA) neurons in the substantia nigra pars compacta of the midbrain. Although the detailed etiology of PD remains unclear, most PD cases were found to be sporadic and can be associated with environmental factors. Only 5–10% of patients result from familial PD. With considerable effort in the past two decades, a number of genes associated with familial PD have been identified and interestingly, many of these genes are involved in regulating and maintaining mitochondrial function. The presenilin-associated rhomboid-like (PARL) gene was found to contribute to mitochondrial morphology and function and was linked to familial Parkinson’s disease (PD). The PARL gene product is a mitochondrial intramembrane cleaving protease that acts on a number of mitochondrial proteins involved in mitochondrial morphology, apoptosis, and mitophagy. To date, functional and genetic studies of PARL have been mainly performed in mammals. However, little is known about PARL function and its role in dopaminergic (DA) neuron development in vertebrates. The zebrafish genome comprises two PARL paralogs: parla and parlb. Here, we show novel information concerning the role of PARL in zebrafish by establishing a loss-of-function mutation in parla and parlb via CRISPR/Cas9- mediated mutagenesis. We examined DA neuron numbers in the adult brain and expression of genes associated with DA neuron function in larvae and adults. We show that loss of parla function, as well as loss of both parla and parlb function result in loss of DA neurons in the olfactory bulb and telencephalon of adult zebrafish brain. Changes in the levels of tyrosine hydroxylase transcripts supported this neuronal loss. Expression of fis1, a gene involved in mitochondrial fission, was increased in parla mutants and in fish with loss of parla and parlb function. Furthermore, we showed that loss of parla and/or parlb function translates into altered locomotion parameters and that loss of parla but not parlb function results in impaired olfaction. Finally, increased susceptibility to neurotoxin exposure was identified in mutants with loss of both parla and parlb function but not with loss of parla or parlb function. These results suggest an evident role for parla in the development and/or maintenance of DA neuron function in zebrafish and confirm the existence of redundant and non-redundant functions for the two paralogs, parla and parlb.

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A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and realtime PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.

Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.

Genetic changes acquired in the pigment producing cells of the skin, called melanocytes, can lead to formation of the deadly cancer melanoma. Mutations or amplifications leading to the activation of the RAS/MAPK pathway occur in more than 90% of melanomas. Melanocyte development and survival requires the stimulation of this pathway by the receptor tyrosine kinase (RTK) KIT. In ~2% of melanomas, oncogenic KIT mutations drive tumor formation; however, the majority of melanomas lose wild-type KIT expression, suggesting that KIT could suppress melanoma formation. In human melanoma patients of The Cancer Genome Atlas (TCGA), we found an association between BRAFV600E mutations and low KIT mRNA expression, so we tested whether KIT loss would affect BRAFV600E-driven tumor onset by crossing a kit(lf) mutant allele into melanoma-prone Tg(mitfa:BRAFV600E); p53(lf) zebrafish. We observed that kit(lf)-mutant zebrafish experienced accelerated tumor onset and their tumors had increased RAS/MAPK pathway activation. In BRAFV600E-mutant melanoma cells, KIT activity reduced RAS/MAPK signaling by promoting activation of wild-type BRAF (BRAFWT). Furthermore, we found that overexpression of BRAFWT delayed tumor onset in Tg(mitfa:BRAFV600E); p53(lf); mitfa(lf) zebrafish, but had no effect in kit(lf); Tg(mitfa:BRAFV600E); p53(lf); mtifa(lf) zebrafish and a cohort of TCGA BRAFV600E-mutant melanoma patients with high KIT expression and high BRAFWT allele ratios experienced a reduced likelihood of metastasis and extended overall survival. These studies indicate that wild-type KIT acts to suppress melanoma formation through activation of BRAFWT, causing reduced signaling output of BRAFV600E-mutant cells.

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Cette thèse est basée sur deux projets principaux: (1) l'étude de la réponse innée antivirale du poisson zèbre, en particulier des voies de signalisation des interférons de type I et (2) l'étude de leucocytes particuliers localisés au voisinage des neuromastes, structures permettant au poisson de percevoir le flux d'eau qu'il traverse et constituant potentiellement des brèches dans la peau de l'animal. La voie des IFN de type I est le principal composant de l'immunité antivirale innée. Dans cette thèse, deux types de protéines de poisson-zèbre capables d'augmenter l'induction des IFN de type I ont été étudiés. Nous avons montré que les deux orthologues chez le poisson zèbre du facteur de transcription à domaine BTB/POZ nommé PLZF (Promyelocytic leukemia zinc finger) augmentent l'induction de l'Ifn par différents stimuli. Ce travail montre que l'implication de PLZF dans la régulation de la voie IFN est ancienne et peut intervenir à différents niveaux de la voie Ifn. Le second modèle étudié est le gène Ftr83 (finTRIM83), qui appartient à un groupe de TRIM très diversifié et spécifique des poissons. L'expression de cette protéine TRIM induit une très forte induction des Ifn de type I et une protection contre différents virus, via la surexpression de différents ISGs. Ftr83 est exprimé dans la peau et dans les branchies, régions très exposées aux pathogènes, et son niveau d'expression est fortement corrélé au niveau d'expression de l'Ifn. Dans cette thèse, une lignée transgénique où les cellules spécifiquement fluorescentes évoquent des leucocytes localisés à proximité des neuromastes a été étudiée. Ces cellules ont été observées, leurs mouvements suivis et leur transcriptome analysé par séquençage profond après tri au FACS. Cette analyse a identifié des marqueurs typiques de cellules myéloides (macrophages, dendritiques); ces observations sont cohérentes avec l'idée de cellules sentinelles autour des neuromastes. / This thesis is based on the studies of two aspects of innate immunity in zebrafish: 1) proteins involved in the regulation of type I interferon (Ifn) and 2) specialized myeloid cells that patrol neuromasts – mechano-sensory organs embed in the skin that could be pathogen entry sites. In this thesis two different proteins are described for the capability to enhance Ifn production. In one part, two zebrafish orthologues of mammalian transcription factor PLZF (Promyelocytic leukemia zinc finger) are shown to augment type I Ifn and ISG in response to double-stranded RNA viruses. PLZF is a BTB/POZ transcription factor that was recently shown to induce a subset of ISG, in human and mouse. Thus, zebrafish Plzf proteins can operate at multiple steps in the Ifn system. Furthermore, their activity was not dependent on the presence of BTB-domain implying that the underlying mechanism is different from the usual mode of action of BTB/POZ transcription factors. In the second part, fish-specific TRIM ubiquitin ligase - Ftr83 (Fish novel tripartite motif protein 83), mounted a strong anti-viral protection through the upregulation of Ifn. Interestingly a strong correlation between the expression of Ftr83 and Ifn was seen in the gills suggesting that Ftr83 might maintain a low basal level of Ifn signalling in organs constantly exposed to pathogens. In the second part, a GFP reporter transgenic line called medaktin:EGFP has been characterized. It marks leukocytes in the skin surrounding neuromasts. Deep sequencing revealed that these cells express several macrophage and dendritic cell markers, including genes involved in autophagy, microbicidial functions and antigen presentation, thus highlighting them as possible sentinel cells.