Orientador: Rafael Henrique Nóbrega / Resumo: Dentre os aspectos hormonais que influenciam o desenvolvimento dos vertebrados, pode-se destacar o papel dos hormônios tireoidianos (Hts). Esses hormônios (T4/T3) mediam funções imprescindíveis para a homeostase dos organismos, modulando processos fisiológicos como a regulação do crescimento, metabolismo e reprodução. Pensando nisso o presente trabalho teve como objetivo avaliar os efeitos do hipotireoidismo in vivo e in vitro na função testicular de machos adultos de D. rerio e a relação dos Hts no eixo hipotalâmico-hipofisário. No presente trabalho nós mostramos que in vivo o hipotireoidismo induzido pelo methimazole afetou a espermatogênese de zebrafish levando ao acúmulo de células pré-meióticas, atraso na diferenciação e meiose, reduzindo a quantidade de espermatozoides formados. in vitro foi observado que os efeitos biológicos do hormônio folículo estimulante (Fsh) na função testicular são dependentes dos hormônios tireoidianos, assim como os efeitos estimulatórios da gonadotropina coriônica humana (hCG) e inibitório do hormônio inibidor de gonadotropina (GnIH) necessitam dos Hts. Além disso, a expressão de genes hipotalâmicos e hipofisários foram afetados de modo antagônico quando na ausência ou presença dos Hts. Portanto nossos resultados indicam que os hormônios tireoidianos são essenciais na manutenção da espermatogênese e do eixo hipotalâmico-hipofisário em adultos de zebrafish. / Mestre

Genus Danio includes zebrafish (Danio rerio) and several other phenotypically diverse species. To understand the history of these species and how they acquired the genetic differences underlying their diverse phenotypes, I performed two phylogenomic studies using Restriction-Site Associated DNA Sequencing and DNA hybridization-based exome enrichment. The results of these studies highlight important methodological considerations applicable to future experiments across taxa. Furthermore, these studies provide detailed understanding of the relationships within Danio including extensive introgression between lineages. The extent of introgression varies across the genome with regions of high recombination at the ends of chromosomes having the most evidence for introgression. Together, this work gives vital insight into the history of a model organism and the evolutionary processes that give rise to phenotypic diversity.

Tobacco addiction is a leading preventable cause of death worldwide and places a heavy social and financial burden on society. There exists a substantial genetic variability in smoking behavior, the mechanisms of which are largely unknown. Despite significant advances in sequencing power, progress in the identification of genetic variants affecting smoking behavior based on human genome wide association studies has been slow. Thus this thesis investigates the utility of zebrafish as a model species in which to search for genetic variants affecting nicotine seeking. The work is based on the premise that as zebrafish are vertebrate with conserved neurochemical pathways and circuitry with humans, and the pathways involved in drug mediated reward and addiction are evolutionarily ancient, homologues of genes affecting zebrafish nicotine-seeking behavior will likely affect human smoking behavior. Thus results in zebrafish can be used to direct human genetic studies. The first result chapter addresses the hypothesis that zebrafish show conserved reward responses to common drugs of abuse. A conditioned place preference assay is used to assess zebrafish reward responses to stimulants, opioids, benzodiazepines and alcohol. The results indicate that, with the exception of benzodiazepines, reward responses are conserved, supporting the use of this model in a screen for genetic variants affecting nicotine preference. The second and third results chapters describe the findings of a pilot screen of ENU-mutagenized zebrafish provided by the Sanger Institute, Cambridge. I demonstrate that nicotine preference is heritable in fish as in Abstract 5 humans and identify 3 mutant lines that show increased or decreased nicotine place preference. Genotyping indicated that one of the families showing increased nicotine preference carries a predicted loss of function mutation in the slit3 gene. The involvement of this gene in nicotine preference was confirmed in a separate line. Further characterization of this line using qPCR showed slit3 mutants to have altered developmental expression of key nicotinic and dopaminergic genes. Having identified the slit3 gene as a locus affecting nicotine seeking in fish, I then tested the hypothesis that results in fish could be used to predict loci that affect human smoking behavior. Cohorts of patients were genotyped for 20 SNPs within the slit3 locus. Results of this analysis identified 1 novel SNP in the slit3 gene associated with smoking behavior in a cohort of individuals that were heavy smokers. This result was validated in cohorts of low and normal smoking prevalence. These data demonstrate the utility of behavioral assays in zebrafish to identify genes affecting human behavior and pave the way for the use of zebrafish to inform human studies exploring the genetic basis of drug seeking and behavioral disease.

The aim of this project was to determine the role of contraction in skeletal muscle development. The role of the initial spontaneous contractions observed in zebrafish embryos from 17 to 24 hours post fertilisation was examined. Genetic and pharmacologic approaches were used to study paralysis-induced disruption of skeletal muscle structure and function and subsequently determine the role of contraction. The structural and functional characteristics of developing skeletal muscles were found to be regulated by a dual mechanism of both movement-dependent and independent processes, in vivo. Novel data demonstrates that contraction controls sarcomere remodelling, namely regulation of actin length, via movement driven localisation of the actin capping protein, Tropmodulin1. Myofibril length was also shown to be linked to the mechanical passive property, stretch, with lengthening leading to an increase of the muscle’s ability to stretch. In addition, myofibril bundling and the myofilament lattice spacing, responsible for active tension generation via cross-bridge formation, were shown to be unaffected by paralysis and thus, movement-independent processes. Furthermore, the mechanism of the contraction-driven myofibril organisation pathway at the focal adhesion complexes (FAC), was shown to be different in zebrafish compared to mammals, with mechanosensing revolving around the Src protein rather than Fak. In summary, the role of contraction was established as a critical driver of myofibril organisation and passive tension in the developing zebrafish skeletal muscle. Passive tension regulates muscle function by determining its operational range ensuring that the needs of locomotion are met. Furthermore, investigation of FAC’s role in the contractiondriven myofibril organisation pathway led to the discovery of a novel function for Src in zebrafish somitogenesis. These two findings (i) that contraction is a driver of myofibril organisation and (ii) that Src is a key protein of the skeletal muscle development provides the potential for new therapeutic approaches in humans.

Orientador: Renata Fracácio Francisco / Resumo: A indústria têxtil é responsável pela maior parte de corantes utilizados comercialmente. Segundo a literatura estes contaminantes representam potencial tóxico a fauna aquática. Neste contexto destaca-se o corante sintético, Índigo Blue, por seu uso intenso no tingimento do “blue jeans”. Antes o corante índigo era obtido da planta Indigofera tinctoria, mas a viabilidade econômica sobrepôs a produção natural e tornou a cor azul acessível a sociedade com a produção sintética do corante. Este estudo objetivou comparar os aspectos ecotoxicológicos do corante Índigo Blue (sintético) e do corante Índigo Natural (obtido da planta Indigofera tinctoria) expondo-se organismos de dois níveis tróficos diferentes à 100 mg/L de cada corante (concentração representativa de corantes em efluntes têxteis). Os organismos-teste utilizados foram: 1) a alga Raphidocelis subcapitata com duração dos testes equivalente a 72h e 96h, avaliando-se o parâmetro crescimento pela contagem do numero de células em relação ao grupo controle ( a concentração estudada não inibiu o crescimento algal) e 2) o peixe Danio rerio, machos e fêmeas mantiveram-se expostos por 21 dias, num total de seis réplicas para cada tratamento e para cada sexo. Sendo 3 organismos-teste em cada uma das quatro réplicas destinadas para a histologia de gônadas de ambos os sexos e duas réplicas com 4 machos destinados para a reprodução e duas réplicas cotendo 2 fêmeas para a reprodução (proporção de dois machos pra cada fêmea). Após o vig... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The textile industry is responsible for most commercially used dyes. According to the literature these contaminants represent toxic potential to aquatic fauna. In this context the synthetic dye, Indigo Blue, stands out due to its intense use in dyeing blue jeans. Before, indigo dye was obtained from the Indigofera tinctoria plant, but economic viability superimposed the natural production and made the blue color accessible to society with the synthetic dye production. This study aimed to compare the ecotoxicological aspects of Indigo Blue (synthetic) and Indigo Natural dye (obtained from the Indigofera tinctoria plant) by exposing organisms of two different trophic levels to 100 mg / L of each dye (representative concentrations of dyes in flakes textiles). The test organisms used were: 1) Raphidocelis subcapitata seaweed with duration of the tests equivalent to 72h and 96h, being evaluated the parameter growth by counting the number of cells in relation to the control group (the concentration studied did not inhibit algal growth) and 2) the Danio rerio fish, males and females remained exposed for 21 days, for a total of six replicates for each treatment and for each sex. Three replicates were used in each of the four replicates for the histology of gonads of both sexes and two replicates with 4 males destined for breeding and 2 replicates with 2 females for reproduction (ratio of two males to each female). After the twenty-first day, two replicates destined for histology were... (Complete abstract click electronic access below) / Mestre

Part I: Target identification studies are one of the most difficult but rewarding challenges in chemical biology. Part I of this thesis describes target identification studies for 5-nitrofuran containing hits. The 5-nitrofurans used in this study were identified in a phenotypic screen for compounds that induced melanocyte cells death in zebrafish. Chapter 1 provides brief overviews on three related areas of the project: 1) the use of zebrafish as a model organism in drug discovery; 2) phenotypic screening using zebrafish and 3) the strategies used in target identification studies. Chapter 2 describes the synthesis of and SAR studies on two series of 5-nitrofuran containing analogues. The design and preparation of biotinylated chemical probes based on the SAR data is also described. These chemical tools are then used in affinity chromatography studies and genetic validation of a potential target (zebrafish Aldh2) of the 5-nitrofuran compounds is reported. Chapter 3 provides a review of the biological and chemical processes that human ALDHs are known to mediate. In addition, small molecules that modulate ALDH2 activity are reviewed. A detailed study of the interaction between 5-nitrofurans and human ALDH2 including in vitro enzymatic assays is described leading to the conclusion that the 5- nitrofurans under study are substrates of human ALDH2. Further mechanism of action investigations using model reactions are also presented. Chapter 4 introduces the use of 5-nitrofuran containing drugs in the clinic and highlights the reported side-effects. Further investigation of the interaction between ALDH2 and 5- nitrofurans in zebrafish and yeast using ALDH2 inhibitors is described. Based on these results, a combination therapy strategy is proposed. Finally, the trypanocidal activity of the newly synthesised 5-nitrofurans is discussed. Experimental details and future work for Part I are presented in Chapters 5 and 6 respectively. Part II: Human sirtuins are associated with various biological functions and diseases, including cancer and neurodegeneration. Previous work from the Westwood Lab has led to the discovery of the tenovins that act as inhibitors of SIRT1 and SIRT2. Part II of the thesis reports the development of potent fixed ring tenovin analogues with high SIRT2 selectivity. Chapter 7 provides a brief review of the biology of human SIRT2 and the reported SIRT2 inhibitors available to date. This is followed by a short summary of the previous work on the tenovins in the Westwood Lab and the design of the fixed ring tenovin analogues. Chapter 8 describes the synthesis of three series of fixed ring tenovin analogues. SAR data is generated based on in vitro enzymatic assays against both SIRT1 and SIRT2 and the prepared analogues showed relatively high potency and selectivity against SIRT2. Further cell-based deacetylation assay are also discussed. All the experimental details are reported in Chapter 9 and Chapter 10 provides with conclusions and proposed future work.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

Injury and degenerative disease of the central nervous system (CNS) are among the major causes for disabilities in humans. They result in permanent damage that is not repaired by regenerative processes. In contrast, anamniotes like fish and amphibia display a striking potential for successful regeneration in the CNS. The zebrafish (Danio rerio) has been established as a model for successful regeneration after spinal cord injury. However, it is yet unknown which factors are involved in regeneration after spinal lesions and other insults to the CNS. Focusing on motor neurons, I asked whether regeneration can also be observed in larval zebrafish. This would allow to take advantage of their accessibility to live imaging, pharmacological and genetic manipulation. It is unknown, whether the loss of a specific cell type in the absence of injury, which is reminiscent of the pathological change observed in neurodegenerative diseases, would be sufficient to induce regeneration. Comparing the regenerative response after spinal lesion to that after selective neuronal cell loss would allow to identify factors that act as a trigger for regeneration, e.g. mechanical injury signals, the extent of cell death or microglia activation. To address these questions, an experimental paradigm in which motor neurons can be selectively ablated without the need to inflict tissue damage would prove useful. Key findings of this work are: · Motor neuron generation ceases during early larval developmental stages. · The Nitroreductase system can be used for successful ablation of motor neurons in the larval spinal cord. · New motor neurons are generated in a regenerative response to both targeted ablation of motor neurons and spinal lesion in larval zebrafish after cessation of developmental generation of MNs. To test whether larval zebrafish can be used to analyse motor neuron regeneration, I carried out a birthdating study to establish a developmental time line for motor neuron generation in the spinal cord. The end of developmental motor neuron generation at an early time point, at around 54 hours post fertilisation, allows for the use of larval zebrafish to assess the regenerative response after insults to the spinal cord. In addition, I could show a time dependent role for Hedgehog signalling during the generation of a motor neuron subpopulation. The influence of Hedgehog is diminished before the end of motor neurogenesis. Utilizing the Gal4/UAS system to combine the Nitroreductase‐mCherry fusion protein expressing Tg(UAS:nfsB‐mCherry) with the motor neuron specific driver Tg(hb9:Gal4), I generated a new transgenic zebrafish line for the genetically targeted ablation of motor neurons. In the resulting transgenic fish, the administration of the prodrug Metronidazole induces apoptotic cell death in ~25% of spinal motor neurons leading to impaired motor performance and increased numbers of microglia in the spinal cord. My work shows that larval animals subjected to motor neuron ablation or spinal lesion display a regenerative response detected by increased numbers of newborn motor neurons. Importantly, this happens after developmental production of motor neurons has ceased, suggesting that progenitor cells are reverting to the generation of motor neurons. The data presented shows that in larval zebrafish, the selective loss of motor neurons is sufficient to induce a regenerative response in the spinal cord. The increased numbers of microglial profiles in the spinal cord after both spinal lesion and targeted cell ablation indicates a role for the immune system in mediating a regenerative response. This new targeted cell ablation paradigm in larval zebrafish will allow to identify and characterize the progenitor cell population forming new motor neurons. One can then further investigate how specific loss of motor neurons is sensed and which factors contribute to the activation of the endogenous stem cell populations. Using larval zebrafish has many benefits, as they are accessible to pharmacological testing with small molecules and live imaging. Moreover, the combination of additional transgenic reporter lines will allow for the investigation of single cell behaviour during regeneration.

RNF4 belongs to the family of SUMO-targeted ubiquitin E3 ligases (STUbLs). The role of STUbLs in maintaining genomic stability was first discovered in yeast. Theyeast STUbL mutants displayed genomic instability, elevated mutation rates, sensitivity to DNA damaging agents and also demonstrated synthetic lethality with other DNA repair genes. Although the role of vertebrate RNF4 in the DNA damage response was not yet established, it could rescue the Schizosaccaromyces pombe STUbL mutant phenotypes, showing that RNF4 is a functional homologue of the yeast STUbL proteins,and that it might be implicated in the vertebrate DNA damage response.A homozygous knockout of RNF4 in the DT40 chicken lymphocyte cell line was generated to investigate the involvement of vertebrate RNF4 in protecting cells against DNA damage. Although the complete loss of RNF4 did not affect cell proliferation or cell cycle distribution, the RNF4 -/- cells exhibited a selective hypersensitivity to some S-phase specific DNA damaging agents. This hypersensitivity could be rescued by introducing an ortholog of RNF4 from another vertebrate species, and this was dependent on a functional ubiquitin E3 ligase activity of RNF4.To explore the physiological function of RNF4 in the context of a wholeorganism, Danio rerio was chosen as an in vivo model. Danio rerio RNF4 sharedsimilar in vitro biochemical characteristics as RNF4 from other vertebrates – it was able to autoubiquitylate itself and also ubiquitylate SUMO2 chains. In Danio rerio, RNF4 is a maternally provided gene and is highly expressed in the adult gonads. In the ovaries, RNF4 expression was restricted to the early stage oocytes, suggesting a possible role in oocyte development. Loss-of-function studies in Danio rerio were performed using morpholino knockdown and zinc-finger knockout technologies, and the depletion of RNF4 in zebrafish did not affect early embryonic development or viability of the animal.The results presented in this thesis suggests that while vertebrate RNF4 is notlikely to be an essential gene in some vertebrates, it plays a role in the DNA damage response and might be implicated in gonad development in Danio rerio. The zinc-finger knockout model has just been established and a more in-depth analysis is necessary to shed more light on the in vivo functions of RNF4.

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.