Melanocytes are pigment-producing cells that colour our hair, skin and eyes. Melanocytes are evolutionary conserved in vertebrates, and in addition to contributing to pigmentation and pattern formation, can contribute to background adaptation (zebrafish) and protection against harmful UV irradiation (humans). Many of the processes involved in melanocyte development – such as migration, proliferation and differentiation - are misregulated in melanoma. Here, I use chemical biology in zebrafish to identify targetable pathways in melanocyte development and regeneration, with a view to how these processes may be misregulated in melanoma and other pigmentation syndromes. We first wanted to address the potential for small molecules to regulate multiple stages of melanocyte development and differentiation. In Chapter 3, I describe my work involved in a small molecule screen for clinically active compounds that alter melanocyte biology (Colanesi et al., 2012). In this work we have identified small-molecules that affect melanocyte migration, differentiation, survival, morphology and number. This is important as it highlights new pathways essential for normal melanocyte development and consequently provides further tools in which to study melanocytes. Identifying the target of small molecules in vivo is a challenge in chemical biology. In Chapter 4, I describe my contributions to understanding how 5-nitrofuran compounds act in zebrafish (Zhou et al., 2012). My work has contributed to understanding the activity of 5-nitrofurans is dependent upon its nitrofuran ring structure. I have also helped confirm a conserved interaction between 5-nitrofurans and ALDH2, which may contribute to the off-target effects observed in the clinic. These results are important as they aid further understand of the 5-nitrofuran class of drugs and give evidence to support combination therapy of 5-nitrofurans with ALDH2 inhibitors as a way to overcome clinical side effects. Additionally I show that NFN1 treatment limits ensuing melanocyte regeneration thereby suggesting a role at the Melanocyte Stem Cell (MSC), which provides me with a key tool to study melanocyte regeneration in zebrafish. How tissue specific cell numbers are specified and maintained is a key question in developmental biology. In Chapter 5, I describe the identification of the MITF gene in the maintenance of cell cycle arrest in differentiated melanocytes (Taylor et al., 2011). We show that the human melanoma mutation MITF4TΔ2B promotes melanocyte division, thereby suggesting a role for melanocyte division in the pathogenesis of melanoma. This work is valuable because it highlights Mitf as a molecular rheostat that controls melanocyte proliferation and differentiation in living vertebrates, and helps us to understand the role of MITF in melanoma progression. Little is known about the pathways that control melanocyte stem cells in animals. To identify new melanocyte stem cell pathways, I used NFN1 as the basis for a small molecule screen for enhancers of melanocyte regeneration (Chapter 6). I find that chemical inhibition of Phosphatase of Regenerating Liver-3 (Prl-3) in zebrafish can enhance melanocyte regeneration. Importantly, I have found that there are an increased number of melanocyte progenitor cells in PRL3-inhibitor treated zebrafish. I propose that PRL-3 may control progenitor cell number in melanocyte regeneration. This is significant because it identifies PRL-3 as a novel molecular target controlling melanocyte progenitor cells, and identifies a new chemical tool with which to study melanocyte differentiation from a progenitor population. In the final chapter, I discuss how this work relates to the larger field of melanocyte developmental biology, and the new insight it provides into the fundamental processes of how organisms control cell number and pattern formation. In addition, I discuss how this work may have implications for understanding and treating melanocyte diseases, such as vitiligo (loss of melanocytes) and melanoma (cancer of the melanocyte).

Cardiac hypertrophy leading to heart failure remains a leading cause of morbidity and mortality in the 21st century despite major therapeutic advances. Improved understanding of novel molecular and cellular processes contributing to cardiac hypertrophy therefore continues to be important. Cyclin-dependent Kinase 9 (CDK9), part of a family of proteins controlling cell cycle and growth, has emerged as one such potential candidate over the last 5 years. CDK9 is the catalytic subunit of the CDK9/CyclinT complex and acts by phosphorylating the carboxy-terminal domain of RNA polymerase II. Hypertrophic signals, such as Endothelin-1 (ET-1) and phenylephrine, have been shown to cause CDK9 activation leading to a hypertrophic response in cultured mouse cardiomyocytes associated with reactivation of the foetal gene program. CDK9 also forms a complex with GATA4 to play a role in differentiation of mouse ES cells into cardiomyocytes. These findings suggest a specific role for CDK9 in controlling growth and differentiation of cardiomyocytes and merits further study in models where cardiomyocyte differentiation and proliferation are key contributors. In contrast to mammals, zebrafish retain a high cardiomyocyte proliferative capacity throughout their life span and can readily repair following injury. I have examined the role of CDK9 on global and cardiac development in the zebrafish embryo. I have also assessed the impact of CDK9 manipulation on response to ventricle injury using a laser-induced injury model developed and validated as part of my thesis. My findings confirm that normal growth of the embryonic ventricle is associated with a rapid increase in cardiomyocyte number, that was of 50% in the period 96-120 hpf, accompanied by increasing chamber trabeculation. This is also characterized by an increase in the gene expression of most of cardiac development relevant transcription factors, i.e. GATA4, 5 and 6, and MEF2c. The significant reduced cardiovascular function (14% of Ejection Fraction compared to 20% in controls) at 2 h post laser injury in the zebrafish embryonic heart promptly recovers at 24 hour post-laser, accompanied by acceleration of cardiomyocyte proliferation, that increased of 49% in injured ventricles compared to 20% in controls in the period 2-24 h post-laser. Pharmacological and genetic inhibition of CDK9 activity also significantly reduced cardiac growth, cardiomyocyte number, ventricle function and impairs functional recovery following laser injury. Conversely, genetic inhibition of LARP7, a CDK9 repressor, resulted in increased cardiomyocyte number and was associated with full functional and cellular recovery following laser-injury. In conclusion, I have provided evidence, in the zebrafish embryonic heart, that CDK9 plays an important role in cardiac growth and development and impacts significantly on cardiomyocyte proliferation. I have also shown that CDK9 manipulation significantly affects cellular and functional recovery following laser-induced injury. Further studies are required to further define the role of CDK9 and LARP7 in the heart and develop therapeutic strategies using this pathway that could contribute to cellular repair mechanisms in the adult mammalian heart.

<p>Background: Organophosphate (OP) pesticides are well-known developmental neurotoxicants that have been linked to abnormal cognitive and behavioral endpoints through both epidemiological studies and animal models of behavioral teratology, and are implicated in the dysfunction of multiple neurotransmitters, including dopamine. Chemical similarities between OP pesticides and organophosphate flame retardants (OPFRs), a class of compounds growing in use and environmental relevance, have produced concern regarding whether developmental exposures to OPFRs and OP pesticides may share behavioral outcomes, impacts on dopaminergic systems, or both. Methods: Using the zebrafish animal model, we exposed developing fish to two OPFRs, TDCIPP and TPHP, as well as the OP pesticide chlorpyrifos, during the first 5 days following fertilization. From there, the exposed fish were assayed for behavioral abnormalities and effects on monoamine neurochemistry as both larvae and adults. An experiment conducted in parallel examined how antagonism of the dopamine system during an identical window of development could alter later life behavior in the same assays. Finally, we investigated the interaction between developmental exposure to an OPFR and acute dopamine antagonism in larval behavior. Results: Developmental exposure to all three OP compounds altered zebrafish behavior, with effects persisting into adulthood. Additionally, exposure to an OPFR decreased the behavioral response to acute D2 receptor antagonism in larvae. However, the pattern of behavioral effects diverged substantially from those seen following developmental dopamine antagonism, and the investigations into dopamine neurochemistry were too variable to be conclusive. Thus, although the results support the hypothesis that OPFRs, as with OP pesticides such as chlorpyrifos, may present a risk to normal behavioral development, we were unable to directly link these effects to any dopaminergic dysfunction.</p> / Dissertation

The present study sought to determine some morphological and physiological critical windows during very early development in zebrafish exposed to crude oil. I hypothesized that exposed zebrafish would present a decrease in survival rate and body mass, and an increase in routine oxygen consumption (ṀO2), and critical oxygen tension (PCrit). To test these hypotheses, zebrafish were acutely exposed (24 h) during different days of development (1 to 6 days post-fertilization, dpf) to different concentrations of high-energy water-accommodated fractions (HEWAFs). The endpoints of survival, body mass, routine oxygen consumption, and critical oxygen partial pressure were measured at 7 dpf. Survival rate decreased based on the exposure concentration but not as a function of the day of crude oil exposure. No significant effects were found in PCrit. Body mass was reduced by the different concentrations of HEWAF, with the size of the effect varying with exposure day, with the effect strongest on when exposure occurred at 2 and 3 dpf. Oxygen consumption (ṀO2) differed significantly depending upon the day of exposure in fish exposed to crude oil. Specifically, HEWAF exposure significantly increased ṀO2 in larvae exposed at 3 dpf (9.081 µmol O2/g/h, ±0.559) versus 2 dpf (6.068 µmol O2/g/h, ±0.652) and 6 dpf (6.485 µmol O2/g/h, ±0.609). Overall, the main effects on body mass and ṀO2 occurred at crude oil exposures during 3 dpf. The presence of a critical window in fish is proposed at this developmental time, which coincides with the hatching period.

Orientador: Luiz Edivaldo Pezzato / Resumo: O zebrafish, teleósteo de água doce, apresenta características biológicas e genéticas favoráveis para pesquisas biológicas, entretanto, faltam informações sobre sua exigência nutricional. Neste sentido este trabalho objetivou determinar sua exigência proteica. Utilizou-se 900 juvenis com 25,2±1,98 mg e 12,95 ± 1,91 mm, distribuídos em 30 aquários de 15 L de volume (30 peixes/aquário) num delineamento inteiramente casualizado com cinco tratamentos e seis repetições. As dietas semipurificadas foram formuladas para conter 27,6; 33,2; 38,2; 43,8 e 49,2% de proteína bruta (PB). Após 62 dias, constatou-se que o teor de 27.6% PB foi mais efetivo (p<0.01) para a taxa de eficiência proteica. Entretanto, não houve diferença entre tratamentos para peso, comprimento, taxa de eficiência alimentar e de crescimento especifico, consumo de ração e sobrevivência. Em relação a utilização dos nutrientes e composição corporal, a dieta contendo 38,2% de PB proporcionou melhores respostas (p<0.01) para retenção e taxa de retenção de nitrogênio, retenção de energia e quantidade de energia corporal. Pode-se concluir que, dieta contendo 27.6% PB foi adequada para obtenção da melhor taxa de eficiência proteica e desempenho zootécnico e 38,2% PB para utilização dos nutrientes e energia pelo zebrafish. / Abstract: The zebrafish, teleost freshwater, shows biological and genetic characteristics tha are propitious for biological research . However, there is a lack of information about their nutritional requirements, considering this the purpose of this study is to determine its protein requirement. Were used 900 fingerlings with 25,2±1,98 mg distributed into 30 fish tanks of 15 liters of capacity (30 fishes/fish tank) in a completely randomized with five treatment and six repetitions. The Semipurified diets were formulated to include 27.6; 33.2; 38.2; 43.8; 49,2% of crude protein (CP). After 62 days, it was found that the percentage of 27.6% CP were more efective (p<0.01) to protein efficiency ratio. Although there was no difference between treatments for weight, length, feed efficiency rate and specific growth, feed intake and survival. In relation the use of nutrients and body composition, diet containing 38.2% crude protein provided better responses (p<0,01) for retention and nitrogen retention rate, energy retention and amount of body energy. We can be concluded that a diet containing 27.6% CP was adequate to obtain the best protein efficiency ratio and growth performance and 38.2% CP for use of nutrients and energy for the zebrafish. / Mestre

Tumor cells often hijack endoplasmic reticulum (ER) mediated signaling to facilitate tumor progression by adapting to the cellular stress evoked by oncogene overexpression and adverse microenvironment. Despite the prevalence of MYC-driven cancers, how the MYC oncoprotein regulates ER stress response pathways during tumorigenesis remains incompletely understood. Here we show that MYC drives continuous upregulation of ubiquitin fusion degradation 1 (UFD1) during T-cell acute lymphoblastic leukemia (T-ALL) development. As the E2 component of an ER-associated degradation (ERAD) complex, UFD1 facilitates the elimination of misfolded/unfolded proteins from the ER. We found that genetic and pharmacological disruption of UFD1 function exacerbates ER stress and activates the unfolded protein response (UPR). Specifically, UFD1 knockdown in human T-ALL cells impairs ERAD and promotes the proapoptotic UPR through the PERK-CHOP-BCL2 axis. This effect is demonstrated by an upregulation of PERK, phospho-PERK and its downstream effector CHOP, as well as a downregulation of BCL2 and BCLxL. Indeed, CHOP inactivation or BCL2 overexpression is sufficient to rescue tumor-cell apoptosis induced by UFD1 knockdown. Allelic loss of ufd1 in zebrafish similarly induces tumor-cell apoptosis and impairs MYC-driven T-ALL progression without affecting general animal health. These studies establish the UFD1-mediated ER stress response as an important mediator of MYC-driven tumor progression and suggest strategies for targeted therapy in T-ALL, and perhaps other MYC-driven cancers. Although UFD1-specific inhibitors have yet to be developed, inhibitors that target the p97 co-factor in UFD1-mediated ERAD are readily available. Importantly, we show that treatment with CB-5083, a selective and oral bioactive inhibitor of p97, can effectively kill human MYC-overexpressing T-ALL patient cells ex vivo and inhibits tumor progression in zebrafish models of MYC-driven T-ALL. Thus, CB-5083 treatment may represent an effective targeted therapy for T-ALL, especially relapsed/refractory ones with gain-of-function NOTCH1 mutations and thus MYC-overexpression.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2017-07-11T14:38:07Z No. of bitstreams: 1 NATALIA_ELTZ_SILVA_DIS.pdf: 1036979 bytes, checksum: 23cdf77b8842f9e9ade0b13e6f7bc111 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-07-28T17:40:18Z (GMT) No. of bitstreams: 1 NATALIA_ELTZ_SILVA_DIS.pdf: 1036979 bytes, checksum: 23cdf77b8842f9e9ade0b13e6f7bc111 (MD5) / Made available in DSpace on 2017-07-28T17:47:24Z (GMT). No. of bitstreams: 1 NATALIA_ELTZ_SILVA_DIS.pdf: 1036979 bytes, checksum: 23cdf77b8842f9e9ade0b13e6f7bc111 (MD5) Previous issue date: 2017-03-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Aging-related diseases are becoming more common. Alzheimer's disease (AD), the most prevalent form of dementia, includes as initial symptoms cognitive deficits that are attributed to the toxic effects of amyloid? peptide (A?) that accumulates in senile plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The amyloid cascade initially proposed to explain the effects of A? pointed to the plaques as the most toxic form of the A? molecule responsible for neuronal dysfunction and death. Recently, several evidences point to the increased toxicity of the soluble forms of the peptide. A better understanding of the dynamics of amyloid aggregation, clearance and toxic potential of the soluble versions may foster significant advances in the understanding AD mechanisms and the identification of potential targets for AD therapies. In this study we used the zebrafish as a model. 24-hour embryos received intracerebroventricular injection of human A?1-42 prepared to have different aggregation potentials: monomeric, oligomeric and plaqueforming. At 5 days post-fertilization (dpf), quantification of A?1-42 levels demonstrated a remnant increase in peptide levels in the animals injected with the solution that favored plaque formation. After monitoring for embryotoxic and teratogenic effects, 5dpf the animals were also evaluated in relation to general physiological aspects and their cognitive ability. Although the injection did not significantly impact animal survival or exploratory ability, the oligomeric solution induced specific cognitive deficits in relation to the vehicleinjected control. Together these results support the revised version of the amyloid cascade in which, although the presence of plaques corresponds to a greater accumulation of A?1- oligomeric forms may induce significant neurotoxic effects and result in cognitive deficits specially at disease?s early stages. / Com o envelhecimento da popula??o, doen?as relacionadas com o envelhecimento v?m se tornando mais comuns. A Doen?a de Alzheimer (DA), a forma prevalente de dem?ncia, inclui como sintomas iniciais deficit cognitivos que s?o atribu?dos a efeitos t?xicos de pept?deo ?- amiloide (A?) que se acumula em placas senis e emaranhados neurofibrilares constitu?dos pela prote?na tau hiperfosforilada. A cascata amiloide inicialmente proposta para explicar os efeitos do A? apontava para as placas de dep?sito de A?1-42 como a forma t?xica da mol?cula respons?vel pela disfun??o e morte neuronal. Recentemente diversas evid?ncias apontam para a toxicidade das vers?es sol?veis do pept?deo antes da agrega??o em placas. O melhor entendimento da din?mica de agrega??o do amiloide, limpeza e potencial t?xico das vers?es sol?veis pode permitir significativos avan?os no conhecimento dos mecanismos da doen?a e a identifica??o de potenciais alvos para terapias da DA. Neste estudo utilizamos o tele?steo zebrafish como modelo para a caracteriza??o destes processos. Embri?es com 24 horas receberam inje??o intracerebroventricular de A?1-42 humano preparado de forma a ter diferentes potenciais de agrega??o: monom?rica, oligom?rica e formadora de placas. Ao atingirem 5 dias p?s-fertiliza??o (dpf), a quantifica??o dos n?veis de A?1-42 demonstrou um aumento remanescente dos n?veis do pept?deo nos animais injetados com a solu??o que favorecia a forma??o de placas. Ap?s monitorarmos eventuais efeitos embriot?xicos e teratog?nicos, ao atingirem 5dpf, os animais foram tamb?m avaliados em rela??o a aspectos fisiol?gicos gerais e sua capacidade cognitiva. Embora a inje??o n?o tenha impactado significativamente a sobreviv?ncia dos animais ou a capacidade explorat?ria, a inje??o da solu??o oligom?rica induziu deficit cognitivos espec?ficos em rela??o ao controle injetado com ve?culo. Juntos, estes resultados suportam a vers?o revisada da cascata amiloide na qual, embora a presen?a de placas corresponda a um maior ac?mulo de A?1-42, a presen?a de vers?es oligom?ricas pode induzir efeitos neurot?xicos significativos e resultar em deficit cognitivos, especialmente nos est?gios iniciais da doen?a.

Submitted by PPG Medicina e Ci?ncias da Sa?de (medicina-pg@pucrs.br) on 2017-12-26T13:35:05Z No. of bitstreams: 1 GIOVANNA_MEDEIROS_TAVARES_DE_OLIVEIRA_TES.pdf: 3690052 bytes, checksum: e6e9b4fea2305637782a214e8f45af2d (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-12-29T10:45:02Z (GMT) No. of bitstreams: 1 GIOVANNA_MEDEIROS_TAVARES_DE_OLIVEIRA_TES.pdf: 3690052 bytes, checksum: e6e9b4fea2305637782a214e8f45af2d (MD5) / Made available in DSpace on 2017-12-29T10:47:48Z (GMT). No. of bitstreams: 1 GIOVANNA_MEDEIROS_TAVARES_DE_OLIVEIRA_TES.pdf: 3690052 bytes, checksum: e6e9b4fea2305637782a214e8f45af2d (MD5) Previous issue date: 2017-08-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Initially used in magnetic resonance imaging in the late 1970s, iron oxide nanoparticles (IONPs) have wide application in the medical field today, in diagnostics, drug delivery, cellular therapies and theragnostic. The high biocompatibility, small size, functionalization and ability to respond to an applied magnetic field gives this nanoparticle great advantage over other nanomaterials. Studies have demonstrated the low toxicity and high applicability of this nanomaterial in the clinic, however some questions remain unanswered. When in contact with the in vivo metabolism, nanomaterials can behave in a way to degrade their coating and release the ions contained in their nucleus. In fact, side effects related to exposure to IONPs are mainly related to the release of its elemental nucleus; which, when endocyted, can be degraded inside the lysosomes and release [Fe] ions. Changes in iron homeostasis can be very damaging to the cell, causing inflammation, lipid peroxidation, and oxidative stress. Organs more sensitive to iron accumulation, such as the heart, may demonstrate apoptosis and tissue degeneration. Such toxic effects are easily detected in studies using zebrafish as an animal model. Protocols with zebrafish embryos for toxicological analysis have the advantage of allowing large-scale screening on development, survival, behavior, gene expression and cardiotoxicity. Under this scenario, this thesis aims to evaluate the toxicity of commertial and "in house" synthesis of Iron Oxide Nanoparticles in zebrafish. Behavioral analysis of locomotion and gene expression of zebrafish larvae exposed to uncoated and dextran-coated iron oxide nanoparticles indicated a toxicity at low concentrations of nanoparticle exposure, contrary to what is reported in the literature. In addition, changes in the apoptotic pathway suggest that this route is closely linked to the behavioral effects found. Subsequent analyzes, targeting cardiotoxicity, suggested that concentrations above 100 ?g/ml are damaging for the heart. Molecular analyzes in the groups exposed to the iron oxide nanoparticle and to iron solution helped to establish a parallel between the toxicity of these nanoparticles and the pathways of iron metabolism. / Inicialmente utilizadas em exames de resson?ncia magn?tica no final dos anos 1970 as nanopart?culas de ?xido de ferro (IONPs) possuem hoje vasta aplica??o na ?rea m?dica, em exames de diagn?stico, sistema de envio de drogas, terapias celulares e como agente teragn?stico. Sua alta biocompatibilidade, pequeno tamanho, facilidade de manipula??o e capacidade de responder a aplica??o externa de campo magn?tico lhe oferece grande vantagem sobre outros nanomaterias. Estudos vem demostrando a baixa toxicidade e alta aplicabilidade deste nanomaterial na cl?nica, entretanto algumas quest?es ainda se encontram sem resposta. Quando em contato com o metabolismo in vivo, nanomateriais podem se comportar de forma a degradar sua estrtutura externa e liberar os ?oins contidos no seu n?cleo. De fato, efeitos adversos relacionado a exposi??o a IONPs est?o majoritariamente relacionadas a libera??o do seu n?cleo elementar; que, quando endocitado, pode ser degradado nos lisossomos e liberar ?ons [Fe]. Altera??es na homeostase de ferro podem ser muito prejudiciais ? c?lula, causando inflama??o, peroxida??o lip?dica e estresse oxidativo. ?rg?os mais sens?veis ao ac?mulo do ferro, como o cora??o, podem apresentar apoptose e degenera??o tecidual. Tais efeitos t?xicos s?o facilmente detectados em estudos utilizando zebrafish como animal modelo. Protocolos com embri?es de zebrafish para an?lise toxicol?gica possuem a vantagem de permitir estudos em grande escala de efeitos no desenvolvimento, sobreviv?ncia, comportamento, express?o g?nica e cardiotoxicidade. Sob esse cen?rio, esta tese tem como objetivo avaliar a toxicidade de Nanopart?culas de Oxido de Ferro (IONPs) de s?ntese pr?pria e comerciais no modelo experimental zebrafish. An?lise comportamental de locomo??o e express?o g?nica de larvas de zebrafish expostas a Nanopart?culas de ?xido de Ferro puras (sem envolt?rio) e revestidas com dextran indicou uma toxicidade em baixas concentra??es de exposi??o ? nanopart?culas, contrario ao que ? relatado na literatura. Al?m disso, altera??es na via apopt?tica sugere que esta rota esteja intimamente ligada aos efeitos comportamentais encontrados. An?lises posteriores, direcionadas ? cardiotoxicidade sugerem efeitos t?xicos acima de 100 ?g/mL. An?lises gen?mica de express?o nos grupos expostos ? nanopart?cula de ?xido de ferro e ? solu??o de ferro met?lico (usado como controle de positivo de excesso de ferro) permitiram a identifica??o de um paralelo entre toxicidade destas nanopart?culas e as vias de metaboliza??o do ferro.

True anophthalmia is the most severe congenital eye malformation. With absence of the eye, optic nerve, chiasm and optic tracts. Identifying the genes that cause genetic true anophthalmia should improve our understanding of the critical processes required for development of the eye. Recessive loss-of-function mutations in SMOC1 have been identified as the cause of Ophthalmo-acromelic syndrome (OAS), a multisystem disorder which has true anophthalmia as a prominent feature with characteristic limb and facial malformations. In order to establish the function of SMOC1 in development I used the zebrafish as a model organism to support a link between SMOC-1 and BMP signalling. As a first step I characterised the genomic structure of zebrafish smoc1 gene. I was able to correct an error in the zebrafish genome (Zv8) that annotated zsmoc1 as two fragmented and rearranged orthologous loci. However, using RTPCR I could show that there is in fact a single intact zsmoc1 transcript. In addition, I was able to identify an un-annotated 5’ coding exon using 5' RACE which showed that the full open reading frame includes a signalling peptide. RT-PCR was also used to identify several novel zsmoc1 splice isoforms. To explore the link between zsmoc1 and bmp signalling I used injection of antisense morpholino oligonucleotide and capped mRNA to examine the effects of loss-of-function and overexpression respectively of smoc1 and genes functioning in the bmp signalling pathway. The resulting embryos were analysed using morphometric analysis (Kishimoto scale), a quantitative assay of dorsalisation/ventralisation and live imaging of reporter transgenic fish. I developed a quantitative RT-PCR assay for expression of dorsal (otx2 and runx3) and ventral (eve1 and gata2) marker genes. I established a reliable system for live imaging of zebrafish development between 8 hpf and 24 hpf. By combining this system with fluorescent transgenic reporters marking the eye field (rx3:gfp reporter) and BMP-signaling (BRE:gfp reporter) I was able to accurately quantitate the effect of smoc1 depletion on eye size and SMAD1/5/8 signalling in the eye. These results support the predictions from the Drosophila homologue Pent that zsmoc1 functions as an antagonist of bmp signalling. Finally, I describe my attempt to produce a zebrafish model for OAS using genome editing technology. I designed, produced and validated transcription activator like effectors nucleases (TALENs) targeted to the zsmoc1 open reading frame using the Voytas Goldengate method. I designed and optimised a novel strategy to demonstrate targeted cutting activity for in vitro validation. Following injections of the in vitro validated TALEN into zebrafish embryos I used Ion Torrent sequencing to assess the in vivo activity of the engineered TALEN pairs. Unfortunately these TALENs were not able to cut the targeted locus in vivo.

A vital ability for an animal is to filter the constant flow of sensory input from the environment to focus on the most important information. Attention is used to prioritize sensory input for adaptive responses. The role of attention in visual search has been studied extensively in human and non-human primates, but is much less studied in other animals. We looked at attentional mechanisms, especially selective and divided attention where animals focus on multiple cues at the same time, using a visual search paradigm. We targeted bumblebee and zebrafish as model species because they are widely used as tractable models of information processing in comparatively small brains. Bees were required to forage from target and distractor flowers in the presence of predators. We found that bees could selectively attend to certain dimension of the stimuli, and divide their attention to both visual foraging search and predator avoidance tasks simultaneously. Furthermore, bees showed consistent individual differences in foraging strategy; ‘careful’ and ‘impulsive’ strategies exist in individuals of the same colony. From the calculation of foraging rate, it is shown that the best strategy may depend on environmental conditions. We applied a similar behavioural paradigm to zebrafish and found speed-accuracy tradeoffs and consistent individual behavioural differences. We therefore continued to test how individuality influences group choices. In pairs of careful and impulsive fish, the consensus decision is close to the strategy of the careful individual. The present thesis provides implications for the study of animal attention, individuality differences based on attentional strategies, the influence of individuality on animal group choices and an exploration of the evolutionary pressures that favour stable individual differences.