Partial Sequence and Characterization of a Growth Hormone Gene of the Red Drum (Sciaenops Ocellatus).

A growth hormone (GH) gene was isolated from nuclear DNA of the red drum (Sciaenops ocellatus). Synthetic oligonucleotides were synthesized based on published cDNA sequences of other perciform fishes. Polymerase chain reaction (PCR) methodology was employed to amplify a nucleotide sequence extending from the 5\sp\prime end of intron I to 15 bases upstream of the 3\sp\prime end of exon VI of the GH gene. Amplified DNA was cloned into a phagemid sequencing vector. Clones were sequenced. Computer analysis of sequence data was used to determine the internal gene arrangement of exons and introns. Homologies of the nucleotide and deduced amino acid sequences with published GH sequences were determined. The GH nucleotide sequence was examined for the presence of regulatory elements. The red drum GH gene is comprised of six exons and five introns, an arrangement like that of the salmonid GH genes but unlike the five exon, four intron arrangement of carp (Cyprinus carpio) and mammalian GH genes. Protein coding regions of the red drum GH gene show a high degree of homology with GH cDNA sequences of other perciform fishes. The 3\sp\prime terminus of exon V of the red drum GH gene shows an 86% similarity with a region of exon V of the human chorionic somatomammotropin gene. A putative glucocorticoid receptor element is present in intron I of the red drum GH gene

The use of molecular biological methods to assess the effects of endocrine disrupting chemicals and natural hormones on growth in the sheepshead minnow (Cyprinodon variegatus)

The work presented in this dissertation examines possible modes of action for growth inhibition by anthropogenic endocrine disrupting chemicals (EDCs) as well as endogenous hormones associated with growth in fish. Using the sheepshead minnow (SHM) (Cyprinodon variegatus) as a model, I developed methods to examine perturbations in the endocrine axis controlling fish growth, and also examined effects of EDCs on the whole fish. I used two relatively new techniques to study the endocrine growth axis, quantitative real-time PCR (TaqMan) and differential display analysis. TaqMan analysis is a highly sensitive method to measure specific sequences from a small amount of total RNA using a fluorescent probe and specific primer pairs. I optimized a TaqMan assay for SHM IGF-I to measure hepatic IGF-I mRNA concentrations. in fish injected with hormones known to influence fish growth (GH, T₃, E₂, insulin, or a carrier control). IGF-I mRNA levels increased in fish injected with GH, T₃ and insulin, peaking at 12 h post-injection. IGF-I mRNA levels decreased significantly at 8 h and 12 h post-injection in fish injected with E₂, suggesting that pharmacological levels of E₂ may affect the GH/IGF-I axis and could have consequences for fish living in waters polluted by EDCs. Differences in growth were observed in fish exposed for 18 weeks to E₂ or chlorpyrifos (an organophsophate). Fish exposed to the highest dose of E₂ grew larger than controls only during the last week of the experiment. Fish exposed to the lower dose of E₂ were not significantly different from controls. The fish exposed to all doses of chloryprifos grew significantly less than controls in a dose-dependent manner. No significant differences were found in hepatic IGF-I mRNA levels in any treatments. To establish patterns of gene up- or down-regulation, I performed differential display analysis on livers of several fish from the previous two experiments. Several genes were identified as being similar to fish including a microsatellite sequence, a choriogenin (vitelline envelope) protein mRNA sequence, a transferrin mRNA sequence and several ribosomal RNA sequences. This technique to evaluate gene expression will become more useful when more fish genes are added to the data bases

Characterization of Endogenous Nucleobindin-2/Nesfatin-1 in Rodents

Whole body energy homeostasis is regulated by the endocrine system. Nesfatin-1 is a newly identified multifunctional metabolic peptide with insulinotropic, endocrine, glucoregulatory, fat reducing and cardiovascular functions. While nesfatin-1 tissue expression and secretion are considered meal responsive, it is unknown whether macronutrients and/or development regulate its secretion. Is endogenous nesfatin-1 critical for energy balance? The central hypothesis of this thesis research is that the tissue specific expression of NUCB2/nesfatin-1 is regulated developmentally, and by nutrients, and that endogenous NUCB2/nesfatin-1 is critical for the maintenance of energy homeostasis. The specific objectives of this research were to determine the developmental, and nutrient regulated expression of NUCB2/nesfatin-1, and to characterize the metabolic phenotype of mice lacking NUCB2/nesfatin-1. Three key findings were made in this research. First, it was found that NUCB2/nesfatin-1 presents an ontogenic pattern of expression in the gastroenteropancreatic tissues and serum of rats. An age-dependent co-expression of related peptides, ghrelin and its processing enzyme, ghrelin-O-acyl transferase (GOAT), and nesfatin-1 processing prohormone convertases were also found in the endocrine pancreas. Second, it was determined that the NUCB2 mRNA expression and NUCB2/nesfatin-1 secretion in mice are influenced by nutrients in a tissue specific manner in vitro and in vivo, and it depends on the duration of exposure to specific diets tested. This research identified nutrients as major regulators of endogenous NUCB2/nesfatin-1. Third, a sexually dimorphic effect of NUCB2/nesfatin-1 disruption in mice was found, with alterations in body weight, food intake, insulin secretion, glucose homeostasis and energy homeostasis. These data support a metabolic role for endogenous nesfatin-1. Together, this research provides important new information on developmental and cell specific regulation of nesfatin-1 expression, nutrient modulation of its expression and secretion, and an essential role for endogenous nesfatin-1 in maintaining energy homeostasis. For example, endocrine pancreas is an abundant source of nesfatin-1. Absence of endogenous nesfatin-1 causes defects in insulin secretion from islet beta cells, and alters glucose homeostasis. Exogenous nesfatin-1 causes a reduction in body weight. NUCB2 gene disruption resulted in abnormal body weight in mice, further confirming that nesfatin-1 indeed influences body mass

Estrategias de alimentación y cultivo de la dorada (sparus aurata). Regulación endocrina y estado inmunopatológico.

RESUMEN En la presente tesis doctoral se abordó la regulación de la somatolactina (SL) y del eje somatotrópico (GH-IGF-I) en tres ciclos de engorde con inicio en distinta época del año (primavera, verano y otoño). Se determinaron variaciones estacionales de GH e IGF-I con picos en el periodo estival y los niveles más bajos en otoño e invierno. Los niveles circulantes de GH estuvieron correlacionados positivamente con las tasas de ingesta de alimento, mientras que los niveles de IGF-I lo estuvieron con las tasas de crecimiento. En la SL también se observó variaciones estacionales, pero el pico se estableció en otoño, una vez los animales hubieron alcanzado un determinado estatus energético. Ello mostró una regulación estacional opuesta entre GH y SL. Los valores de SL no coincidieron con los aumentos de cortisol. Como hipótesis de trabajo se estableció que los incrementos de GH actuarían movilizando energía en momentos de déficit energético promoviendo el crecimiento, mientras que los incrementos de SL, una vez el animal hubiera alcanzado cierta disponibilidad energética, servirían para movilizar las reservas energéticas y disparar procesos fisiológicos como el ayuno invernal y la reproducción. Para comprobar esta hipótesis, se llevó a cabo un modelo de alimentación restringida anual. Se utilizó el mismo diseño experimental de tres engordes del capítulo anterior con dos grupos de alimentación, uno ad limitum (saciedad visual) y otro con alimentación restringida. El valor medio de la restricción en todos los engordes fue del 20%. La restricción de la alimentación comportó un aumento de la amplitud del pico de GH, corroborando que esta hormona es utilizada por el animal para movilizar energía en situaciones de déficit energético. En el caso de la SL, la restricción ocasionó un retraso del pico otoñal, corroborando la hipótesis de que se trata de una señal de adiposidad. Estos cambios hormonales se produjeron cuando existieron también cambios en el contenido graso de los animales. Desde un punto de vista práctico, la alimentación restringida comportó una ligera disminución de la biomasa final (<7%), sin embargo, mejoró la tasa de conversión del alimento en todos los grupos (15%) y disminuyó el grado de adiposidad de los animales. Los beneficios de esta alimentación restringida fueron mayores cuanto más alejado estuvo el ciclo de engorde del modelo natural de la dorada en estado salvaje. También se estudió el efecto de esta alimentación restringida sobre el estado inmunopatológico de los animales, ya que experiencias previas pusieron de manifiesto la relación entre la adiposidad y este estado inmunopatológico en otras especies de peces. En este caso, la alimentación restringida comportó una menor tasa de mortalidad, menos alteraciones histopatológicas y una menor prevalencia de infección para la mayoría de los parásitos detectados. Asimismo, esta restricción parece mejorar el estado de inmunocompetencia en algunos aspectos (estallido respiratorio). Otro de los aspectos abordados fue la caracterización del receptor de GH (RGH) en dorada, como pieza clave dentro del eje somatotrópico y para la determinación más precisa del estado nutricional de los animales en cultivo. La secuencia nucleotídica (1726 nt de longitud) codifica para una proteína de 575 aminoácidos de pauta abierta de lectura. El análisis de dicha secuencia se completó por 3 y 5RACE, encontrándose un péptido señal de 38 aminoácidos, seguido de una región extracelular de 227 aminoácidos, una región transmembrana de 24 aminoácidos y el resto perteneciente a la región intracelular. El alineamiento de las secuencias aminoacídicas del RGH de dorada con el de otros receptores de vertebrados superiores e inferiores mostró la conservación de 7 cisteínas, 5 sitios N-glicosilación (1 de ellos exclusivo de peces) y el motivo FGEFS en la región extracelular. En la región intracelular se encontraron conservados los Dominios 1 y 2 citoplasmáticos y 8 tirosinas (1 de ellas exclusiva de peces). La búsqueda de isoformas del RGH de dorada por Northern blot, 3RACE y screening mediante RT-PCR del extremo 3 no reflejó la existencia de un procesamiento alternativo del ARN mensajero. El análisis de cross-linking también abogaron por la existencia de una única proteína con distinto grado de glicosilación. El análisis de la estructura genómica del RGH de dorada y rodaballo mostró un alto grado de conservación de la estructura exón-intrón, con la presencia de un intrón exclusivo de peces que, en la chopa, posibilita la existencia de una forma larga con una inserción de 93 pb en la región intracelular. En el caso del rodaballo, la región divergente y el 3UTR de la forma truncada anclada a membrana están codificados por el extremo 5 del intrón 9/10. Todo ello pone de manifiesto que la presencia de isoformas y los mecanismos de generación de los RGHs de peces y vertebrados superiores son marcadamente específicos. ____________________________________________________________________________________________________Aquaculture production of gilthead sea bream has largely increased over the past decade, and research in this sector is being directed towards the improvement of feeding practice. In this scenario, the most suitable approach for culturing fish would be the use of appropriate feeding standards aimed not only to improve economic returns, but also to develop a lasting cohabitation of sustainable aquaculture and a cleaner environment. In this way, available data indicate that the potential benefits of high-energy diets can be compromised in gilthead sea bream by unrestricted feeding. In addition, the growth hormone spurts of summer is concurrent with up-regulation of circulating levels of growth hormone (GH) and insulin-like growth factor-I (IGF-I). This endocrine arrangement provides an integrated signal for growth and perhaps immune status at times of increasing daylength and temperature. Furthermore, there is a direct evidences for the involvement of somatolactin (SL), a new member of GH and prolactin family. This hormone is up-regulated by the increase of ration size, and their rise and fall is a consistent event after the summer repletion of energy reserves. By other side, restricted feeding regime had lower percent mortality, lower prevalence of infection of the most of the detected parasites, less histopathological alterations than those fed under visual satiety, and seemed to be enhanced respiratory burst activity. Finally, molecular characterization of GH receptor in gilthead sea bream, genomic organization of this receptor in sea bream and turbot and season expression were focused

Zebrafish liver and pancreas development: 1. Roles of rbp4 in early liver formation; 2. Genetic ablation to deduce pancreas cell lineage

Ph.DDOCTOR OF PHILOSOPH

Cryopreservation of zebrafish germ cells: technological improvements and methodological standardization for gene banking and management

Due to the increasing number of zebrafish (Danio rerio) mutant and transgenic lines, there is a high demand for assisted reproductive techniques to support facility management. Efficient zebrafish sperm cryopreservation is a pressing necessity to manage and preserve the valuable zebrafish genetic resources. Although zebrafish sperm cryopreservation was first attempted more than 30 years ago, protocols still lack standardization, which translates into high variability in post-thaw sperm quality and in vitro fertilization success. Therefore, the present thesis aims to improve the current methodologies used for zebrafish sperm cryopreservation and broodstock management towards the standardization of procedures in this species. The introductory context of the present thesis is approached in chapter 1. In this chapter the relevance of zebrafish model is discussed as well as this species sperm cryopreservation usefulness. The main factors affecting sperm quality and the application of reliable quality analysis are discussed in this chapter. The final objective of sperm cryopreservation is to obtain high quality offspring and therefore in vitro fertilization, early development and offspring quality analysis are important tools for the optimization of sperm cryopreservation methodologies. The current knowledge in sperm cryopreservation fundamentals is approached in this chapter, as well as the main advances and bottlenecks in zebrafish sperm cryopreservation. In chapter 2, the zebrafish sperm motility activation was assessed under different conditions of water temperature and conductivity. The environmental conditions present in the fertilization microenvironment are responsible for the mechanism of spermatozoa motility activation and metabolic modulation that influence the probability of fertilization success. Zebrafish is commonly reared at 28°C, but with variable water conductivity conditions among facilities. However, sperm motility analysis is routinely performed with distilled water at room temperature. We aimed to understand the effect of water temperature and conductivity on sperm motility and fertilization ability. Water at 28°C with lower water conductivity (0 and 700 μS/cm) improve sperm motility parameters. Standardization of the water conditions (of system water and activation médium used for motility analysis) among facilities is highly relevant to improve the reproducibility of sperm quality analysis and thus, to predict with higher accuracy fertilization ability. Successful cryopreservation depends on high quality sperm, which depends on the quality of breeders. Consequently, broodstock selection and management is a priority to improve sperm cryopreservation. The broodstock diet has a preponderant effect on gamete quality, particularly in phospholipids and antioxidants content which are known to promote spermatogenesis. Therefore, in chapter 3 we aimed to determine the effects of a tailor-made purified diet supplemented with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the zebrafish reproductive performance, gamete quality and larval skeletal malformations. Both dietary supplementations with phospholipids improved sperm motility and eggs quality, however PC increased the incidence of skeletal malformations on the offspring, as previously observed in other teleosts. Although dietary phospholipids classes have a role in the ossification process of the vertebral column in teleosts, its mechanisms are still to be understood. Therefore, the development and use of a standardized diet for zebrafish broodstock is essential to reduce the variability of the reproductive performance among facilities. In chapter 4, the selection of optimal age and minimum sperm collection frequency was evaluated, since these factors are essential to obtain high quality samples. Our results indicate that young males (6-8 months) showed higher sperm quality and require a minimum of 14 days between sperm collections to recover sperm plasma membrane viability. An important bottleneck in cryopreservation is the liquid nitrogen requirement for storage. Therefore, it was established in chapter 5 a new cryopreservation method using an electric ultrafreezer (-150°C) as an alternative to liquid nitrogen, for the first time in a teleost species. This protocol reaches a fast cooling rate (-66°C/min) in one single step and yields higher sperm viability and DNA integrity in comparison to the traditional methods (-20°C/min in liquid nitrogen). The synergy obtained by the combination of cryoprotectants is a successful cryopreservation strategy that can be beneficial in the optimization of zebrafish sperm cryopreservation. Therefore, it was selected the most adequate cryoprotectant combination that generates offspring with normal skeletogenesis. Data show that 15% of DMF with 50 mM of bicine or 10% of egg yolk is beneficial for the quality of zebrafish offspring sired by cryopreserved sperm. To the best of our knowledge, this is the first report on skeletal development of zebrafish offspring sired by cryopreserved sperm performed with different extender compositions. Zebrafish is especially useful to investigate some of the most prominent human diseases such as diabetes. Among other consequences, diabetes (type I and II) causes disturbances in the male reproductive system, since glucose metabolism is an important event not only in spermatogenesis but also in mature spermatozoa metabolism. In chapter 6 we aimed to validate zebrafish as a useful model organism to investigate male reproductive dysfunctions mechanisms caused by type I diabetes. In this chapter, sperm cryopreservation was applied to a relevant zebrafish model of type I diabetes. The transgenic zebrafish under diabetic conditions shows higher levels of insulin a (insa), insulin receptor a (inra) and glucose carrier 2 (slc2a2) transcripts in spermatozoa when compared to the controls. This is because gametogenesis occurred under diabetic conditions, changing transcription in the germline. Consequently, spermatozoa carry the imprinted transcripts that will be transmitted during fertilization. Sperm quality (motility, viability and DNA integrity) was lower in the transgenic fish under (transient) diabetic state as observed in human and mouse model. Sperm cryopreservation affects sperm quality of fish both under diabetic and non-diabetic conditions. However, diabetic conditions were detrimental in sperm freezability, which can be explained by the lower initial sperm quality. In this chapter zebrafish was validated as a useful model organism to investigate male reproductive dysfunctions mechanisms caused by type I diabetes. Relevant differences between different zebrafish lines are evidenced in terms of sperm quality and susceptibility to damage, which suggests that it is an important factor to consider while establishing sperm cryopreservation protocols. This thesis offers new insights and a set of guidelines on breeder’s management and sperm cryopreservation to improve zebrafish husbandry practices. Keywords: Zebrafish, sperm quality, cryopreservation, ultrafreezer, sperm motility activation, diet, type I diabetesO peixe zebra (Danio rerio) tornou-se inquestionavelmente num dos organismos modelo mais proeminentes da atualidade, devido às suas características favoráveis para investigação. O desenvolvimento de técnicas de edição genética e a sequenciação do genoma desta espécie possibilitou o desenvolvimento de milhares de linhas transgénicas e mutantes. Consequentemente, a gestão dos numerosos genótipos trouxe desafios na manutenção de espaço e gestão destes recursos genéticos. A criopreservação de sémen é uma ferramenta valiosa para a gestão destes valiosos recursos genéticos, que pode solucionar este problema. No entanto, apesar do primeiro protocolo de criopreservação de sémen de peixe zebra ter sido desenvolvido há mais de 30 anos, ainda requer otimização e estandardização. Consequentemente, existe elevada variabilidade na qualidade do sémen e sucesso da fertilização in vitro entre biotérios. O desenvolvimento de uma técnica de criopreservação de sémen eficiente é atualmente um dos maiores desafios da comunidade de peixe zebra. O objetivo principal da presente tese foi a otimização das técnicas de gestão de reprodutores e criopreservação de sémen de peixe zebra, no sentido da estandardização das práticas e maior reprodutibilidade dos resultados científicos nesta espécie. A introdução ao contexto da presente tese é abordada no capítulo 1. Neste capitulo a importância do peixe zebra como organismo modelo é abordado assim como a utilidade da criopreservação do sémen nesta espécie. Os factores que afetam a qualidade do sémen assim como a aplicação de análises de qualidade robustas são discutidos neste capítulo. O objetivo final da criopreservação de sémen é a produção de progenia com elevada qualidade. Consequentemente, neste capítulo a fertilização in vitro, o desenvolvimento emb rionário e a análise da qualidade da progenia é discutida. Neste capítulo são explorados os fundamentos de criobiologia, principais avanços e dificuldades no desenvolvimento de protocolos de criopreservação de sémen de peixe zebra. As condições do ambiente de fertilização são responsáveis pela ativação da mobilidade dos espermatozóides e pela modulação do seu metabolismo, afetando consequentemente o sucesso da fertilização. O peixe zebra é estabulado a 28°C com parâmetros de condutividade da água variáveis entre biotérios. No entanto, as análises de mobilidade espermática são realizadas rotineiramente com água destilada a temperatura ambiente. Consequentemente, no capítulo 2 o objetivo do nosso trabalho foi caracterizar o efeito da temperatura e condutividade da água na mobilidade de sémen de peixe zebra. Adicionalmente, foi estudado o efeito da condutividade da água no sucesso da fertilização. A água a 28°C e com baixa condutividade (0 e 700 μS/cm) melhorou os parâmetros de mobilidade. A estandardização das condições da água (dos sistemas de cultivo e do meio de ativação usado na análise da mobilidade) entre biotérios é essencial para a otimização das análises de qualidade, reprodutibilidade científica e maior precisão na estimação do potencial de sucesso de fertilização de uma amostra de sémen. O sucesso da criopreservação depende da qualidade do sémen que, por sua vez, depende da qualidade dos reprodutores. Consequentemente, a seleção e gestão de reprodutores é uma prioridade, de forma a assegurar o sucesso do método de criopreservação. Um dos factores mais importantes na gestão de reprodutores é a sua dieta. A nutrição dos reprodutores tem um importante efeito na qualidade dos gametas já que afeta a gametogénese, particularmente a composição da dieta em fosfolípidos e antioxidantes. O objetivo do capítulo 3 foi determinar o efeito de dietas purificadas suplementadas com fosfatidilcolina (PC) e fosfatidiletanolamina (PE). A suplementação em fosfolípidos melhorou a mobilidade do sémen; no entanto, a suplementação em PC provocou um aumento da incidência de malformações esqueléticas na progenia. Estes resultados estão de acordo com estudos de nutrição anteriores em teleosteos. O desenvolvimento e utilização de dietas estandardizadas nos reprodutores de peixe zebra é essencial para optimizar a performance reprodutiva e reduzir a variabilidade entre biotérios. A seleção da idade ótima dos machos e a frequência mínima adequada para recolha de sémen é essencial para obter amostras com elevada qualidade. No capítulo 4 foi determinado o efeito da idade e da frequência de extração na qualidade do sémen. O nosso estudo mostrou que machos jovens (6-8 meses) de peixe zebra revelam maior qualidade de sémen e necessitam de um mínimo de 14 dias de repouso para recuperarem a viabilidade da membrana plasmática dos espermatozóides. Uma das maiores desvantagens da criopreservação é a necessidade de azoto líquido para armazenamento de amostras. Considerando esta questão, foi desenvolvido no capítulo 5 o primeiro protocolo de criopreservação de sémen de teleósteos utilizando um ultracongelador (-150°C). Este protocolo é realizado num só passo, sem a utilização de azoto líquido, sendo as amostras criopreservadas a - 66°C/min. Este protocolo melhorou a viabilidade e integridade do ADN dos espermatozóides em comparação com o método convencional (-20°C/min armazenado em azoto líquido). A combinação de diferentes crioprotetores é uma estratégia de criopreservação com elevado sucesso. Consequentemente, um dos objetivos do nosso trabalho foi selecionar a combinação de crioprotetores mais adequada para o protocolo de criopreservação estabelecido anteriormente. Os resultados deste trabalho indicam que a utilização de 15% de DMF com 50 mM de bicina ou 10% de gema de ovo produzem sémen de elevada qualidade do sémen e maior sucesso em fertilizações in vitro, assegurando também o adequado desenvolvimento esquelético da progenia. Este foi o primeiro estudo de caracterização de malformações esqueléticas desenvolvidas na progenia de peixe zebra produzido com sémen criopreservado com diferentes composições de crioprotetores. O peixe zebra é particularmente útil na investigação de doenças humanas com elevada prevalência na população mundial tal como a diabetes. Entre outras complicações geradas por esta patologia, a diabetes tipo I e II afeta o sistema reprodutor masculino. Estas perturbações ocorrem devido à alteração do metabolismo da glucose, essencial durante a espermatogénese e no metabolismo dos espermatozóides. Comparando com outros modelos, o peixe zebra tem gerações mais curtas, consequentemente seria uma ferramenta útil para esta investigação. O objetivo do capítulo 6 foi validar o peixe zebra como organismo modelo para o estudo dos mecanismos de ação da diabetes tipo I pelos quais afetam o sistema reprodutor masculino. Tal como observado em humanos e no organismo modelo de diabetes roedor, a qualidade do sémen (mobilidade, viabilidade, integridade do ADN) é reduzida na estirpe transgénica sob estado transiente de diabetes tipo I em relação ao controlo. O sémen do modelo transgénico em estado diabético revela aumento dos níveis de transcriptos de insulina a (insa), receptor de insulina a (inra) assim como de um transportador especifico de glucose GLUT 2 (slc2a2). Este facto é devido a uma alteração dos níveis de transcrição destes genes na linha germinal durante a gametogénese. O sémen criopreservado de ambos os tratamentos (controlo e diabético) revelou um decréscimo na qualidade, tal como esperado. O tratamento diabético aumentou a susceptibilidade das células à criopreservação, o que se pode dever à sua qualidade seminal inicial inferior. Assim, evidenciamos neste modelo transgénico para a diabetes tipo I os mesmos efeitos na qualidade seminal observados em humanos e em rato, validando desta forma esta linha para a investigação dos efeitos desta doença no sistema reprodutor masculino. A presente tese propõe o estabelecimento de medidas de seleção e maneio de reprodutores e análise de qualidade seminal. Adicionalmente propomos um método inovador de criopreservação de sémen, prático e económico, através do uso de um ultracongelador. Verificou-se o impacto de diferentes combinações de crioprotectores na qualidade e na esqueletogénese da progénie gerada com sémen criopreservado. Resumindo, esta tese propõe procedimentos e metodologias para a gestão de reprodutores de peixe zebra relevantes para o estabelecimento de medidas de estandardização, promovendo desta forma a reprodutibilidade de metodologias científicas.Patricia Diogo was the recipient of a doctoral grant SFRH/BD/97466/2013 from the FCT. This work was partly founded by the FCT and the European Commission (ERDF-COMPETE) through PEst-C/MAR/LA0015/2011 project and by the FCT through UID/Multi/04326/2019, and by project LARVAMIX-17925 funded by Portugal and by the European Union through FEDER, COMPETE 2020 and CRESC Algarve 2020, in the framework of Portugal 2020

Zinc Signaling in Physiology and Pathogenesis

The essential trace element zinc plays indispensable roles in multiple cellular processes. It regulates a great number of protein functions, including transcription factors, enzymes, adapters, and growth factors as a structural and/or catalytic factor. Recent studies have highlighted another function of zinc as an intra- and intercellular signaling mediator, which became recognized as the “zinc signal”. Indeed, zinc regulates cellular signaling pathways, which enable conversion of extracellular stimuli to intracellular signals, and controls various intracellular and extracellular events, and thus zinc mediates communication between cells. The zinc signal is essential for physiology, and its dysregulation causes a variety of diseases, such as diabetes, cancer, osteoarthritis, dermatitis, and dementia. This Special Issue focuses on crucial roles of zinc signaling in biological processes in molecular and physiological basis, addressing the future directions and questions underlying this unique phenomenon. Because there is growing interest and attention in physiopathological contribution of zinc signal, we believe this Special Issue will provide very timely information on it and thus should appeal to a wide range of readers