Thèses sur le sujet « Danno e plasticità »

2008/2009
L’obiettivo dello studio è stata la valutazione del comportamento di elementi strutturali in muratura sottoposti a sollecitazioni di taglio e compressione, sia attraverso prove sperimentali, che mediante analisi numerica: in particolare, nell’ambito della modellazione agli elementi finiti è stato valutato l’utilizzo di legami costitutivi di danno e plasticità per descrivere il comportamento non lineare della muratura. L’approccio utilizzato nelle analisi numeriche proposte è la macromodellazione, dove il materiale è rappresentato da un continuo omogeneo, le cui proprietà meccaniche globali sono dedotte dai risultati di prove su elementi rappresentativi della muratura. A partire dai risultati sperimentali su pannelli sollecitati a taglio e compressione ottenuti negli anni ’80 allo Swiss Federal Institute of Technology (Ganz e Thürlimann) e da quelli su fascia di piano nel biennio 2007-2008 presso l’Università degli Studi di Trieste, è stata inizialmente verificata la possibilità di impiego di un modello di danno e plasticità isotropo (Lubliner et. al 1989, Lee & Fenves 1998) per determinare la risposta di elementi strutturali sottoposti a carico monotono e ciclico. Quindi sono state valutate le ragioni della differenza tra i risultati delle prove sperimentali e quelli delle analisi numeriche, evidenziando sia le difficoltà costruttive, sia quelle derivanti dalla descrizione matematica del comportamento del materiale. E’ stato inoltre proposto lo sviluppo di alcune parti del modello isotropo verso una formulazione ortotropa, sulla base di un legame costitutivo anisotropo (Voyiadjis et al. 2007), prestando particolare attenzione ai parametri non lineari di modello, nello specifico l’angolo di dilatanza.
XXII Ciclo
1982

The aim of this thesis was to investigate respiratory plasticity in zebrafish (Danio rerio) confronted with long-term changes in water gas composition (hypoxia, hyperoxia or hypercapnia) either as embryos or adults. The ventilatory responses to acute changes in water gas composition (hypoxia, hypercapnia or cyanide) were assessed using a non-invasive technique (Altimiras and Larsen, 2000) to continuously monitor breathing frequency (f R) and relative breathing amplitude. The ventilatory response to acute hypoxia consisted of an increase in fR while the response to acute hypercapnia was an increase in relative breathing amplitude. (Abstract shortened by UMI.)

Le gène NR4A2 est un membre de la super famille des récepteurs nucléaires qui sont des facteurs de transcription activés par un ligand, tel que hormone stéroïde, rétinoïde ou hormone thyroïdienne. Cependant au sein de cette super famille, un groupe de facteurs, dont NR4A2 fait parti, ne possèdent pas de ligand identifié, c’est pourquoi ils sont dits orphelins. Le récepteur NR4A2, qui est un gène à réponse immédiate, induit par différents stimuli, est largement exprimé dans le cerveau. La description de son expression pendant le développement chez la souris montre que sa distribution anatomique va de pair avec certains neurones à dopamine. Afin d’étudier sa fonction, des souris (NR4A2 -/-) ont été générées. L’analyse de ces souris a montrée que l’absence de ce gène altère le développement et le maintien des neurones dopaminergiques de la substance noire et de l’aire tegmentaire ventrale, ainsi que l’expression de plusieurs gènes marqueurs du phénotype dopaminergique. Ces résultats ont placés NR4A2, au rang des gènes d’intérêt dans l’étude de la neuro-dégénérescence des neurones dopaminergiques impliquée dans la maladie de Parkinson. Pourtant, la description de l’expression de ce facteur chez le poisson, montre, que bien qu’il soit exprimé dans des régions dopaminergiques, il est aussi largement exprimé dans des régions qui ne sont pas dopaminergiques. L’analyse de NR4A2 par perte de fonction transitoire chez le poisson Danio rerio, sujet de ce mémoire, a permit de mettre en évidence certains aspects inconnus des fonctions de ce récepteur nucléaire. Ainsi, le gène NR4A2 régule l’expression d’un gène inhibiteur de CDK et l’expression d’un inhibiteur de neurogenèse. Par ce biais NR4A2 régule l’expression de gènes dits proneuraux, car exprimés dans les progéniteurs des neurones. Ces gènes proneuraux sont connus pour participer à la spécification cellulaire, à la sous spécification et à la différenciation de plusieurs phénotypes neuronaux. Ainsi, le gène NR4A2 induit chez Danio rerio le phénotype dopaminergique, mais il participe aussi à l’induction du neuro-phénotype NPY, du neuro-phénotype sérotoninergique et du neuro-phénotype Gabaergique. Ces nouvelles données in vivo font de NR4A2 un acteur important de la neurogénèse dont le rôle s’avère beaucoup plus large que ce qui était supposé jusqu’à présent
The gene NR4A2 is a member of the great family of the nuclear receptors which are factors of transcription activated by a ligand, such as hormone steroid, rétinoide or thyroid hormone. However within this great family, a group of factors, NR4A2 of which makes left, does not possess an identified ligand, this is why they are said orphans. The receptor NR4A2, an immediate early gene, induced by different stimuli, is widely expressed in the brain. The description of its expression during the development to the mouse shows that its anatomical distribution goes together with certain dopaminergics neurons. To study its function, mice (NR4A2-/-) were generated. The analysis of these mice showed that the absence of this gene alters the development and the preservation of dopaminergics neurons in substancia nigra and in ventral tegmental area, as well as the expression of several genes markers of the dopaminergic phenotype. These results placed NR4A2, to the rank of the genes of interest in the study of the neuro-degeneration of dopaminergics neurons involved in the Parkinson's disease. Nevertheless, the description of the expression of this factor in fish, shows, that although it is expressed in dopaminergics regions, it is also widely expressed in regions which are not dopaminergic. The analysis of NR4A2 by loss of function in the fish Danio rerio, the subject of this thesis, has allowed bringing to light certain unknown aspects of the functions of this orphan nuclear receptor. So, the gene NR4A2 regulates the expression of an inhibitive gene of CDK and the expression of an inhibitor of neurogenesis. By this way NR4A2 regulates the expression of said proneuraux genes, because expressed in neuron’s progenitors. These proneural genes are known to participate in the cellular specification, in sub-specification and in the differentiation of several neuronal phenotypes. So, the gene NR4A2 leads to the dopaminergic phenotype in Danio rerio, but it also participates in the induction of the neuro-phenotype NPY, the serotoninergic neuro-phenotype and the neuro-phenotype Gabaergique. These new in vivo data make of NR4A2 an important actor of the neurogenesis whose role turns out much wider than what was supposed until now

Experience of stress and/or cortisol, the end-product of activation of the hypothalamic-pituitary-interrenal (HPI) axis, may serve as a cue to trigger developmental plasticity. In fish, most research in this area has focused on effects of maternal stress or maternal cortisol levels on development, particularly with respect to the HPI axis and stress responses, and little attention has been paid to the effects of an endogenous stress response during early life. In the current study, zebrafish (Danio rerio) at four developmental stages (4, 7, 15 or 35 days post fertilization, dpf) were subjected to an air exposure stressor twice a day for two days. Individuals stressed early in life exhibited decreased survival and growth, increased whole-body Na+ and Ca2+ concentrations, and altered HPI axis activity associated with changes in anxiety-related behaviour at 7 to 35 dpf, with most effects diminishing with increasing age. Stress at 7 dpf was particularly effective at eliciting phenotypic changes, suggesting this age represents a critical window for cortisol to influence development. Finally, stress at 35 dpf induced masculinization, suggesting that cortisol influences sexual differentiation in zebrafish. These findings demonstrate that early-life stress in zebrafish triggers developmental plasticity, with effects on physiology and behaviour mediated by the HPI axis in an age-dependent manner.

O confinamento de pilares de concreto por meio de camisas de aço ou compósitos possui uma função importante na preservação, recuperação e reforço de estruturas, pois proporciona aumento de resistência e ductilidade desses elementos estruturais. Porém, grande parte dos modelos existentes apresenta limitações na previsão do comportamento do concreto confinado, principalmente por serem dependentes do tipo de confinamento. Portanto, este trabalho apresenta um modelo para descrição do comportamento tensão-deformação do concreto submetido a qualquer tipo de confinamento uniforme, ativo ou passivo, e confinado com diferentes materiais confinantes - aço ou compósitos. O modelo constitutivo associa plasticidade e dano a fim de prever adequadamente a resistência, deformabilidade e redução de rigidez elástica do concreto confinado. O modelo é desenvolvido para um processo incremental explícito de implementação, permitindo, portanto, o seu desenvolvimento em qualquer tipo de planilha. Finalmente, o modelo foi validado por meio de um conjunto representativo de experimentos encontrados na literatura.
Confinement of concrete columns through steel or composites jackets has an important function in the preservation, recovery and strengthening of structures, because it provides increased strength and ductility of these structural elements. However, most of the existing models have limitations in the prediction of the behavior of confined concrete, mainly because they are dependent on the type of confinement. This work presents a model for the description of the stress-strain behavior of the concrete submitted to any type of uniform confinement, active or passive, and confined with different confinement materials, steel or composites. The constitutive model associates plasticity and damage in order to predict with accuracy the strength, ultimate strain and reduction of elastic stiffness of the confined concrete. The model is developed by an explicit incremental implementation process allowing, therefore, its development in any type of spreadsheet. Finally, the model was validated through a representative set of experiments found in the literature.

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The work is a contribution to the mechanical numerical modeling of concrete structures with steel fibers using constitutive models based on Damage Mechanics behavior. The same, presents the formulation of a model proposed Damage admits that the concrete as initially isotropic elastic material, but with the evolution of the damage process, the material exhibits plastic deformations, anisotropy and bimodularidad induced damage. The incorporation of fibers in the modeling is performed through a homogenization procedure. The constitutive model for the concrete as well as the homogenization technique for dealing with the case of fiber concrete are implemented on a structural analysis program bar finite element laminated in layers. The parametric identification of the constitutive model together with the proposed homogenization is carried out using experimental results from the literature. Finally, numerical analysis of reinforced concrete beams reinforced with steel fibers and subjected to bending are conducted to assess the applicability of the constitutive model considered in this work. An attempt is thus contribute to the study of the deformation of fibrous concrete beams in service, in order to aggregate results and discussions in a future proposal of Brazilian technical standard for this type of structure.
O trabalho trata de uma contribuição à modelagem numérica do comportamento mecânico de estruturas de concreto com fibras de aço utilizando modelos constitutivos baseados na Mecânica do Dano. O mesmo, apresenta a formulação de um modelo de Dano proposto que admite o concreto como material inicialmente isótropo e elástico, mas com a evolução do processo de danificação o material exibe deformações plásticas, anisotropia e bimodularidade induzidas pelo dano. A incorporação das fibras na modelagem é efetuada através de um procedimento de homogeneização. O modelo constitutivo para o concreto, assim como a técnica de homogeneização para tratar do caso do concreto com fibras são implementados em um programa para análise de estruturas de barras com elementos finitos estratificados em camadas. A identificação paramétrica do modelo constitutivo, juntamente com a proposta de homogeneização, é realizada utilizando resultados experimentais encontrados na literatura. Por fim, análises numéricas de vigas de concreto armado reforçado com fibras de aço e sujeitas à flexão são conduzidas no sentido de avaliar a aplicabilidade do modelo constitutivo tratado neste trabalho. Procura-se, assim, contribuir para o estudo da deformabilidade de vigas de concreto fibroso em serviço, com o intuito de agregar resultados e discussões numa futura proposta de norma técnica brasileira para este tipo de estrutura.

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The development of new materials, in addition to the increasing industrial demand for more efficient numerical tools capable of predicting defects in metal forming processes, has stimulated research on new material models. In this context, the Continuum Damage Mechanics has proved to be able of successfully predicting ductile failure onset in metal forming operations. The main objective of this work is the study of a thermo-elastic-plastic formulation and thermo-mechanical coupling schemes aiming at prediction of mechanical degradation of ductile materials. The material internal degradation is described using a modified version of Lemaitre s (1985) damage model, in which the void opening and void closure effects associated to tensile and compressive stress states are accounted for. The mechanical and thermal problems are formulated using the Finite Element Method. Coupling of thermal effects is defined by a sensitivity factor included in the yield function and by a component describing the energy generated due to dissipation of plastic work. Two coupling procedures are addressed in this work: staggered scheme and iterative scheme. Accuracy of the iterative coupling scheme is assessed by the analysis of the load increment size. In this case, the results show that the iterative procedure is more accurate than the staggered scheme. The study of the coupled thermal and mechanical effects is discussed by the analysis of the influence of the temperature and the heat transfer coefficient based upon the simulation of tensile tests of U-notched specimens. The results show that the internal degradation of the material is strongly affected by its temperature and heat transfer coefficient, i.e., higher temperatures increase the material capacity to deform with smaller rates of material degradation.
O surgimento de novos materiais, aliado ao aumento da demanda industrial por ferramentas numéricas capazes de prever o aparecimento de defeitos em processos de conformação mecânica, tem estimulado o desenvolvimento de novos modelos materiais. A Mecânica do Dano Contínuo, em cujo contexto este trabalho está inserido, provou ser uma abordagem capaz de prever o início da fratura dúctil em operações de conformação mecânica. O principal objetivo deste trabalho é o estudo da formulação termo-elastoplástica de problemas com acoplamento termomecânico visando a sua aplicação na predição da degradação mecânica de materiais dúcteis. A descrição da degradação interna do material é feita através da modificação do modelo de dano de Lemaitre (1985) para incluir efeitos de abertura e fechamento de vazios relacionados a estados de tensão trativos e compressivos. Os problemas térmico e mecânico são formulados utilizando o método de Elementos Finitos. O acoplamento dos efeitos térmicos é definido através da inclusão de um fator de sensibilidade na função de escoamento e da geração de calor por dissipação plástica. Dois métodos de acoplamento foram abordados: método particionado e método iterativo. A avalição da precisão do método de solução iterativo do problema acoplado é feita através da análise de influência do incremento de carga. Neste caso, os resultados obtidos mostraram que o método iterativo é mais preciso que o método particionado. O estudo dos efeitos térmico e mecânico acloplados é feito através da análise da influência da temperatura e do coeficiente de troca de calor na simulação de um ensaio de tração usando um corpo de prova cilíndrico. Os resultados mostram que a degradação interna do material é fortemente influenciada pela temperatura do material e pelo coeficiente de troca de calor, ou seja, quanto maior a temperatura, maior é a capacidade do material de se deformar plasticamente com uma redução da taxa de degradação. interna do material.

Este trabalho apresenta uma contribuição ao emprego do Método dos Elementos Finitos Generalizados (MEFG) na análise tridimensional não-linear de sólidos. A análise numérica em campo não-linear, com modelos de dano e plasticidade, é original. O MEFG é uma formulação não-convencional do Método dos Elementos Finitos (MEF), que resulta da incorporação a este último de conceitos e técnicas dos denominados métodos sem malha, especialmente o enriquecimento da aproximação inicial (partição de unidade) por funções convenientes. Apresenta-se uma breve revisão bibliográfica dos métodos sem malha e do método dos elementos finitos generalizados, bem como suas principais características. Apresenta-se, com base no MEFG, a formulação de elementos tetraédricos e hexaédricos. Três modelos constitutivos são considerados visando análises não-lineares: o de plasticidade (perfeita ou com encruamento isótropo linear) com critério de plastificação de von Mises; o de dano frágil em concreto sob carregamento monótono crescente (modelo de Mazars) e o de dano e plasticidade acoplados (modelo de Lemaitre), próprio para materiais metálicos. São apresentados detalhes do código computacional, baseado no MEFG e nos modelos constitutivos acima mencionados, bem como resultados de análises numéricas. Esses resultados ressaltam algumas das vantagens do MEFG aplicado à análise não-linear, tais como: o enriquecimento da aproximação inicial limitado a regiões de interesse no domínio, como por exemplo, as que exibem elevados gradientes de deformação e tensão; uma definição mais precisa da distribuição de grandezas como a variável de dano e a tensão equivalente de von Mises, evitando a necessidade de alterações na malha; e a superação do travamento volumétrico associado a modelos de plasticidade
This work presents a contribution to the generalized finite element method (GFEM) employment in three-dimensional nonlinear analysis of solids. The nonlinear numerical analysis conduced with damage and plasticity models is original. GFEM is a nonconventional formulation of finite element method (FEM) which results from the addition to the latter of concepts and techniques of the so called Meshless methods, specially the enrichment of the initial approximations (partition of unity) by customized functions. A brief review of Meshless methods and generalized finite element method bibliography is presented, as well as their main features. Based on GFEM, the formulation of tetrahedral and hexahedral elements is shown. Three material laws are considered aiming nonlinear analysis: plasticity (perfectly plastic or linear isotropic hardening), with von Mises yield criterion; brittle damage on concrete under monotonic increasing loading (Mazars model) and damage coupled with plasticity (Lemaitre model), a suitable model for metals. Details of the computational code, based on GFEM and material laws mentioned above, are presented, as well as results of numerical analysis. These results emphasize some of the advantages of GFEM applied to nonlinear analysis, such as: enrichment of the basic approximations limited to some regions of interest in the domain, for instance, those exhibiting high strain and stress gradients; an accurated definition of the distributions of quantities like damage variable and von Mises equivalent stress, avoiding remeshing; and overcoming of volumetric locking associated to plasticity models

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The adoption of different ecological strategies is an important factor to determine the establishment and persistence of species in local communities. In general, the Cerrado is characterized by high fire frequency and poor soils. Generally under conditions of low fertility and high fire frequency the filtered species tend to have characteristics that represent adaptations to these environmental stresses. Considering that savanna species evolved under severe environmental filters, our aim was to evaluate how the adoption of different ecological strategies can determine the performance of the functional traits, the structure of the communities, and the relationship between a focal plant and its neighborhood. In this thesis work, which is divided into three chapters, we use three different scales to evaluate how species ecological strategies can determine the performance and establishment in local communities. In the first chapter, which is based on habitat scale, we evaluated how ecological strategies of generalist and specialist species of seasonal forest and savannas are fundamental for the establishment and persistence of the species in these habitats with marked differences in frequency in fire frequency and nutrient availability. In this chapter, we discuss that the different strategies adopted by species are in accordance to the limiting factors of the species occurrence in each of these environments. In the second chapter, which is based on community scale, we seek to understand how environmental gradients can determine different ecological strategies related to functional traits and density of individuals. We showed that the changes in trait values and density of individuals were more evident in the fertility gradient than toxicity, and that seasonal forest communities were more sensitive to changes savanna communities in both gradients. We also observed that species with conservative traits were associated with poor soils and species with acquisitive traits with more fertile soils. In the third chapter, which was developed at the individual scale, we discussed whether the characteristics and phylogenetic relationship of the neighboring plants influence leaf damage in trees and shrubs of savannas. In this chapter, we showed that the ecological and evolutionary distance between individual plants and neighboring plants does not determine the level of leaf damage by herbivores. We discussed that the dominance of generalist herbivores, co-evolution between plants and specialist herbivores, and preferential consumption of young leaves may be more important to determine the level of leaf damage than the neighboring context in which a given plant is inserted.
A adoção de diferentes estratégias ecológicas é um fator importante para determinar o estabelecimento e a persistência de espécies em comunidades locais. De maneira geral, o cerrado é caracterizado por uma alta frequência de fogo e solos pobres em nutrientes. Geralmente em condições de baixa fertilidade e alta frequência de fogo as espécies filtradas tendem a possuir características que representam adaptações a estes estresses ambientais. Considerando que as espécies de Cerrado se desenvolvem sob a atuação destes filtros ambientais, nosso objetivo foi avaliar como a adoção de diferentes estratégias ecológicas podem determinar a performance dos atributos funcionais, a estrutura das comunidades e a relação entre uma planta focal e sua vizinhança. Neste trabalho de tese, que está dividido em três capítulos, nós utilizamos três diferentes escalas para avaliar como estratégias ecológicas das espécies podem determinar seu desempenho e estabelecimento em comunidades locais. No primeiro capítulo que está baseado em uma escala de habitat, nós avaliamos como as estratégias ecológicas de espécies generalistas e especialistas de floresta estacional e cerrado sentido restrito são fundamentais para o estabelecimento e a persistência das espécies nestes habitats com diferenças marcantes em relação à frequência de fogo e disponibilidade de nutrientes. Neste capítulo, nós discutimos que as diferentes estratégias adotadas pelas espécies estão de acordo com os fatores limitantes da ocorrência de espécies em cada um destes ambientes. No segundo capítulo, que está baseado em escala de comunidades, nós buscamos compreender como os gradientes ambientais podem determinar diferentes estratégias ecológicas relacionadas aos atributos funcionais e a densidade de espécies. Nós demonstramos que as mudanças nos valores de atributos e densidade de espécies foram mais claras no gradiente de fertilidade do que toxicidade, e que comunidades de floresta estacional foram mais sensíveis a mudanças do que comunidades de cerrado sentido restrito em ambos os gradientes. Nós observamos também que espécies com atributos conservativos foram associados à solos pobres e espécies com atributos aquisitivos associado à solos mais férteis. Já no terceiro capítulo, que foi desenvolvido na escala de indivíduo, nós discutimos se as características e relação filogenética das plantas vizinhas influenciam o dano foliar em árvores e arbustos do cerrado. Neste capítulo, demonstramos que a distância ecológica e evolutiva entre plantas individuais e as plantas vizinhas não determina o nível de consumo foliar por herbívoros. Nós discutimos que a dominância de herbívoros generalistas, a co-evolução entre plantas e herbívoros especialistas, e o consumo preferencial de folhas jovens podem ser mais importante para determinar o nível de dano foliar do que o contexto de vizinhança em que uma dada planta está inserida.

In most organisms the cellular response to hypoxia is mediated by the master regulator hypoxia-inducible factor-1 (HIF-1). Zebrafish embryos can also arrest development (suspended animation) to tolerate low oxygen. I tested the hypothesis that induction of HIF-1 and associated target genes (eg. erythropoietin) during embryonic development would alter the hypoxia tolerance phenotype of larval and adult fish. I exposed zebrafish embryos at 3 developmental stages to acute (4 h) bouts of hypoxia (5% dissolved oxygen, DO) or anoxia (<0.5% DO). I found that embryos that mount a HIF-1 response have a greater hypoxia tolerance as larvae. Additionally, populations that experienced embryonic HIF-1 induction show an increase in the proportion of males (~70% male), that are more hypoxia tolerant than female fish, compared to control populations (~45% male). Overall, induction of HIF-1 during ontogeny alters the larval and adult zebrafish phenotype to better tolerate future hypoxic bouts.
NSERC

Στην παρούσα εργασία, για πρώτη φορά, τίθεται η πιο πρόσφατη εισαχθείσα τεχνολογία των –omics, η μεταβολομική, στην υπηρεσία της μοριακής βιολογίας για την μελέτη της θερμοκρασιακής μεταβολικής πλαστικότητας. Με τον όρο θερμοκρασιακή μεταβολική πλαστικότητα αναφερόμαστε στην ιδιότητα ενός γονότυπου να παράγει ποικίλους φαινότυπους σε επίπεδο μεταβολισμού, ως απόκριση στις θερμοκρασιακές συνθήκες στις οποίες υπόκειται. Σκοπός, λοιπόν της παρούσας ερευνητικής εργασίας ήταν η μελέτη της επίδρασης της πρώιμης θερμοκρασίας ανάπτυξης στο μεταβολικό πρότυπο ενήλικων θηλυκών και αρσενικών zebrafish σε γονάδες, εγκέφαλο και μυϊκό ιστό. Στα πλαίσια του σκοπού αυτού, τρεις πληθυσμοί zebrafish τοποθετήθηκαν στους 22οC, 28οC και 32οC από 0-20 ημερών, κατά την 20η ημέρα και οι τρεις πληθυσμοί αναπτύχθηκαν σε κοινή θερμοκρασία 28οC μέχρι την πάροδο εφτά μηνών, οπότε και πραγματοποιήθηκε δειγματοληψία. Ακολούθησε η ανάκτηση των μεταβολικών προτύπων, μετά την αριστοποίηση του πρωτοκόλλου για κάθε ιστό, χρησιμοποιώντας αέριο χρωματογράφο-φασματόμετρο μάζας. Τα αποτελέσματα της πολυπαραμετρικής στατιστικής ανάλυσης έδειξαν να μη διαφαίνεται θερμοκρασιακή μεταβολική πλαστικότητα στις γονάδες και στο μυϊκό ιστό, μιας και η ομαδοποίηση των μεταβολικών προτύπων έγινε ως προς το φύλο. Συγκεκριμένα και στις δυο κατηγορίες ιστών παρατηρήθηκε μεγάλη μείωση της μεταβολικής ενεργότητας στα θηλυκά ως προς τα αρσενικά zebrafish. Εξαιρετικής σημασίας, όμως, είναι το γεγονός ότι, για πρώτη φορά, αποδείχθηκε η επίδραση της πρώιμης θερμοκρασίας ανάπτυξης, στο μεταβολικό πρότυπο ενήλικων θηλυκών και αρσενικών zebrafish μέσω της δραματικής μείωσης της ενεργότητας του κεντρικού μεταβολισμού των εγκεφάλων 22ο C και 32ο C ως προς τους 28ο C.
With the term temperature induced metabolic plasticity one refers to the feature of genotype to produce different phenotypes on a metabolic level, due to the temperature conditions of its environment. The aim of this thesis is to study the effect of temperature on the metabolic profile during the early growth stage of adult male and female zebrafish in three tissues: gonads, brain and muscle. For this end three populations of zebrafish were bread at 22οC, 28οC and 32οC from 0 to 20 post-fertilization (pf) days. After the 20th pf day all populations were bread at 28οC for seven months. The metabolic profiles of all tissues were acquired using gas chromatography-mass spectrometry; in this thesis the protocol of metabolic profile acquisition was optimized for each tissue separately. Multivariate statistical analysis of the profiles showed no temperature plasticity in gonads and muscles. However, for the first time worldwide it was shown temperature induced plasticity in the brains metabolism of adult male and female zebrafish. Characteristically, a decline of the metabolic activity was observed in the 22ο C and 32ο C grown zebrafish compared to the 28ο C grown.

Ο όρος φαινοτυπική πλαστικότητα χαρακτηρίζει την ιδιότητα ενός γονότυπου να παράγει ποικίλους φαινότυπους, ως απόκριση στις περιβαλλοντικές συνθήκες στις οποίες υπόκειται (Pigliucci et al. 2006). Αυτή η απόκριση μπορεί να εκφράζεται σε μορφολογικό, βιοχημικό, φυσιολογικό ή αναπτυξιακό επίπεδο (οντογενετική πλαστικότητα), καθώς και στα πρότυπα συμπεριφοράς. Δεδομένης της σημασίας της φαινοτυπικής πλαστικότητας για τις λειτουργικές αποκρίσεις των ατόμων και των πληθυσμών (π.χ. αναλογία φύλου και πληθυσμιακή δομή), η μελέτη του φαινομένου στην ομάδα των ψαριών θεωρείται σημαντική τόσο για τους φυσικούς πληθυσμούς, σε οικολογικό ή εξελικτικό χρόνο, όσο και για τους εκτρεφόμενους. Η ολοκληρωμένη μελέτη της φαινοτυπικής πλαστικότητας, με έμφαση στους υποκείμενους μοριακούς και αναπτυξιακούς μηχανισμούς, μπορεί να δώσει πιο τεκμηριωμένες απαντήσεις στα ερωτήματα που σχετίζονται με τον τρόπο δράσης του φαινομένου στην οικολογία και στην εξέλιξη των ειδών, αλλά και σε πιο πρακτικά ζητήματα, όπως αυτά που αφορούν την εκτροφή ψαριών. Ο κύριος περιβαλλοντικός παράγοντας, υπεύθυνος για την εμφάνιση φαινοτυπικής πλαστικότητας στα ψάρια, φαίνεται ότι είναι η θερμοκρασία ανάπτυξης, η οποία έχει βρεθεί ότι επιδρά στο μεταβολισμό, την ανάπτυξη (Johston 1996, Stickland et al. 1998, Guderley 2004) και τη δομή των μυών (Guderley and Johston 1996, Ochi and Westerfield 2007), τους μεριστικούς χαρακτήρες (Lindsey 1998, Georgakopoulou et al. 2007), το σχήμα του σώματος (Loy et al. 1996, Georgakopoulou et al. 2007), την κολυμβητική ικανότητα (Fuiman and Batty 1997) και την αναλογία φύλου (Pavlidis et al. 2000, Koumoundouros et al. 2002a). Η θερμοκρασία ανάπτυξης, φαίνεται ότι επηρεάζει σημαντικά το ρυθμό αύξησης και διαφοροποίησης των ψαριών, τροποποιώντας το σωματικό μέγεθος όπου επιτελούνται τα διάφορα αναπτυξιακά γεγονότα, φαινόμενο γνωστό ως οντογενετική πλαστικότητα (Koumoundouros et al. 2001, Sfakianakis et al. 2004). Στην παρούσα εργασία εξετάστηκε η επίδραση της θερμοκρασίας πρώιμης ανάπτυξης, στη φαινοτυπική πλαστικότητα του είδους Danio rerio. Εξετάστηκε η αναλογία του φύλου και το σχήμα του σώματος των ψαριών, καθώς και ο ρυθμός ανάπτυξης των νυμφών και των ιχθυδίων κατά τη διάρκεια της εφαρμογής των διαφορετικών θερμοκρασιακών συνθηκών. Παράλληλα έγινε μια προσπάθεια προσδιορισμού του ευαίσθητου στην επίδραση της θερμοκρασίας, οντογενετικού σταδίου. Σύμφωνα με τον πειραματικό σχεδιασμό που ακολουθήθηκε, εξετάστηκε η επίδραση της θερμοκρασίας κατά τη διάρκεια δύο διαφορετικών πρώιμων οντογενετικών περιόδων, διάρκειας 280οd η κάθε 100 μία (28-308οd και 280-560οd). Στην πρώτη οντογενετική περίοδο, τα έμβρυα αφέθηκαν για είκοσι τέσσερις ώρες στους 28οC και στη συνέχεια διαχωρίστηκαν σε τρεις πληθυσμούς ψαριών οι οποίοι υποβλήθηκαν σε τρεις διαφορετικές θερμοκρασιακές συνθήκες (22, 28, 32οC) για μια περίοδο 280οd (28-308od, πρώτη υπό εξέταση οντογενετική περίοδος, ΟΠ1). Στη δεύτερη οντογενετική περίοδο, τα έμβρυα και οι νύμφες διατηρήθηκαν σε κοινές συνθήκες (28οC) μέχρι την 10η ημέρα μετά τη γονιμοποίηση. Στη συνέχεια διαχωρίστηκαν σε τρεις πληθυσμούς ψαριών, οι οποίοι υποβλήθηκαν σε διαφορετικές θερμοκρασιακές συνθήκες (22, 28 και 32οC) μέχρι την 560οd (280-560od, δεύτερη υπό εξέταση οντογενετική περίοδος, ΟΠ2). Και στις δύο περιπτώσεις, μετά το πέρας της περιόδου των 280οd με διαφορετική θερμοκρασιακή επίδραση, η ανάπτυξη πραγματοποιήθηκε σε κοινές συνθήκες (28οC). Τα πειράματα πραγματοποιήθηκαν εις διπλούν. Όλοι οι πειραματικοί πληθυσμοί, προήλθαν από κοινό απόθεμα αυγών. Η διατήρηση και η εκτροφή των νυμφών και των ενήλικων ψαριών πραγματοποιήθηκε βάσει μεθοδολογίας που περιγράφεται στο «The Zebrafish Book» (Westerfield 1995). Το φύλο των ενήλικων ατόμων προσδιορίστηκε μακροσκοπικά, έχοντας ως βάση τους χαρακτήρες της διογκωμένης κοιλιάς των θηλυκών ατόμων και του κίτρινου χρώματος των αρσενικών (Νεοφύτου 2003). Προκειμένου να επιβεβαιωθεί ο μακροσκοπικός προσδιορισμός του φύλου, ακολούθησε ο εγκλεισμός τριάντα συντηρημένων δειγμάτων (Εγκλεισμός σε Τechnovit 7100, τμήση 1-3 μm, χρώση με Polychrome I και II κια μικροσκοπική παρατήρηση των τομών). Για τη μελέτη της επίδρασης της θερμοκρασίας ανάπτυξης στην εξωτερική μορφολογία του σώματος των ενήλικων zebrafish, επιλέχθηκαν τυχαία 30 άτομα ανά φύλο και πειραματικό πληθυσμό, τα οποία υποβλήθηκαν σε ανάλυση γεωμετρικής μορφομετρίας. Με την ίδια μέθοδο αναλύθηκε και το σχήμα του σώματος των νυμφών της πρώτης και της δεύτερης υπό εξέταση οντογενετικής περιόδου, κατά το τέλος της εφαρμογής των διαφορετικών θερμοκρασιακών συνθηκών, στις 308od και στις 560od, αντίστοιχα. Στις νύμφες της πρώτης οντογενετικής περιόδου, ελήφθησαν επιπλέον δείγματα δέκα ημέρες μετά από το τέλος της εφαρμογής των θερμοκρασιακών συνθηκών. Για τη μελέτη του ρυθμού διαφοροποίησης, ελήφθησαν δείγματα νυμφών από κάθε πειραματικό πληθυσμό, μετά από τη λήξη της εφαρμογής των διαφορετικών θερμοκρασιακών συνθηκών. Συγκεκριμένα, από τους πληθυσμούς της ΟΠ1 δείγματα ελήφθησαν στις 588°d, ενώ από τους πληθυσμούς ΟΠ2, στις 560°d. Επίσης, εξετάστηκαν οι μεριστικοί χαρακτήρες των νυμφών της πρώτης και της δεύτερης οντογενετικής περιόδου, αφού προηγήθηκε διπλή χρώση των δειγμάτων με 101 Αλιζαρίνη (Alizarin Red S) και Κυανό της Αλσατίας (Alcian Blue) (Park and Kim 1984). Τα δείγματα φωτογραφήθηκαν ατομικά και μετρήθηκε το μεσουραίο μήκος (FL) του κάθε ατόμου. Στη συνέχεια, μετρήθηκε (1) ο αριθμός των κοιλιακών πλευρών, (2) ο αριθμός των πτερυγιοφόρων και των λεπιδοτριχίων του ραχιαίου και εδρικού πτερυγίου, και (3) ο αριθμός των δερματοτριχίων και των λεπιδοτριχίων του ουραίου πτερυγίου. Ο έλεγχος των διαφορών της αναλογίας φύλου μεταξύ των διαφορετικών πληθυσμών, έγινε με G-test (Sokal and Rohlf 1981). Για τη σύγκριση του σχήματος μεταξύ των δύο φύλων και των διαφορετικών πειραματικών πληθυσμών, εφαρμόστηκε ανάλυση κανονικών συνιστωσών (Canonical Variate Analysis, λογισμικό Statistica, έκδοση 6.0). Τέλος, ο έλεγχος των διαφορών του μέσου μήκους του σώματος μεταξύ των διαφορετικών πληθυσμών, πραγματοποιήθηκε με Mann-Whitney test (μη παραμετρικός έλεγχος), αφού προηγήθηκε ο έλεγχος ομοιοσκεδαστικότητας και κανονικής κατανομής. Η επίδραση της θερμοκρασίας στην αναλογία φύλου αποδείχθηκε στατιστικά σημαντική μόνο κατά την πρώτη οντογενετική περίοδο (p < 0.05, G-test), και όχι κατά τη δεύτερη (p > 0.05, G-test). Ο κατά ζεύγη έλεγχος των διαφορών στην αναλογία φύλου μεταξύ των διαφορετικών θερμοκρασιακών συνθηκών, έδειξε στατιστικά σημαντική επίδραση στην αναλογία φύλου μόνο μεταξύ των 22 και 28οC της ΟΠ1 (28-308οd), όπου η μέση συχνότητα των θηλυκών ατόμων ευνοείται στους 22°C (52,3% έναντι του 37,0% των 28°C, p < 0.05, G-test). Η ίδια συχνότητα εμφανίστηκε αυξημένη και στους 32°C (44,9% έναντι του 37,0% των 28°C), χωρίς ωστόσο να επιβεβαιώνεται και στατιστικά (p > 0.05, Gtest). Σε ότι αφορά τη δεύτερη οντογενετική περίοδο (280-560°d), η μέση συχνότητα των θηλυκών ατόμων στους 22°C (51,9%) και στους 32°C (46,1%), εμφανίζεται αυξημένη σε σχέση με αυτή των 28°C (44,5%), χωρίς ωστόσο οι διαφορές αυτές να είναι στατιστικά σημαντικές (p > 0.05, G-test). Η ύπαρξη μη στατιστικά σημαντικής διαφοράς της αναλογίας φύλου μεταξύ των 28 και 32°C και στατιστικά σημαντικής διαφοράς μεταξύ των 22 και 28°C, υποδεικνύει ότι το πρότυπο απόκρισης της αναλογίας φύλου στην θερμοκρασία πρώιμης ανάπτυξης εμφανίζει ανεξαρτησία στο εύρος 28-32°C και θηλυκοποίηση στους 22°C. Σε όλα τα οντογενετικά παράθυρα που εξετάστηκαν και σε όλες τις πειραματικές επαναλήψεις, τα αποτελέσματα της ανάλυσης των κανονικών συνιστωσών, έδειξαν ότι το φύλο και η θερμοκρασία πρώιμης ανάπτυξης επέδρασαν σημαντικά στο σχήμα του σώματος των ενήλικων ατόμων zebrafish. Η κατά φύλο γεωμετρική ανάλυση του σχήματος, έδειξε ότι η θερμοκρασία ανάπτυξης κατά την ΟΠ1 (28-308od) και ΟΠ2 (280-560od) επιδρά σημαντικά στο σχήμα 102 του σώματος και των δύο φύλων, με τους τρεις πληθυσμούς κάθε οντογενετικής περιόδου και επανάληψης να διαχωρίζονται πλήρως μεταξύ τους κατά μήκος των δύο κανονικών μεταβλητών. Η εξέταση του σχήματος του σώματος των νυμφών της ΟΠ1 (28-308οd), στις 308οd και στις 588 οd, και της ΟΠ2 (280-560οd), στις 560οd, έδειξε ότι η θερμοκρασία ανάπτυξης επηρέασε το σχήμα του σώματός τους. Σε όλες τις, οι διαφορές στο σχήμα οφείλονται τόσο στις ομοιόμορφες συνιστώσες του σχήματος, όσο και στις μη ομοιόμορφες, με τις πρώτες να συμβάλλουν στη νωτοκοιλιακή διεύρυνση ή συμπίεση του σώματος των νυμφών, ανάλογα με την περίπτωση. Ενώ όμως η επίδραση της θερμοκρασίας στο σχήμα του σώματος είναι σημαντική, δε φαίνεται να υπάρχει κάποιο καθορισμένο – σταθερό πρότυπο επίδρασης των κανονικών συνιστωσών. Αυτό πιθανότατα οφείλεται στο ότι δεν είχαν φτάσει όλες οι νύμφες στο ίδιο στάδιο ανάπτυξης κατά τις ημέρες των δειγματοληψιών. Τα δείγματα των νυμφών που λήφθηκαν μετά την έκθεση των πληθυσμών στις τρεις διαφορετικές θερμοκρασιακές συνθήκες, κατά την πρώτη (28-308οd) ή τη δεύτερη (280- 560οd) οντογενετική περίοδο, διαφοροποιήθηκαν σημαντικά ως προς το μέσο μεσουραίο μήκος (FL) των ατόμων. Συγκεκριμένα, η έκθεση στους 22οC οδήγησε σε σημαντική μείωση του FL των ατόμων (p<0.05, Mann Whitney U-test), παρά το ότι τα δείγματα λήφθηκαν στο ίδιο θερμικό άθροισμα. Τα αποτελέσματα έδειξαν, ότι η ανάπτυξη των ψαριών που αφέθηκαν στους 22οC, κατά τη δεύτερη υπό εξέταση οντογενετική περίοδο (280-560οd), καθυστέρησε σε σχέση με τους πληθυσμούς των 28 και 32οC. Αυτό φαίνεται από τα διαγράμματα που αφορούν τον αριθμό των πτερυγιοφόρων και των ακτινών του ραχιαίου πτερυγίου, τον αριθμό των πτερυγιοφόρων και των ακτινών του εδρικού πτερυγίου, καθώς και τον αριθμό των κοιλιακών πλευρών. Συγκεκριμένα, η ολοκλήρωση της ανάπτυξης των πτερυγιοφόρων του ραχιαίου πτερυγίου, των ακτινών του εδρικού και των κοιλιακών πλευρών, καθυστερεί στους 28οC, σε σχέση με τους 32 οC. Η εμφάνιση των ακτινών του ραχιαίου πτερυγίου, καθυστερεί στους 28 οC, σε σχέση με τους 32 οC. Τέλος, η εμφάνιση των πτερυγιοφόρων του εδρικού πτερυγίου, καθυστερεί στους 22 οC, σε σχέση με τους 28 οC. Σε ότι αφορά τα άτομα της πρώτης οντογενετικής περιόδου, οι χαρακτήρες που μελετήθηκαν είτε είχαν εμφανιστεί νωρίτερα από τη λήψη των δειγμάτων, είτε είχε ολοκληρωθεί αργότερα η ανάπτυξή τους.
The term phenotypic plasticity characterizes the property of a genotype to produce different phenotypes in response to distinct environments (Pigliucci et al. 2006). This response can express itself in a morphological, biochemical, physiologic or developmental level (developmental plasticity), or even through changes in the models of behavior. Due to its great importance among individuals in a population at a certain time period or in future generations (e.g. proportion of sex and demographic structure), the study of the phenotypic plasticity in the group of fish is considered significant not only for the natural populations, at the ecological or evolutionary level, but for the cultured populations too. The complete study of phenotypic plasticity, including the underlying molecular and developmental mechanisms, can answer questions related with the way the phenomenon acts in the ecology and species development, but also in more practical applications, such as those that concern aquaculture. One of the most important environmental factors responsible for the appearance of plasticity in fish appears to be the developmental temperature, which affects metabolism, muscle growth (Johston 1996, Stickland et al. 1998, Guderley 2004) and structure (Guderley and Johston 1996, Ochi and Westerfield 2007), meristic characters (Lindsey 1998, Georgakopoulou et al. 2007), body shape (Loy et al. 1996, Georgakopoulou et al. 2007), swimming performance (Fuiman and Batty 1997) and sex ratio (Pavlidis et al. 2000, Koumoundouros et al. 2002a). The developmental temperature influences considerably growth rate and differentiation of fish, modifying the body size as development proceeds. This phenomenon is known as developmental plasticity (Koumoundouros et al. 2001, Sfakianakis et al. 2004). The present study examined the effect of precocious developmental temperature, in phenotypic plasticity of Danio rerio. The Sex ratio and body shape of fish was analyzed, as well as the growth rate of larvae and juveniles during the application of different temperature conditions. At the same time the most sensitive developmental period in the effect of temperature was determined. According to the experimental design, the effect of temperature was examined during two different early developmental periods of 280°d, each (28-308°d and 280-560°d). Two experimental groups of fish were subjected in duplicate to three different temperatures. More specifically, in the first developmental period the embryos were left for twenty four hours in 28°C and then separated in three populations, which were submitted to three different temperature conditions (22, 28 and 104 32°C) for a period of 280°d (28-308°d, first developmental period DP1). In the second developmental period the embryos and the larvae were maintained under common conditions (28°C) up to the 10th day post fertilization. Then they were separated in three populations, which were submitted to three different temperature conditions (22, 28 and 32°C) up to the 560th °d (280-560°d, second developmental period DP2). In both developmental periods that were examined, after the end of the 280°d of temperature effect, fish growth was completed under common conditions (28°C). All the eggs of the experimental populations were obtained from a broodstock of the same genetic origin. The maintenance and the culture of larvae and adult fish were as described in "The Zebrafish Book" (Westerfield 1995). The sex of adult individuals was determined macroscopically using the characters of the swelled abdomen of females and the yellow color of males (Neophytou 2003). In order to confirm the macroscopic determination of sex, thirty fixed samples of zebrafish were enclosed for histology sections (Technovit 7100, sections of 1-3μm and staining with Polychrome I and II) and microscopy. To study the temperature effect on growth and body shape of adult zebrafish, 30 individuals per sex and experimental population were selected randomly and were submitted in geometric morphometrics analysis. The body shape of the larvae of the first and the second developmental period, was analyzed with the same method, at the end of the different temperature conditions, in 308°d and 560°d, respectively. Additional samples were selected in the first developmental period, ten days after the end of the three different temperature regimes. To study the temperature effect on growth rate and differentiation, samples of larvae were obtained from each experimental population, following completion of the different temperature periods. From the populations of DP1, samples were taken at 588°d, while from DP2 samples were obtained at 560°d. The meristic characters of the larvae from the first and second developmental period were examined, after staining of the samples with Alizarin Red and Alcian Blue (Park and Kim 1984). Larvae were photographed and the fork length of each individual was measured (FL). The meristic characters that were examined are (1) the number of ribs (2) the number of pterygiophors and lepidotrichia of the dorsal and the anal fin, and (3) the number of pterygiophors and lepidotrichia of the caudal fin. Sex ratio differences between populations were studied with G-test (Sokal and Rohlf 1981). Canonical Variate Analysis was applied for the comparison of the body 105 shape between the two sexes and between the different experimental populations (Statistica, 6.0). Finally, the differences of medium total length between the different populations was examined with the Mann-Whitney test (non parametric). The sex ratio was significantly affected only at the first developmental period (p < 0.05, G-test), and not at the second (p > 0.05, G-test). The paired control of changes in the sex ratio between the different temperature conditions at DP1 (28-308°d), showed a significant effect only between the populations of 22°C and 28°C (52.3% females in 22°C vs. 37.0% in 28°C, p < 0.05, G-test). The female/male frequency was also increased in the 32°C group (44.9% females in 32°C vs. 37.0% females in 28°C), without however being confirmed as statistically significant (p > 0.05, G-test). Regarding the second developmental period (280-560°d), the medium frequency of the female individuals in the 22°C (51.9%) and 32°C (46.1%) groups, was also increased compared to 28°C (44.5%), although these differences were not confirmed as statistically significant (p > 0.05, G-test). The significant differences in sex ratio between the 22 and 28°C, and the non significant differences between 28 and 32°C indicate that the model of response in sex ratio due to the different developmental temperature shows autonomy in the range of 28-32°C and feminization at 22°C. In both ontogenetic windows that were examined and in all experimental repetitions, the results of the canonical variables showed that sex and developmental temperature significantly affected the bodyshape of the adult individuals. The body shape analysis in accordance with the gender of the individuals, was significantly affected from the developmental temperature at DP1 (28-308°d) and DP2 (280-560°d). The three populations of each ontogenetic period were totally separated amongst them, along the two axes of canonical variables CV1 and CV2. The body shape analysis of the larvae at DP1 (28-308οd), in 308οd and in 588 οd, and at DP2 (280-560οd), in 560οd, indicates that developmental temperature significantly affected their body shape. In all cases, the differences in body shape are contributed from uniform, as much as from non uniform components of shape. Despite the significant effect of developmental temperature in larval body shape, there is not a stable model of the way that canonical variables effect, probably because larvae were not at the same stage of growth during sampling. The total (TL) or the fork length (FL) of the larvae that were analyzed following development at the three different temperatures during the first (28-308°d) or the second (280-560°d) developmental period, was significantly differentiated. The exposure at 22°C 106 led to important reduction of the individuals FL (p < 0.05, Mann-Whitney test), despite the fact that all samples were of the same age. These results showed that the growth of fish that were submitted to 22°C during DP2 (280-560°d) was delayed relative to the populations at 28 and 32°C. This is noticeable from the diagrams that refer to the number of pterygiophors and lepidotrichia of the dorsal fin, the number of pterygiophors and lepidotrichia of the anal fin and the number of ribs. The completion of growth of pterygiophors of the dorsal fin, of lepidotrichia of the anal fin and the abdominal ribs, are also delayed at 28°C vs. 32°C. The appearance of lepidotrichia of the dorsal fin is delayed at 28°C vs. 32°C, as well. Finally, the appearance of pterygiophors of the anal fin is delayed at the 22°C vs. 28°C. Regarding the individuals of DP1, the development of the meristic characters that were studied was completed either earlier or later than the time of sampling, making it difficult to evaluate differences within various groups.

Σκοπός της εργασίας ήταν να μελετήσει την επίδραση του ενδοειδικού ανταγωνισμού, κατά τη διάρκεια της μεταμόρφωσης, στο ρυθμό αύξησης, στην αναλογία φύλου και στο σχήμα του σώματος του zebrafish. Εφαρμόσθηκαν 4 πειραματικές συνθήκες σε τρεις επαναλήψεις. Οι πειραματικοί πληθυσμοί αποτελούνταν από 200 άτομα μήκους 5,6–6,5 mm FL («αμυνόμενα», ηλικίας 15 ημερών μετά τη γονιμοποίηση, dpf), τα οποία ήταν υποκείμενα στην επιθετική συμπεριφορά κανενός, ενός, τριών ή πέντε ατόμων με μέσο FL 12,6-13,3 mm FL («επιτιθέμενα», ηλικίας 30 dpf). Κάθε πειραματικός πληθυσμός βιντεοσκοπείτο δυο φορές την εβδομάδα (λήψη διάρκειας 15 λεπτών, 2 ώρες μετά το τάισμα). Τριάντα ημέρες μετά την έναρξη των πειραμάτων (50 dpf για τα «αμυνόμενα άτομα») τα «επιτιθέμενα» άτομα απομακρύνθηκαν από τους πειραματικούς πληθυσμούς. Η εκτροφή των «αμυνόμενων» ατόμων συνεχίστηκε μέχρι την ενηλικίωσή τους (150-190 dpf). Η ανάλυση των βιντεοσκοπήσεων έδειξε ότι ο αριθμός των «επιτιθέμενων» ατόμων σε κάθε ενυδρείο επηρέασε σημαντικά τον αριθμό των επιθέσεων που δέχονταν τα «αμυνόμενα» άτομα (p<0,05, Kruskall-Wallis), αλλά μόνο στην οντογενετική περίοδο των 9,5-12,5 mm FL. Η ένταση της επιθετικότητας επηρέασε σημαντικά το ρυθμό αύξησης των «αμυνόμενων» ατόμων, όπως αυτός εκφράστηκε από το FL στην ηλικία των 50 dpf (p<0,05, ANOVA). Ως προς την αναλογία φύλου, τα αποτελέσματα δεν έδειξαν κάποια σημαντική επίδραση (p>0,05, G-test) του εξεταζόμενου παράγοντα. Τέλος, αναφορικά με το σχήμα του σώματος των ενήλικων «αμυνόμενων» ατόμων, τα αποτελέσματα έδειξαν σημαντική επίδραση της έντασης της επιθετικότητας (p<0,05, MANOVA). Η γεωμετρική μορφομετρική ανάλυση έδειξε σημαντικές διαφορές στο σχήμα σώματος των θηλυκών μεταξύ των συνθηκών με το ένα «επιτιθέμενο» άτομο και αυτών με τα τρία και πέντε. Στα αρσενικά σημαντικές διαφορές παρατηρήθηκαν ανάμεσα στις συνθήκες με το ένα και τρία «επιτιθέμενα» άτομα. Τα πιο σημαντικά landmarks τα οποία συνέβαλαν στη μεταβολή τους σχήματος ήταν το πρόσθιο άκρο της βάσης του κοιλιακού πτερυγίου για τα θηλυκά, ενώ για τα αρσενικά το πρόσθιο άκρο της βάσης του κοιλιακού πτερυγίου και το κάτω άκρο του βραγχιακού επικαλύμματος. Στην παρούσα εργασία, για πρώτη φορά δείχθηκε η επίδραση της ενδοειδικής επιθετικότητας κατά τη φάση της μεταμόρφωσης, στο σχήμα του σώματος των ενήλικων ψαριών.
The present study examined the effect of intraspecific competition during metamorphosis, in the specific growth rate, in the ratio sex and on the adult body shape zebrafish (Danio rerio). Four experimental conditions were conducted in three replicate. The experimental populations were constituted by 200 individuals which length was 5,6-6,5 mm FL (“defendants”, age of 15 days post fertilization, dpf) and they were subjects in aggressive behavior of no, three or five individuals with mean FL 12,6-13,3 mm FL (“aggressors”, age 30 dpf). Each experimental population videotapped two times a week (time 15 minutes, 2 hours after feeding). Thirty days after the beginning of experiments (50 dpf for the “defendant individuals”) the “aggressor” individuals were removed by the experimental populations. The stockfarming of “defendant” individuals was continued up to adultness (150-190 dpf). The analysis of videos showed that the number of “aggressor” individuals in each aquarium influenced considerably the number of attacks that accepted the “defendant” individuals (p<0,05, Kruskall-Wallis), but only in the ontogenetic period 9,5-12,5 mm FL. The intensity of aggressiveness influenced considerably specific growth rate of “defendant” individuals, as this was expressed by the FL in the age of 50 dpf (p<0,05, ANOVA). As for ratio of sex, the results did not show important effect (p>0,05, G-test) in the examined factor. Finally, as far as the intensity of aggressiveness is concerned, the adult body shape of “defendant” individuals affected significantly (p<0,05, MANOVA). The geometric morphometrics analysis showed the important differences in female body shape between conditions with one “aggressor” individual and these with three and five. Important differences were observed between the conditions with one and three male “aggressor” individuals. Most important landmarks that contributed in males body shape were anterior base of anal fin, while for females were the base of anal fin and the base of gill arch. For first time was shown that intraspecific aggressiveness, at the phase of metamorphosis, affects the adult body shape.

In order to ascertain the degree of compatibility in developmental restructuring and behavioral plasticity between two fish species frequently made subject of laboratory research (Metriaclima zebra & Danio rerio), alternative trophic niche exposure experiments utilizing novel three-prong feeding treatments were conducted to obtain morphometric data, which demonstrated both species do bear some degree of plasticity. The results are somewhat complicated by differences in locality of detectable restructuring, which may be due to disparity in the form-function relationship for each species’ lineage. Each is notable in the manner of respective species’ jaw protrusion, as it is driven by anterior kinethmoid rotation in D. rerio. as opposed to force imparted upon the rostral cartilage of the premaxilla’s articular process in M zebra. Each is markedly distinct in the pharyngeal jaw as well, as zebrafish (also toothless at the oral jaw) bear teeth only on the lower set at the posterior of the mouth, while cichlids bear teeth on all jaws and additionally possess a unique, fused lower pharyngeal jaw. However, accounting for this difference in experimental models does allow for direct comparison, both at the morphological/behavioral and potentially the genetic level, though additional research is necessary. The evidence provided here also provides encouragement that more nuanced approaches to laboratory trophic niche exposure experiments could elucidate further evidence on the nature of phenotypic plasticity.

Η θερμοκρασία ανάπτυξης αποτελεί παράγοντα μεγάλης σημασίας στην οντογένεση των ιχθύων, αφού ως ποικιλόθερμοι οργανισμοί είναι συνεχώς εκτεθειμένοι στις μεταβολές του περιβάλλοντός τους. Έχει παρατηρηθεί πως η θερμοκρασία ανάπτυξης δύναται να προκαλέσει μετατόπιση του χρονοδιαγράμματος των οντογενετικών γεγονότων και πλαστικότητα σε μορφολογικούς και φυσιολογικούς χαρακτήρες (πχ στο μυοσκελετικό και το καρδιαγγειακό σύστημα). Ωστόσο, μέχρι σήμερα δεν έχει μελετηθεί η επίδραση της θερμοκρασίας ανάπτυξης στο πρότυπο της γονιδιακής έκφρασης του zebrafish και σκοπός της παρούσας εργασίας είναι η μελέτη του ολικού μεταγραφικού προτύπου νυμφών zebrafish. Για το λόγο αυτό σχεδιάστηκαν δύο πειράματα, όπου τρεις θερμοκρασιακές συνθήκες ανάπτυξης (22, 28 και 32oC) εφαρμόστηκαν στην πρώιμη οντογενετική περίοδο: για το διάστημα 0-20 dpf* (1ο πείραμα) και 10-20 dpf (2ο πείραμα). Πραγματοποιήθηκε απομόνωση ολικού RNA από τα άτομα ηλικίας 20 dpf όλων των πληθυσμών και από τα άτομα ηλικίας 10 dpf του πληθυσμού των 28oC του 2ου πειράματος και ακολούθησε υβριδοποίηση σε ολιγονουκλεοτιδικές μικροσυστοιχίες Affymetrix, με 15.509 αντιπροσωπευτικές αλληλουχίες γονιδίων (probe sets). Τα 21 μεταγραφικά προφίλ επεξεργάσθηκαν με τα εξειδικευμένα προγράμματα ανάλυσης μικροσυστοιχιών, dChip και MeV (v.4.5.1). Η κανονικοποίηση και το φιλτράρισμα των δεδομένων των μικροσυστοιχιών απέδωσε μεταγραφικά πρότυπα με 9.488 probe sets. Με τεχνικές πολυπαραμετρικής στατιστικής ανάλυσης (HCL και PCA) πραγματοποιήθηκαν οι συγκρίσεις των μεταγραφικών προτύπων μεταξύ των πειραματικών πληθυσμών για κάθε θερμοκρασία ανάπτυξης και οντογενετικό στάδιο. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων μεταξύ των δύο πειραμάτων, ii) σαφή διαχωρισμό των προτύπων των 28oC και 32oC ως προς αυτά των 22oC και στα δύο πειράματα, και iii) σαφή διαχωρισμό των προτύπων των 28oC διαφορετικού οντογενετικού σταδίου (ηλικίας 10 dpf vs 20 dpf). Θα αναμέναμε τα πρότυπα έκφρασης των 28oC να παρουσιάζουν παρόμοιο πρότυπο, κάτι που δεν παρατηρείται. Αυτό οφείλεται στο πειραματικό σφάλμα που υπεισέρχεται από το διαφορετικό χρόνο πραγματοποίησης των δύο πειραμάτων και τις ρυθμίσεις κατά την υβριδοποίηση. Έτσι πραγματοποιήθηκε η κανονικοποίηση των “28”, που απαλείφει το πειραματικό σφάλμα. Οι HCL και PCA αναλύσεις έδειξαν i) σαφή διαχωρισμό των μεταγραφικών προτύπων των 28oC και 32oC ως προς αυτά των 22oC ως αποτέλεσμα της επίδρασης της θερμοκρασίας ανάπτυξης, ii) σαφή διαχωρισμό των προτύπων των 22oC των δύο πειραμάτων ως αποτέλεσμα της επίδρασης της περιόδου εφαρμογής και διάρκειας της θερμοκρασιακής αγωγής και iii) σαφή διαχωρισμό των πρότυπων των 28oC (ηλικίας 10 dpf vs 20 dpf) ως αποτέλεσμα της επίδρασης του οντογενετικού σταδίου. Ακολούθως, πραγματοποιήθηκε ανάλυση σημαντικότητας (SAM) και λειτουργική γονιδιωματική ανάλυση (λογισμικό DAVID) των στατιστικώς σημαντικών γονιδίων που διαφοροποιούν τα πρότυπα. Η ανάλυση των γονιδίων, ως προς την ιστοειδική έκφραση ανέδειξε γονίδια που σχετίζονται με την ανάπτυξη του ματιού, των θωρακικών πτερυγίων και του εγκεφάλου στους πληθυσμούς των 22oC έναντι των 28oC και 32oC. Η παρούσα εργασία αποδεικνύει την επίδραση της θερμοκρασίας ανάπτυξης και της διάρκειας της θερμοκρασιακής αγωγής, καθώς επάγει μηχανισμούς πλαστικότητας στο επίπεδο της γονιδιακής έκφρασης. Τέλος, υποδεικνύεται πως η πρώιμη οντογενετική περίοδος τείνει να είναι περισσότερο θερμοευαίσθητη, καθώς παρατηρείται εντονότερη επίδραση της θερμοκρασίας (στην περίπτωση των 22οC)στην ανάπτυξη του ατόμου. *dpf: days post fertilization
Developmental temperature plays a principal role in the ontogeny of fish. It is known that developmental temperature may shift the initiation time of the ontogenetic stages and induce plasticity in morphological and physiological characters e.g. the musculoskeletal and the cardiovascular system. However, its effect on the gene expression pattern has not previously been attempted for zebrafish. In the present study, zebrafish Affymetrix microarrays of 15,509 probe sets were used to map the transcriptome profile of: a) 20 dpf* old zebrafish larvae at three developmental temperatures, i.e. 22oC, 28oC and 32oC (1st experiment) and b) 20 dpf old zebrafish larvae, which were all grown at 28oC for the first 10 days and subsequently divided into three groups, which were grown at 22oC, 28oC and 32oC, respectively; the profile of 10 dpf old larvae was also measured (2nd experiment). We have isolated total RNA from the above populations and then, hybridization of RNA samples has been done on oligonucleotide Affymetrix microarrays of 15,509 probe sets. All 21 profiles were normalized and filtered (dChip software), and multivariate statistical analysis techniques were used on the normalized 9,488 probe set expression profiles (TM4 MeV software). Hierarchical Clustering (HCL) and Principal Component Analysis (PCA) on expression profiles indicated: a) clear separation of the two experiments based on their transcriptomic patterns, b) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC in both experiments and c) clear separation of the 28oC profiles based on the developmental stage. We would expect expression profiles of 28oC to be clustered together, though this was not observed because of experimental parameters during the hybridization, as the two experiments were carried out independently on different dates. So the normalization of “28” profiles took place, in order to eliminate the experimental noise. HCL and PCA, then, indicated: a) clustering of the 28oC and 32oC profiles of the 20 dpf old larvae separately from those at 22oC, as the effect of developmental temperature, b) clear separation of 22oC profiles of the two experiments, based on the effect of the period and duration of thermal conditions and c) clear separation of the 28oC profiles based on the developmental stage. Then, Significant Analysis of Microarrays (SAM) and Functional Genomic Classification Analysis (DAVID software) of statistically significant genes was carried out. Analysis of genes based on tissue-specific expression indicated characteristic genes for the development of the eye, pectoral fins and brain in 22oC profiles versus 28oC and 32oC profiles. The present study has proved that thermal effect is determinative among the early ontogenetic stage, especially in the case of longer cold thermal period, and developmental temperature may induce plastic response of gene expression, that could affect the fate of fish. *dpf: days post fertilization