Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.

Glutamate AMPA receptors (AMPARs) are major excitatory receptors in the vertebrate CNS. In many biological systems there are changes in the properties of AMPARs during development that are essential for providing an increase in efficiency of information transfer between neurons and a refinement of motor co-ordination and sensory perception and cognition. It is not surprising that improper development or loss of function of AMPARs can lead to many neurological disorders such as epilepsy and amyotrophic lateral sclerosis. Thus, determining the mechanisms by which AMPARs mature is of particular importance. The objectives of my thesis were to characterize the developmental changes in AMPAR-mediated currents in zebrafish Mauthner cells and to determine the mechanisms underlying any changes. The major findings reported in this thesis are that (1) there are developmental changes in the properties of AMPAR-currents as the Mauthner cell matures; (2) the mechanism underlying these changes is a switch in the composition of AMPA receptor subtypes; and (3) PKC is necessary for the developmental switch in AMPAR subtypes from slow receptors to fast receptors. These findings provide valuable insights into the mechanism underlying the development of AMPARs. In addition, they provide the first instance of a signalling link (PKC) required for the developmental subunit switch and the developmental speeding of AMPAR kinetics. / Physiology, Cell Biology and Developmental Biology

In vertebrates, the otic placode forms inner ear and epibranchial placodes produce sensory ganglia within branchial clefts. Fibroblast growth factor (FGF) family of protein ligands from the surrounding tissues are responsible for otic and epibranchial placode induction. Members of pax2/5/8 family of transcription factors function as mediators during otic induction. To understand the temporal and spatial requirements of Fgf and their interaction with pax2/8 for otic induction, we used heat shock inducible transgenic lines of zebrafish to misexpress fgf3/8 and pax2a/8 under the control of hsp70 promoter. Loss of function studies were done to examine the functions of pax2/8 genes in regulating otic and epibranchial development. We show that global transient activation of hs:fgf3 or hs:fgf8 at mid-late gastrula stages (7-8 hpf) severely impairs otic induction, in part by disrupting formation of the principal signaling centers in the hindbrain. Additionally, mosaic studies show that high-level misexpression blocks otic fate cell-autonomously, whereas low to moderate levels promote otic development. At later stages high-level Fgf misexpression, both globally and locally does not inhibit otic fate, but rather causes a dramatic expansion of endogenous otic domains. Misexpression of hs:pax2a or hs:pax8 also expands endogenous otic domains but is not sufficient to bypass the requirement for Fgf signaling. Co-misexpression of Fgf with pax2a or pax8 leads to production of ectopic otic tissue in a broad range of cranial ectoderm. These data show that changes in timing, distribution and level of Fgf signaling and its downstream effectors influences otic induction. We show that otic and epibranchial placodes are induced at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 along with pax2a/pax2b downregulate foxi1 expression in otic cells, which is necessary for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Blocking functions of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These results support the model whereby the otic placode forms first and induces epibranchial placodes through pax2/8-dependent Fgf24 signaling.

Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish ??you-type?? mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant ??you??. you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 44-52).

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

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.

Orientador: Vanderlan da Silva Bolzani / Coorientador: Emerson Ferreira Queiroz / Banca: Nivaldo Boralle / Banca: Cristiano Soleo de Funari / Resumo: A espécie Tetrapterys mucronata (Malpighiaceae), uma das espécies ocasionalmente utilizados na Ayahuasca, despertou há algum tempo o interesse do grupo de pesquisa NuBBE para a realização do estudo químico e avaliação das atividades biológicas devido a sua utilização nos rituais religiosos conhecidos como "Santo Daime". Este estudo revelou resultados interessantes nos ensaios preliminares voltados para a identificação de potenciais inibidores de acetilcolinesterase, e mostrou que a espécie T. mucronata tem alcaloides e compostos fenólicos como principais constituintes químicos. Estes resultados levaram ao estudo de outras espécies de Tetrapterys ainda sem estudo químico, incluindo espécies que pertenciam a esse gênero e recentemente foram reclassificados como Niedenzuella, como no caso da Niedenzuella multiglandulosa, espécie selecionada para esta pesquisa de mestrado. Esta espécie é conhecida devido aos efeitos tóxicos que causam a morte de rebanhos bovinos, e de outros mamíferos, em geral, causando prejuízos substanciais na balança comercial brasileira. Este trabalho tem como objetivo a busca de compostos tóxicos presentes nas folhas de Tetrapterys multiglandulosa. Essa espécie foi selecionada por serem relatados na literatura surtos de intoxicação do gado (em 2004 e 2005) em áreas altamente infestadas pela planta, causando morte de vacas e aborto de bezerros no estado do Mato Grosso do Sul. Estudos recentes encontrados na literatura indicam a presença de monofluoracetato (... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tetrapterys mucronata (Malpighiaceae) species, one of the species occasionally used in Ayahuasca, has become of interest of NuBBE research group to perform a chemical study for an evaluation of the biological activities due to its use in religious rituals known as "Santo Daime". This study revealed interesting results in preliminary assays aiming to identify acetylcholinesterase potential inhibitors, and that this species has alkaloids and phenolic compounds as its major chemical compounds. These results led our research group to study other species from Tetrapterys genus without further chemical studies, including species that used to belong to this genus and that recently has changed to Niedenzuella genus, such as Niedenzuella multiglandulosa, the selected species for this master's research. This species is known for causing toxic effects that may cause death in cattle and other mammals in general, causing substantial losses in Brazilian trade balance. The objective of this research is to search for toxic compounds in Niedenzuella multiglandulosa leaves. This species was chosen for having reports in the literature of outbreaks of cattle intoxication (in 2004 and 2005) in which the pasture was highly infested with it, causing death of cows and led to abortion of calves in Mato Grosso do Sul state. Recent studies found in the literature indicate the presence of monofluoracetate (MFA), a well-known toxic substance, in this species, but so far there were no phytochemical and to... (Complete abstract click electronic access below) / Mestre

Accumulating evidence from epidemiological, wildlife, and laboratory studies indicates that abnormalities of reproduction, development and physiology can be ascribed to environmental contaminants with biological activities. Many such contaminants disrupt essential hormone-regulated processes by virtue of their ability to interact with nuclear hormone receptors (endocrine disrupting chemicals, EDC). Of particular concern are chemicals that mimic or block estrogen signaling (xenoestrogens, XE) through their direct interaction with estrogen receptors (ER). The current model of XE action focuses on disrupted transcriptional activity, as measured by changes in the expression of ER-regulated genes. However, transcription is tightly coupled to splicing, by which a single target gene transcript is processed to multiple structurally and functionally different mRNAs. In theory, any XE that interacts with ER to regulate transcription has the potential to disrupt splicing, thereby affecting not only mRNA quantity but also quality. To address this hypothesis, alternative splicing of the gene encoding ER alpha (esr1), itself an estrogen responsive gene, was investigated. In these studies, killifish (Fundulus heteroclitus), an environmentally relevant species, and zebrafish (Danio rerio), an advantageous laboratory fish model, were used. First, the occurrence of ER alpha splice variants in adult tissues, in developing embryos and in response to estrogens in the two species was documented. Additionally, the effects of long-term, multigenerational XE exposure on ER alpha splicing were examined in two killifish populations, one from an estrogenic (polluted) site and a second population from a reference (unpolluted) site. A subset of ER alpha variants from killifish was expressed in cell culture to document their transcriptional activities. To determine the in vivo relationship between estrogen responsiveness and an ER alpha splice variant of interest, esr1 splicing was experimentally altered in living embryos by microinjecting morpholino oligonucleotides, and changes in induction of a panel of estrogen responsive gene targets were measured as markers of effect. These results provide evidence that dysregulation of mRNA processing is also a mechanism of XE action, and suggest that resultant ER alpha splice variants mediate the short-term effects of estrogen disruption and are also part of the adaptive response to long-term, multigenerational XE exposures in the natural environment.

The loss of neurons throughout aging, due to trauma or neurodegenerative diseases, was considered irreversible until recent discoveries demonstrated the capacity for the postnatal mammalian brain to generate new neurons in discrete niches capable of integrating into existing neural circuitry. Adult neurogenesis is highly dynamic and modulated by numerous factors. However, the temporal patterns of adult neurogenesis, kinetics of cell proliferation, and migration remain poorly understood. Zebrafish, a model used in this investigation, is a diurnal vertebrate with a circadian clock and clock-controlled processes organized similar to humans. Importantly, zebrafish display active neurogenesis. The studies comprising this dissertation demonstrate for the first time that a diurnal vertebrate displays robust circadian, i.e. near-24-h, patterns of adult neurogenesis. It proceeds as an orderly transition of cells from G1 to S phase of the cell cycle throughout the day, followed by nighttime progression of G2 phase, culminating with M phase in the early morning. While all five neurogenic niches studied reveal a common circadian pattern, each niche demonstrates a distinct S length and timing of the G1-S transition. Further investigation into kinetics of adult neurogenesis focused on the events occurring in the neurogenic niches over several days. Both experimental and mathematical modeling approaches determined a consistent number of neural stem cells (NSCs) dividing daily. These approaches also elucidated the predominant modes of division for transient amplifying cells, the neural progenitors (NPCs), the pace of migration and survival of their progeny. Finally, this dissertation addressed age-related changes in adult neurogenesis in zebrafish, supporting its gradual decline with age. Developing a pathological aging model, based on excessive nutrition throughout development and maturation, revealed a major decline in the number of dividing NSCs and extreme modification of the pattern of division and survival of NPCs. Together the results of the studies presented in this dissertation reveal that adult neurogenesis undergoes predictable dynamic changes over hours, days, and years. Future studies using a high-throughput zebrafish model should provide needed insights into the role of specific factors in adult neurogenesis and help develop therapeutic strategies to benefit human patients.