Thyroid hormones (THs) are essential signaling molecules for the postembryonic development of all vertebrates. THs are capable of initiating a diverse set of developmental programs across multiple tissues. The role of TH in regulating gene expression is well-known, but the initiation of TH signaling is still not fully understood. In amphibians, THs are the sole hormones required for the metamorphosis from tadpole to juvenile froglet. Amphibians are a useful model for studying TH signaling, as they undergo extensive, tissue-specific response programs in response to exogenous TH. The metamorphosis of the American bullfrog, Rana [Lithobates] catesbeiana is temperature sensitive. R. catesbeiana tadpoles do not undergo metamorphosis at cold temperatures (4-5 °C) even in the presence of THs that should otherwise prompt it. However, tadpoles undergo metamorphosis at an accelerated rate when returned to warm temperatures (24-25 °C) forty days after their initial TH exposure. R. catesbeiana tadpoles possess a “molecular memory” of TH exposure which establishes the TH signal at cold temperatures and prompts accelerated metamorphosis after a return to warmer temperatures. The mechanisms of the molecular memory which allow it to uncouple the initiation of TH signaling from the execution of the TH response program are not fully understood. Previous research has established that transcripts encoding transcription factors are a substantial component of the TH-dependent transcriptomic response of cultured tailfin (C-Fin) at cold temperatures. However, not all of these putative transcripts encoding transcription factors required active transcription and translation for their induction, which suggests that the initiation of a TH signal involves mechanisms other than regulating gene expression. Herein, we used quantitative polymerase chain reaction (qPCR) and RNA-Sequencing (RNA-Seq) to investigate the TH-dependent transcriptomic response of the back skin, a tissue that undergoes extensive remodeling during metamorphosis. Cultured back skin (C-Skin) was TH-responsive in warm, cold and temperature shift conditions. Forty-four transcripts underwent significant changes in abundance in response to TH in cold temperatures under which the molecular memory is established. Seven of these transcripts encoded putative transcription factors. Surprisingly, the only TH-responsive transcript significantly changed at 4 °C in both the C-Skin and the previously studied C-Fin was thyroid hormone-induced basic leucine zipper-containing protein (thibz). Thibz has been found to be TH-responsive at cold temperatures in the liver, lung, liver, brain, tailfin and back skin of whole animals, which suggests it may be an important regulator of initiating TH signaling. The lack of overlap in the transcriptomic responses of C-Skin and C-Fin may suggest that even the early initiation of TH signaling has tissue-specificity. Alternately, the molecular memory may include mechanisms that do not require active transcription and translation. Transcripts associated with epigenetic modifications and post-transcriptional changes to mRNA stability were also significantly expressed at 4 °C within the C-Skin. Previous investigation of the putative transcription factors in C-Fin revealed that active transcription and translation was not always required for changes in transcript abundance. Multiple mechanisms may be at play in the TH response at different temperatures. In cold temperatures, TH may modulate mRNA stability to influence transcript abundance as a part of initiating TH signaling without executing metamorphosis. Further research is needed to explore potential alternative mechanisms of establishing the molecular memory and the accelerated metamorphic response. The temperature sensitivity of R. catesbeiana’s TH response is incredibly valuable in investigating mechanisms of early TH signaling during postembryonic vertebrate development. / Graduate

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Leopard frogs (Rana pipiens) rely on vision to recognize behaviorally meaningful aspects of their environment. The optic tectum has been shown to mediate the frog's ability to recognize and respond to moving prey and looming objects. Nonetheless, atectal frogs are still able to appropriately respond to non-moving aspects of their environment. There appears to be independent visual systems operating in the frog: one system for recognizing moving objects; and another system for recognizing stationary objects. Little is known about the neural mechanisms mediating the recognition of stationary objects in frogs. Our laboratory showed that a retino-recipient area in the anterior lateral thalamus--the NB/CG zone--is involved in processing visual information concerning stationary aspects of the environment. This thesis aims to characterize the frog's responses to a range of stationary stimuli, and to elucidate the thalamic visual system that mediates those responses. I tested leopard frogs' responses to different stationary stimuli and found they respond in stereotypical ways. I discovered that leopard frogs are attracted to dark, stationary, opaque objects; and tested the extent of this attraction under different conditions. I found that frogs' preference to move toward a dark area versus a light source depends on the intensity of the light source relative to the intensity of ambient light. Unilateral lesions applied to the NB/CG zone of the anterior lateral thalamus resulted in temporary deficits in frogs' responses to stationary stimuli presented in the contralateral visual field. Deficits were observed in response to: dark objects, entrances to dark areas, light sources, and gaps between stationary barriers. However, responses to moving prey and looming stimuli were unaffected. Interestingly, these deficits tended to recover after about 6 days in most cases. Recovery time ranged from 2 - 28 days. The NB/CG zone is anatomically and functionally connected to a structure in the posterior thalamus called the "PMDT." The PMDT has no other connections in the brain. Thus, I have discovered a "satellite" of the NB/CG zone. Preliminary evidence suggests that the PMDT is another component of the visual system mediating stationary object recognition in the frog. / Biology

Anthropogenic disturbances can alter the abiotic composition of freshwater systems. These compositional changes can act as physiological stressors towards system inhabitants. However, little is known about how these altered abiotic factors influence stress hormones (corticosterone) in larval amphibians. Throughout the following chapters, I examined the effects of several abiotic factors on baseline and stress-induced corticosterone levels in the larvae of four amphibian species: Jefferson salamander (Ambystoma jeffersonianum), spotted salamander (A. maculatum), wood frog (Rana sylvatica), and grey treefrog (Hyla versicolor). Chapter II examined corticosterone level differences throughout development in A. jeffersonianum and R. sylvatica larvae under field, mesocosm, and laboratory venues. Baseline corticosterone levels in R. sylvatica increased near metamorphic climax in all venues, but not in A. jeffersonianum. Rather, baseline corticosterone levels differed with respect to venue throughout development in A. jeffersonianum. Chapter III examined corticosterone level differences among free-living A. jeffersonianum populations and possible abiotic factors underlying these hormone differences. Corticosterone levels significantly differed across populations. Increased baseline corticosterone levels significantly correlated to low pH. There was also a trend for increased baseline corticosterone levels to be positively correlated with chloride levels and negatively correlated with conductivity. Chapter IV examined the effects of laboratory manipulated pH on corticosterone levels in A. jeffersonianum, A. maculatum, R. sylvatica, and H. versicolor. There was a significant correlation between increased baseline corticosterone levels to low pH in all four species. Prey consumption (in both Ambystoma species) and survival (in A. jeffersonianum, A. maculatum, and R. sylvatica) were also negatively correlated to low pH. Chapter V examined the effects of increased conductivity on corticosterone levels in A. jeffersonianum, R. sylvatica, and H. versicolor. Increased conductivity exposure significantly correlated to increased baseline corticosterone levels in A. jeffersonianum and R. sylvatica. Prey consumption in A. jeffersonianum was also negatively correlated to increased conductivity. My dissertation shows that abiotic factors, such as pH and conductivity, can influence corticosterone levels in larval amphibians. These results suggest that corticosterone levels in larval amphibians may be a suitable biomarker reflective of altered freshwater habitat quality. However, my results also suggest that one should use a high degree of caution when using corticosterone levels in larval amphibians as a means to infer the health status of a population. / Ph. D.

Allochthone Wasserfrösche haben in Deutschland und vielen anderen Ländern Europas zu Faunenverfälschungen geführt. Sie konkurrieren mit einheimischen Tieren und stellen aus genetischer Sicht eine Bedrohung der Bestände dar. In dieser Arbeit wurden die Genotypen von 447 Wasserfroschproben aus Deutschland sowie 460 aus anderen Teilen Europas untersucht. Im Gesamtdatensatz konnten 56 ITS2- und 65 ND3-Genotypen nachgewiesen werden. Unter den 20 in Deutschland gefunden ITS2-Allelen wurden zwei Rana ridibunda-ähnliche Genotypen als autochthon und fünf als allochthon erkannt (Häufigkeit 7 %), der Status weiterer fünf ITS2-Allele war nicht klar zu belegen. Unter den 14 mitochondrialen Genotypen der R. ridibunda-Gruppe befanden sich drei autochthone, zehn allochthone (Häufigkeit 18 %) sowie eine Variante mit unklarem Status. Allochthone Genommerkmale wurden vor allem in Südwestdeutschland entlang des Rheins und im Ruhrtal nachgewiesen. Im Raum Karlsruhe konnte ein mitochondrialer Genotyp mit hohem Anteil festgestellt werden, der typisch für eine bisher nur aus Italien bekannte Art ist (R. bergeri). Da allochthone ITS2-Allele häufig heterozygot mit autochthonen Varianten auftreten, gibt es eindeutige Hinweise auf Hybridisierungen zwischen einheimischen und eingeschleppten Wasserfröschen. Aufzuchtsexperimente zeigten keine reduzierte Überlebenswahrscheinlichkeit von F1-Hybriden aus Kreuzungen zwischen autochthonen R. ridibunda und allochthonen R. cf. ridibunda aus Anatolien. Um die Rolle allochthoner Wasserfrösche bei der Verbreitung von Krankheitserregern beurteilen zu können, wurden Nachweistests für die Amphibien-Chytridiomykose durchgeführt. Die Nachweistests ergaben eine Prävalenz des Erregers (Batrachochytrium dendrobatidis) von 6,3 % unter deutschen Proben. Da die Chytridiomykose überwiegend in Populationen auftrat, in denen auch allochthone Wasserfrösche vorkamen, wird ein Zusammenhang zwischen Einschleppungsereignissen und dem Auftreten des Erregers vermutet. / Allochthonous water frogs have been introduced into Germany and other European countries. They compete with autochthonous water frogs and threaten the genetic integrity of native populations. In the present work the genotypes of 447 water frogs collected in Germany as well as 460 samples from various European countries have been determined and compared. In the complete dataset 56 ITS2- and 65 ND3-genotypes were identified. Among the 20 ITS2-alleles found in Germany, two indigenous and five introduced Rana ridibunda-like genotypes (relative frequency 7%) occurred. Five R. ridibunda alleles could not assigned as either indigenous or exotic. Among the 14 mitochondrial genotypes of the R. ridibunda-group three autochthonous and ten introduced (relative frequency 18%) variants could be identified, whereas the status of one mt-genotype remained unclear. Exotic alleles were mainly found in southwest Germany along the river Rhine and along the Ruhr in the Ruhr area. The wide distribution of a mitochondrial genotype previously known from Italian water frogs (R. bergeri) was ascertained in the region surrounding Karlsruhe. Allochthonous ITS2-alleles often occur heterozygously combined with autochthonous alleles. This is a strong evidence for cases of hybridisations between indigenous and introduced water frogs. Crossing experiments between autochthonous R. ridibunda and Anatolian water frogs (R. cf. ridibunda) revealed no reduced viability among the F1-hybrids. To evaluate the possible role of introduced water frogs on the dispersal of infectious diseases, detection tests of the amphibians-chytridiomycosis were carried out. The detection tests for its pathogen, Batrachochytrium dendrobatidis, on the tissue samples collected in Germany showed a prevalence of 6.3%. Most of the infected frogs were found in populations influenced by non-native water frogs. This points towards a possible relationship between introduction events and the occurrence of the pathogen.

Ling, Ka Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-157). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of content --- p.ix / List of figures --- p.xiii / List of tables --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Food authentication --- p.1 / Chapter 1.1 --- Introduction and definition --- p.1 / Chapter 1.2 --- Importance of species identification in food authentication --- p.3 / Chapter 1.3 --- Methods for species identification in food authentication --- p.8 / Chapter 1.4 --- Legislation --- p.17 / Chapter 1.5 --- Objectives --- p.19 / Chapter 2 --- Molecular authentication and antitussive bioassay of Stemona root (Baibu) and root of Asparagus filicinus (Xiao-baibu) --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.22 / Chapter 2.3 --- Results --- p.34 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusions --- p.44 / Chapter 3 --- Rapid detection of oilfish and escolar in fish steaks: a tool to prevent keriorrhea episodes --- p.45 / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Materials and methods --- p.49 / Chapter 3.3 --- Results --- p.59 / Chapter 3.4 --- Discussion --- p.69 / Chapter 3.5 --- Conclusions --- p.75 / Chapter 4 --- Widespread adulteration of crocodile meat with python and water monitor meats --- p.76 / Chapter 4.1 --- Introduction --- p.76 / Chapter 4.2 --- Materials and methods --- p.78 / Chapter 4.3 --- Results --- p.89 / Chapter 4.4 --- Discussion --- p.96 / Chapter 4.5 --- Conclusions --- p.100 / Chapter 5 --- Authentication of dried and ready-to-eat hashima products --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Source species of hashima --- p.103 / Chapter 5.3 --- Materials and methods --- p.106 / Chapter 5.4 --- Results --- p.116 / Chapter 5.5 --- Discussion --- p.119 / Chapter 5.6 --- Conclusions --- p.121 / Chapter 6 --- General conclusions --- p.122 / Chapter 6.1 --- Key findings --- p.122 / Chapter 6.2 --- Applications and implications --- p.123 / Chapter 7 --- References --- p.124

Made available in DSpace on 2014-12-17T14:16:33Z (GMT). No. of bitstreams: 1 EvertonNA_DISSERT.pdf: 1792705 bytes, checksum: 2091882fa52265fca623e2d01c142cfa (MD5) Previous issue date: 2013-06-10 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Natural oils have shown a scientific importance due to its pharmacological activity and renewable character. The copaiba (Copaifera langsdorffii) and Bullfrog (Rana catesbeiana Shaw) oils are used in folk medicine particularly because the anti-inflammatory and antimicrobial activities. Emulsion could be eligible systems to improve the palatability and fragrance, enhance the pharmacological activities and reduce the toxicological effects of these oils. The aim of this work was to investigate the antimicrobial activity of emulsions based on copaiba (resin-oil and essential-oil) and bullfrog oils against fungi and bacteria which cause skin diseases. Firstly, the essential oil was extracted from copaiba oil-resin and the oils were characterized by gas chromatography coupled to a mass spectrometry (GC-MS). Secondly, emulsion systems were produced. A microbiological screening test with all products was performed followed (the minimum inhibitory concentration, the bioautography method and the antibiofilm determination). Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Candida albicans, C. parapsilosis, C. glabrata, C. krusei and C. tropicalis American Type Culture Collection (ATCC) and clinical samples were used. The emulsions based on copaiba oil-resin and essential oil improved the antimicrobial activity of the pure oils, especially against Staphylococcus e Candida resistant to azoles. The bullfrog oil emulsion and the pure bullfrog oil showed a lower effect on the microorganisms when compared to the copaiba samples. All the emulsions showed a significant antibiofilm activity by inhibiting the cell adhesion. Thus, it may be concluded that emulsions based on copaiba and bullfrog oils are promising candidates to treatment of fungal and bacterial skin infections / Os ?leos naturais v?m chamando aten??o da comunidade cient?fica devido a suas atividades farmacol?gicas e seu car?ter renov?vel. Os ?leos de copa?ba (Copaifera langsdorffii) e de r?-touro (Rana catesbeiana Shaw) ganham destaque, especialmente devido a ampla utiliza??o na medicina popular como anti-inflamat?rios e antimicrobianos. Sistemas emulsionados a base destes ?leos podem ser produzidos com a finalidade de melhorar o odor e a palatabilidade dos mesmos, al?m de potencializar a??o e reduzir toxicidade dos seus componentes. O objetivo deste trabalho foi investigar a atividade antimicrobiana de emuls?es contendo ?leos de copa?ba (?leo-resina e ?leo essencial) e ?leo de r?-touro frente a cepas de micro-organismos causadores de infec??es cut?neas. Inicialmente, foi realizada a extra??o de ?leo essencial e sua caracteriza??o qu?mica por cromatografia gasosa acoplada a espectroscopia de massas. Em seguida, foram produzidas emuls?es, ensaios de triagem microbiol?gica, microdilui??o para determina??o de concentra??o inibit?ria m?nima, bioautografia para determina??o dos componentes antimicrobianos e avalia??o da atividade antibiofilme. Foram utilizadas cepas da American Type Culture Collection (ATCC) e cl?nicas de Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Candida albicans, C. parapsilosis, C. glabrata, C. krusei e C. tropicalis. Foi observado que os sistemas emulsionados de ?leo-resina de copa?ba e ?leo essencial de copa?ba contribu?ram na potencializa??o da atividade antimicrobiana, especialmente contra cepas do g?nero Staphylococcus e Candida resistentes aos az?is. A emuls?o de ?leo de r?-touro assim como o ?leo puro, apresentou menor atividade que as amostras de copa?ba, por?m exibiu significativa a??o antibiofilme, demonstrando que este sistema ? um potencial inibidor da ades?o de micro-organismos. Deste modo, pode-se inferir que as emuls?es a base destes ?leos s?o promissores sistemas para o tratamento de infec??es cut?neas f?ngicas e bacterianas

Phenotypic plasticity is the ability of an organism to express different phenotypes depending on the biotic or abiotic environment. In many cases, the response to the environment is beneficial to the individual and can therefore represent adaptive phenotypic plasticity. An example includes morphological and life-historical response to predators. Because there has been a weak interest in the effect of these morphological changes, and it often assumes, but rarely shown, that predator-induced changes are adaptive. I tested how different variables, such as maximum acceleration and velocity, were related to each other in different Gosner stages. Eggs and tadpoles of the species Rana temporaria were exposed to various predator enclosures, a control, a stalking predator (European Perch, Perca fluviatilis), and an ambush predator (dragonfly larvae). The tadpoles mean weight, maximum acceleration and velocity, mobility, boldness, and morphological changes in various Gosner stages were analyzed. My tests showed a significant difference in tadpole weight and their morphology, however, there were no differences in speed, acceleration, boldness or mobility. The morphometric analysis of the tadpoles showed a significant change of tadpoles in the perch treatment compared to the tadpoles in the control. The tadpoles in the perch treatment showed a higher TH/TL (tail height/tail length) ratio, and were overall heavier, than the tadpoles in the control, but no significant change compared to the tadpoles in the dragonfly treatment. This shows that the kairomones and alarm cues triggered a large morphological change, but the effect of the change requires more research.

Amphibian populations are declining world-wide and one of the suggested reasons is environmental pollutants. Studies of long-term effects on the reproductive system in frogs following larval exposure to environmental pollutants are scarce. It is therefore important to develop methods to study developmental reproductive toxicity in amphibians. In this thesis the usefulness of Xenopus tropicalis (the West African clawed frog) as a model species for a test system was investigated. Effects on the reproductive system after larval exposure to the pharmaceuticals ethynylestradiol (EE2) and clotrimazole were evaluated. The susceptibility to EE2 exposure was compared between the model species and a wild species, the European common frog (Rana temporaria). Larval exposure to EE2 caused female-biased sex ratios in both examined frog species, indicating male-to-female sex-reversal. In adult Xenopus tropicalis, male frogs that were not sex-reversed had reduced fertility and decreased amount of mature spermatozoa in the seminiferous tubules. The proportion of frogs with ovaries but lacking oviducts increased with increasing EE2-concentrations. A female frog without oviducts is sterile. The development of ovaries in sex-reversed male frogs was implied to be similar to control females. The combination of a reduced number of males, due to sex-reversal, and impaired fertility could have severe effects on frog populations. Larval exposure to clotrimazole modulated aromatase activity in gonads and brain in Xenopus tropicalis. Brain aromatase activity was decreased at the time for gonadal differentiation and gonadal aromatase activity was increased at metamorphosis. The findings in this thesis indicate that reproduction in wild frogs might be impaired by estrogenic compounds in the environment. The results combined with the short generation time supports the use of Xenopus tropicalis as a model species when evaluating long term effects of endocrine disruptors on the reproductive system in amphibians.