Mycobacteriosis is a common disease of laboratory zebrafish (Danio rerio). Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more wide spread and not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture, followed by molecular or biochemical identification is the traditional approach for species identification, but recently it has been shown that DNA of diagnostic value can be retrieved from paraffin blocks. Type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection were investigated as parameters to determine if the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21 and 45 days. Subsequently, fish were evaluated by H&E and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective, and obtained PCR product from 75% and 88% of the M. chelonae and M. marinum infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test. Regarding natural transmission, zebrafish are capable of contracting mycobacterial infections by feeding on infected fish tissue, but other natural routes have not been clearly elucidated. Free living amoebae have been shown to be vectors for mycobacteria and their virulence is enhanced when residing in these protozoans. Paramecium caudatum are commonly used as a first food for zebrafish, and I investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 days post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 days post hatch) showed 31% incidence (14/45). Adult fish fed gelatin diets containing bacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure: M. marinum OSU 214; in paramecia 47% (21/45; 3.5 x 10⁵ dose/fish/day), M. marinum CH in paramecia 47% (9/19; 3.6 x 10⁵ dose/fish/day), M. chelonae in paramecia 38% (5/13; 3.5 x 10⁵ dose/fish/day). I investigated the ability of mycobacteria to persist within paramecia, as this has previously been demonstrated in amoebae. Gram negative bacteria ingested by paramecia were processed within an hour. In contrast, I determined using GFP-labeled Mycobacterium marinum that mycobacteria can persist within paramecia digestive vacuoles. The concentration of M. marinum at 1 hour was similar to that at the time of ingestion. Twenty-four hours post-ingestion and later there was significant decline in M. marinum concentrations compared to time of ingestion, but M. marinum continued to persist inside digestive vacuoles for up to one week. My results demonstrate for the first time that Paramecium caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from April 2, 2013 - April 2, 2015

Biological invasions are among the leading causes of species diversity loss; however, the impacts of invasion are context-dependent and can vary with the local environment. The mechanisms governing variation in impact and their relationship to specific abiotic and biotic factors remain largely unexplored. Recent local declines in native unionid mussels have been attributed to the invasion of North American lakes and rivers by the Eurasian zebra mussel ( Dreissena polymorpha), as a result of intense fouling of unionid shells by zebra mussels. My research investigated the role of abiotic and biotic factors in mediating the impact of zebra mussels on native mussels. I examined the impact of zebra mussels on unionids in a habitat thought to be suboptimal for zebra mussels and compared this to the impact observed in other invaded habitats. A predictive model relating fouling intensity to local environmental variables (calcium concentration, sediment particle size) was developed, and a predator-exclusion experiment was conducted to investigate the role of predation in mediating fouling intensity. Overall, I found that two abiotic factors of the local environment were significant predictors of fouling intensity and that relationships used to predict the impact of zebra mussels could be extended to a broader range of habitats.

No description available.

Tuberculosis (TB) is still a global epidemic disease despite its discovery over 100 years ago. It is caused by Mycobacterium tuberculosis, which invades and replicates within macrophages, key cells of the innate immune system. The hallmark of tuberculosis is the granuloma which is an accumulation of Mycobacterium-infected cells surrounded by immune cells, and the containment of the bacteria is assured as long as the host immune response remains intact. Despite a well-developed immune response in the infected host, reactivation of latent tuberculosis infection (LTBI) may occur through the introduction of other bacterial pathogens, re-infection with M. tuberculosis or due to other immunosuppression, e.g. AIDS or cancer. The zebrafish–M. marinum model provides an ideal system for examining the pathogenesis of tuberculosis and the associated immune response of the host due to its vertebrate-like immune system, and the close phylogenetic relationship of M. marinum to M. tuberculosis. Granuloma formation and immune response to M. marinum have been investigated mainly in zebrafish embryos or larvae, which lack an adaptive immune response, and little work has been performed in adult fish. This complicates the transfer of findings in these models to chronic, latent or re-activated disease stages in humans, where adaptive immunity plays an important part. The aim of the research presented here was to investigate the immune response of the adult zebrafish to M. marinum infection, with the focus on the kidney as one of the major immune organs in fish. The results obtained support further use of the adult zebrafish-M. marinum model for human tuberculosis infections in the future. In the present study, adult zebrafish were infected with low doses of M. marinum (NCIMB 1297 or NCIMB 1298) and the kidney was investigated for histopathological changes in the form of granulomas over a period of two months(Chapter 3). No granulomas were detected in the fish infected with M. marinum NCIMB 1298 while in zebrafish infected with NCIMB 1297, macrophage aggregation and granuloma formation were detected as early as day 11 post-infection. Occurrence and severity of granulomas and the presence of replicating bacteria increased over time, resulting in a high density of non-caseating and caseating granulomas in the head and posterior kidney after two months of infection. Interleukin 1 beta (IL-1β), Interleukin-12 (IL-12), Tumor necrosis factor alpha (TNFα) and Interferon gamma (IFNγ) have been shown to be important cytokines functioning in defence against tuberculosis, especially IFNγ which is considered to play an important part in acute, chronic and latent tuberculosis. Changes in gene expression of these immune genes in adult zebrafish were investigated over the first two weeks of infection with M. marinum NCIMB 1298 and NCIMB 1297. The results obtained in the first week after infection were inconclusive for both strains investigated. In agreement with the results presented in Chapter 3, no specific immune response was detectable in fish infected with M. marinum NCIMB 1298. However, after 14 days, a high-fold change in IL-12 and TNFα expression were detected in fish infected with M. marinum NCIMB 1297, while IL-1β showed no changes compared to the control fish. Furthermore, no IFNγ expression was detectable over the first two weeks of infection. The delay in the expression of IL-12 and the lack of IFNγ expression can be explained by the ability of M. marinum to manipulate the host immune response, as described for M. tuberculosis and other intracellular bacteria. Besides in vivo investigations of the host-pathogen interactions, in vitro primary macrophage cultures from individual zebrafish kidneys were developed to investigate macrophage-specific gene expression to M. marinum infection (Chapter 4). Although the results looked promising, further optimization is required before the results of the in vitro assays can be fully compared to the in vivo results. Our understanding of reactivation in latent tuberculosis infection (LTBI) both in healthy and immune compromised individuals is insufficient and is delaying the development of treatments for the disease. Therefore, the transcriptome profile of long-term infections (26 weeks) with M. marinum NCIMB 1297 in adult zebrafish was investigated to determine whether the gene expression in this model is comparable to LTBI in humans or other vertebrate model organisms (Chapter 5). In addition, transcriptome profiling was investigated in a group of long-term infected zebrafish exposed to stress to induce re-activation of the disease. Expression profiles in the long-term infected fish and the infected plus stressed fish differed from each other and displayed similar gene profiles to those found in the latent or re-activated disease states, respectively, in human and other vertebrate models. Infected fish displayed a profile highlighted by IFNγ, TNFα, NOS2b and IL-8 expression alongside activating and regulatory T cell responses, including involvement of cytotoxic T cells (CTLs). The transcriptome profile of the group of fish that had been infected and then stressed was distinguished by the lack of IFNγ expression and reduction in TNFα and NOS2b expression, as well as a lack of T cell response compared to the infected fish. In conclusion, the results obtained from Chapters 3 and 4 showed that M. marinum NCIMB 1298 is non-pathogenic to zebrafish. Infection with M. marinum NCIMB 1297, on the other hand, resulted in a similar immune response to that described for human and other mammalian vertebrate models (Chapters 3-5). These results support the use of the adult zebrafish-M. marinum model to investigate LTBI and disease reactivation, and will aid our understanding host-pathogen interactions for tuberculosis in the future.

Le facteur de transcription ZEBRA (EB1) du virus d'Epstein-Barr joue un rôle essentiel dans l'initiation de l'infection lytique et la production virale. L'ubinucléine a été identifiée comme un partenaire cellulaire de ZEBRA, capable de l'empêcher de se fixer à ses séquences d'ADN cibles. Le rôle de l'ubinucléine dans la cellule demeure inconnu, ainsi que les conséquences de son interaction avec ZEBRA dans les cellules infectées par l'EBV. Notre travail a permis, d'abord, de mieux caractériser l'ubinucléine dans la cellule épithéliale en l'identifiant comme une protéine des jonctions serrées. L'ubinucléine a été proposée comme un nouveau membre de la famille des protéines NACos (nuclear and adhesion complex components) possèdant une double localisation, les noyaux et les jonctions des cellules. Afin de mieux comprendre son rôle dans la cellule épithéliale, nous avons étudié par une approche protéomique couplée à la spectrométrie de masse les partenaires de l'ubinucléine et identifié les protéines LYRIC et RACK-1. Nos résultats suggèrent que l'ubinucléine est impliquée dans différents processus biologiques tels que la régulation de la prolifération et de l'adhésion cellulaires. Enfin, dans les cellules épithéliales infectées par l'EBV, les fonctions de l'ubinucléine semblent dépendre de sa localisation cellulaire. Au niveau nucléaire, l'ubinucléine régule négativement le cycle lytique et la production de particules virales en empêchant ZEBRA et d'autres facteurs cellulaires de se fixer à leurs promoteurs de type AP-1. Lorsqu'elle est séquestrée dans les jonctions serrées, l'inhibition de ZEBRA est levée permettant ainsi le bon déroulement du cycle lytique du virus.

How so many species coexist while utilizing the same resources is both of ecological interest and important for the management of wildlife communities and parks. This thesis endeavours to understand how grazing herbivores co-exist, with special emphasis on understanding the mechanisms of competition and facilitation over temporal and spatial scales. I compared the dry season use of grasslands, grass species, grass height and grass greenness by white rhino and three other ungulate species, zebra, wildebeest, and impala. I was specifically interested in the extent to which white rhinos, with their capacity to graze both short and tall grass, either competed with or facilitated other grazers. In Chapter Two, I clarify the mechanisms of facilitation and competition in terms of temporal and spatial scales, and discuss why there is little evidence in the literature to support these mechanisms. I conclude that evidence for facilitation through stimulation of grass re-growth during the growing season appears stronger than that for increased resource access through removal of obstructing grass structures during the dormant season. Facilitation may benefit the nutritional gains obtained by certain species in the short term, but these benefits do not appear to be translated into the expected population consequences. In collaboration with co-author Norman Owen-Smith, we suggested this could be due to seasonal tradeoffs between facilitation and competition, as well as to restrictions on the spatial extent of trophic overlap. In Chapter 3 Norman Owen-Smith and I compared the grass height use in relation to body size. We expected that the grass height favoured would increase with the body size of the herbivore species, as suggested from past studies of resource partitioning among large mammalian herbivores. Instead we found that the largest of these species, white rhino, concentrated on the shortest grass, while the smallest species, impala, favoured grass heights intermediate between those grazed by wildebeest and zebra. Results suggest that the scaling of mouth width relative to body size, and hence metabolic demands, is the primary factor governing grass height selection, rather than body size alone. Hence grazing successions governed by body size differences may not be a typical feature of their ecology, contrary to past suggestions. Furthermore, there was considerable overlap in grass height grazed among these four species, indicating that niche separation by grass height is inadequate alone to explain their coexistence. More attention needs to be paid to other aspects such as the grass species selected and habitat structure favoured. Chapter Four compared the overlap in grassland use and grass species use, as well as grass height and grass greenness of swards utilized by the herbivores as the dry season advances. I show that all species prefer grazing lawns during times of abundance, and that zebra leave grazing lawns before other species, and wildebeest leave grazing lawns before white rhino and impala. This suggests zebra and wildebeest may be competitively excluded from grazing lawns through a reduction in grass height, by white rhino and impala, during the dry season. However, white rhino are also potentially the ‘supreme’ facilitator increasing the availability of nutritious grazing lawns, as well as increasing the quality of those lawns through grazing in the wet season. A better understanding of the trade-off between “habitat facilitation” and competitive exclusion by white rhino, allows us to better understand how grazing herbivores co-exist.

Orientador: Marcelo Campos / Banca: Claudiana Lameu Gomes / Banca: Camila Nomura Boscolo / Resumo: O BaP é um contaminante ambiental capaz de causar inflamação e desregulação de vias celulares. Pela ação da CYP1a1 e CYP1b1, é convertido a metabólitos mais reativos. A literatura mostra que o BaP aumenta a expressão de algumas citocinas próinflamatórias, como a IL-1, porém, são bem contraditórios os relatos sobre o efeito do BaP no cFOS, o qual apresenta papel importante na proliferação, na formação de tumores e, possivelmente, na inflamação. O objetivo deste estudo foi de elucidar a participação do receptor purinérgico P2X7 sobre a expressão dos genes IL- 1 e cFOS, durante exposição ao BaP. Foi empregado as técnicas de qPCR para quantificação de expressão gênica, e testes de correlação e regressão entre IL-1 e cFOS. A exposição ao BaP induziu a expressão dos dois genes, além das enzimas do seu metabolismo. Quando bloqueado o receptor P2X7, além de uma menor indução das CYPs, os níveis de IL- 1e cFOS caíram abaixo dos níveis controle, sugerindo a participação do P2X7. Os testes de correlação e regressão mostraram uma relação forte direta entre IL-1e cFOS, reforçando o papel do cFOS na inflamação / Abstract: BaP is an environmental contaminant capable to cause inflammation and impair cellular pathways. CYP1a1 and CYP1b1 convert it to more reactive metabolites. Studies show that BaP enhances some proinflammatory citokines expression, like IL-1, yet reports about BaP affecting cFOS, which plays important role in proliferation, tumor formation and inflammation, are controversial. This work aimed to elucidate whether P2X7 purinergic receptor plays a role in IL-1 and cFOS expression during BaP exposure. We applied qPCR techniques to quantify gene expression, correlation and regression assays. Our results showed that BaP raised both IL-1 and cFOS genes expression, besides CYPs ones. Morevoer, when blocking P2X7 receptor, IL-1 and cFOS expression dropped under normal levels, which suggest P2X7 participation, in addition to a smaller enzymes induction. Correlation and regression assays exhibited a strong straight relationship between IL-1 and cFOS expression, reinforcing the role of cFOS in inflammation / Mestre

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T13:23:57Z No. of bitstreams: 1 DIS_DEBORA_DREHER_NABINGER_PARCIAL.pdf: 1830529 bytes, checksum: 7313ef6fa47ca8352c8b7c238d077733 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T13:24:07Z (GMT) No. of bitstreams: 1 DIS_DEBORA_DREHER_NABINGER_PARCIAL.pdf: 1830529 bytes, checksum: 7313ef6fa47ca8352c8b7c238d077733 (MD5) / Made available in DSpace on 2017-06-30T13:24:16Z (GMT). No. of bitstreams: 1 DIS_DEBORA_DREHER_NABINGER_PARCIAL.pdf: 1830529 bytes, checksum: 7313ef6fa47ca8352c8b7c238d077733 (MD5) Previous issue date: 2017-02-13 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Metals are some of the more toxic substances in the environment. Nickel is one of them and this heavy metal is naturally present in the earth?s crust. However, excessive levels of nickel lead to environmental contamination and can cause serious and irreversible health problems. The aim of this study was to evaluate the toxicological effects of nickel exposure on cognition and behavior, in larvae and adult zebrafish (Danio rerio). Larvae and adult zebrafish were exposed to four different concentrations (0.025, 2, 5, and 15 mg/L NiCl2) or water (control group) in two treatments: acute and subchronic. Larvae were exposed to NiCl2 for 2 hours (acute treatment: 5-day-old larvae treated for 2 hours) or 11 days (subchronic treatment: 11-day-old larvae treated since fertilization) and adults were exposed for 12 hours (acute treatment) or 96 hours (subchronic treatment). In both treatments, for larvae and adults, exposed animals had a significant concentration-dependent increase in nickel levels compared to control group. For larvae, the survival rate was similar in both treated groups compared to control. However, a significant delay of hatching, a decreased heartbeat rate and morphological alterations (decrease of body length and ocular area at 5 and 8 days post-fertilization, dpf) were observed in subchronically-treated animals. Aversive and exploratory behavior showed no significant differences among doses in acute treatment. In contrast, larvae analyzed at 5 dpf in subchronic treatment displayed differences in exploratory behavior, showing decrease in distance traveled and mean speed, at 0.025 mg/L, whereas there was an increase at same parameters in higher doses (5 and 15 mg/L). Over the 11 days of treatment, the locomotor behavior decreased significantly, at 15 mg/L, at 8 and 11 dpf. Furthermore, subchronic-treated larvae showed impaired aversive long-term memory in the inhibitory avoidance task in high doses analyzed (2, 5 and 15 mg/L). For adults, acute treatment did not alter the locomotor activity. Besides, animals submitted to the concentration of 15.0 mg/L, in subchronic treatment, showed anxiogenic effects. The social behavior was not altered by treatments. However, the exposure to NiCl2 caused a decrease in aggressive behavior (subchronic treatment) and impaired memory (acute and subchronic treatments) in all doses compared to controls. In order to evaluate if nickel exposure produced alterations in the hematological system, we analyzed different blood cells in adult animals. The results showed that treated animals submitted to the concentration of 2.0 mg/L, in acute and subchronic treatment, presented an increase of monocytes. Furthermore, to verify if the behavioral alterations observed in treated animals were related to mechanisms of neuronal death, analyzes of apoptotic death in larvae and specific markers of neuronal death in larvae and adults were performed. The results of apoptotic death demonstrated a significant increase in cell death in acutely treated larvae at a concentration of 15 mg/L. In the subchronic treatment, at 5 dpf, there was an increase in apoptotic death at concentration of 5 mg/L, at 8 dpf, in concentrations of 5 and 15 mg/L and at 11 dpf, in concentrations of 2 and 5 mg/L. In the analysis of specific markers of neuronal death, the results suggest that NiCl2 concentrations tested in larvae and adults did not alter the protein levels of cell death markers. These results suggest that prolonged exposure to nickel in early life stages of zebrafish development leads to morphological and physiological alterations and cognition and locomotor deficits, whereas it may cause anxiogenic effects, impaired memory and decrease aggressive behavior in adult stage. These morphological and behavioral alterations may be associated to neurotoxic effects damage caused by this metal. / Metais s?o algumas das subst?ncias mais t?xicas de grande preocupa??o ambiental. O n?quel ? um destes agentes, sendo que este metal pesado ? naturalmente presente na crosta terrestre. Entretanto, n?veis excessivos de n?quel levam ? contamina??o ambiental e podem causar s?rios e irrevers?veis problemas de sa?de. O presente estudo visa avaliar os efeitos t?xicos da exposi??o aguda e subcr?nica ao n?quel, em peixe-zebra (Danio rerio), no est?gio larval e adulto. Os animais foram expostos a quatro diferentes concentra??es (0,025, 2, 5 e 15 mg/L NiCl2) ou ?gua (grupo controle) e submetidos a dois tratamentos: agudo e subcr?nico. As larvas foram expostas por 2 horas (tratamento agudo: larvas de 5 dias p?s-fertiliza??o - dpf - tratadas por 2 horas) ou 11 dias (tratamento subcr?nico: larvas tratadas do momento da fertiliza??o at? 11 dpf). Os animais adultos foram expostos por 12 horas (tratamento agudo) ou 96 horas (tratamento subcr?nico). Nos dois tratamentos, larvas e adultos expostos apresentaram um aumento dependente da concentra??o nos n?veis de NiCl2, comparados com animais do grupo controle. A taxa de sobreviv?ncia das larvas e embri?es n?o foi alterada pelos tratamentos. Entretanto, atraso na eclos?o dos ovos, diminui??o dos batimentos card?acos e altera??es morfol?gicas (diminui??o do comprimento corporal e ?rea ocular aos 5 e 8 dpf) foram observados em animais tratados subcronicamente. O comportamento aversivo e explorat?rio n?o mostrou diferen?as significativas entre as concentra??es de NiCl2, no tratamento agudo. Diferentemente, larvas subcronicamente tratadas, analisadas aos 5 dpf, apresentaram diferen?as no comportamento explorat?rio, exibindo diminui??o na dist?ncia percorrida e na velocidade m?dia, na concentra??o de 0,025 mg/L, enquanto houve um aumento dos mesmos par?metros em altas concentra??es (5 e 15 mg/L). Ao longo dos 11 dias de tratamento, o comportamento explorat?rio diminuiu significativamente, na concentra??o de 15 mg/L, aos 8 e 11 dpf. Al?m disso, larvas submetidas ao tratamento subcr?nico com altas concentra??es de NiCl2 (2, 5 e 15 mg/L) apresentaram comprometimento no comportamento aversivo. Em animais adultos, o tratamento agudo n?o promoveu altera??es na atividade locomotora. Por outro lado, animais expostos ? concentra??o de 15 mg/L no tratamento subcr?nico demonstraram efeito ansiog?nico. A intera??o social n?o foi alterada pelos tratamentos. Entretanto, a exposi??o ao NiCl2 levou a uma diminui??o do comportamento agressivo (tratamento subcr?nico) e comprometimento de mem?ria (tratamento agudo e subcr?nico), em todas as concentra??es testadas, quando comparados ao grupo controle. No sentido de avaliar se a exposi??o ao n?quel produziu altera??es no sistema hematol?gico, avaliamos diferentes c?lulas leucocit?rias em animais adultos. Os resultados encontrados mostraram um aumento significativo de mon?citos em animais expostos de forma aguda e subcr?nica na concentra??o de 2 mg/L comparado aos controles. Al?m disso, para verificar se as altera??es comportamentais observadas em animais tratados estavam relacionadas com mecanismos de morte celular e neuronal, foram realizadas an?lises de morte apopt?tica em larvas e de marcadores espec?ficos de morte neuronal em larvas e adultos. Os resultados de morte apopt?tica, demonstraram um aumento significativo de morte celular em larvas tratadas de forma aguda na concentra??o de 15 mg/L. No tratamento subcr?nico, aos 5 dpf, houve um aumento de morte apopt?tica na concentra??o de 5 mg/L, em 8 dpf nas concentra??es de 5 e 15 mg/L e em 11 dpf nas concentra??es de 2 e 5 mg/L. Na an?lise de marcadores espec?ficos de morte neuronal, os resultados sugerem que as concentra??es de NiCl2 testadas em larvas e adultos n?o alteraram os n?veis de prote?na dos marcadores de morte celular. Estes resultados sugerem que a exposi??o prolongada ao n?quel, em est?gios iniciais do desenvolvimento do peixe-zebra, leva a altera??es morfol?gicas, fisiol?gicas e d?ficits na cogni??o e locomo??o, enquanto que, em animais adultos, foi observado um efeito ansiog?nico, comprometimento de mem?ria e diminui??o do comportamento agressivo. Estas altera??es morfol?gicas e comportamentais podem estar associadas a efeitos neurot?xicos causados por este metal.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:09:29Z No. of bitstreams: 1 TES_STEFANI_ALTENHOFEN_PARCIAL.pdf: 820184 bytes, checksum: 93540eb7fece868ec872a31e8f894852 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T18:09:36Z (GMT) No. of bitstreams: 1 TES_STEFANI_ALTENHOFEN_PARCIAL.pdf: 820184 bytes, checksum: 93540eb7fece868ec872a31e8f894852 (MD5) / Made available in DSpace on 2017-06-30T18:09:44Z (GMT). No. of bitstreams: 1 TES_STEFANI_ALTENHOFEN_PARCIAL.pdf: 820184 bytes, checksum: 93540eb7fece868ec872a31e8f894852 (MD5) Previous issue date: 2017-03-06 / Neurotoxic agents, such as pesticides, organic solvents and metals, are capable to alter normal CNS activity. Manganese (Mn) is a metal found in the environment, which when in excess, accumulates in the CNS making it a potential hazard to health. In addition to Mn, other neurotoxic agents can cause changes, leading to deficits in neurotransmission systems. The growth of crop pests has exponentially increased the use of agrochemicals to maintain plantations. The fungicide tebuconazole is a triazole used in crops, such as barley, wheat, peanuts and orchard fruits and related to behavioral and oxidative changes. The insecticide dichlorvos, belongs to organophosphate family, acts through the acetylcholinesterase (AChE) inhibition, leading to hyperstimulation of cholinergic receptors. Cholinergic system is characterized by the action of acetylcholine (ACh) on muscarinic and nicotinic receptors. The level of this molecule is regulated by acetylcholinesterase (AChE), which catalyzes degradation of ACh into choline and acetate. Purinergic system is characterized by the action of ATP and adenosine on purinoreceptor P2 and P1, respectively. The levels of these molecules are regulated by ectonucleotidases, which constitute the extracellular cascade for ATP hydrolysis to adenosine. Adenosine can be subsequently deaminated to inosine by action of adenosine deaminase (ADA). Dopamine (DA), synthesized from L-DOPA, is the neurotransmitter of the dopaminergic system and it acts through its D1-D5 receptors, controlling the start and execution of movement. This study aims to evaluate the effects of exposure to manganese and pesticides, such as dichlorvos and tebuconazole, on behavioral parameters in larval and adult zebrafish, as well as its effects on purinergic, cholinergic and dopaminergic signaling. Exposure to MnCl2 in the early stages of development (1 hpf - 5 dpf) was able to reduce the distance traveled, absolute turn angle and mobile time of the larvae at 5, 7 and 10 dpf. In addition, it caused an increase in TH levels at 5 and 7 dpf, followed by a reduction at 10 dpf. There was also an increase in markers of cell death. In adult animals, exposure during 96 hours caused a reduction in distance traveled, number of line crossings and absolute turn angle, and an inhibition on memory acquisition through the inhibitory avoidance task. In addition, adult animals exposed to MnCl2 showed increased levels of markers of cell death, TH and DOPAC (3,4-dihydroxyphenylacetic acid) in the brain. The 96-hour exposure to this metal was also able to inhibit the NTPDases and ecto-ADA activities and to reduce ADA2.1-mRNA transcripts, besides for increasing ADP levels and reducing the amount of AMP, ADO and INO in the incubation medium. Tebuconazole was able to increase the ocular distance of larvae at 5 dpf and reduce the exploratory behavior of these animals, and still decrease the locomotor activity of 96 hours exposed adult zebrafish. Moreover, this fungicide decreased AChE activity in both 5 dpf larvae and brains of adult animals. Exposure to dichlorvos (1 hpf - 7 dpf) reduced body length, heartbeat rate and larval surface at 7 dpf as well as affected the escape capacity of larvae at 7 and 14 dpf. Further, it reduced exploratory behavior at 7 and 14 dpf, maintaining this pattern when animals became young adults at 30 dpf, and adults at 70 dpf. However, the analysis performed at 120 dpf showed a recovery of behavioral activity equal to the control. In addition, the analysis of exposure to dichlorvos in the early stages of development at 120 dpf on the enzymes of the purinergic system, showed that this insecticide is able to elevate the ecto-5'-nucleotidase activity and reduce the ecto-ADA activity. The results showed that neurotoxic agents, such as Mn, dichlorvos and tebuconazole, play a role in the larval and adult behavior of zebrafish, and affect different neurotransmission systems. Such behavioral changes may be associated with the deficits observed in neurochemical and molecular mechanisms investigated. / Agentes neurot?xicos, tais como pesticidas, solventes org?nicos e metais, s?o capazes de alterar a atividade normal do Sistema Nervoso Central (SNC). O mangan?s (Mn) ? um metal encontrado no ambiente que, quando em excesso, acumula-se no SNC, tornando-se um perigo potencial para a sa?de. Al?m do Mn, outros agentes neurot?xicos podem causar altera??es, levando a d?ficits em sistemas de neurotransmiss?o. O crescimento de pragas de lavoura tem aumentado exponencialmente o uso de agrot?xicos para manuten??o de planta??es. O fungicida tebuconazol ? um triazol utilizado em culturas como a cevada, trigo, amendoim e frutas do pomar e relacionado com altera??es comportamentais e oxidativas. O inseticida dicl?rvos, pertence a fam?lia dos organofosforados atuando atrav?s da inibi??o da acetilcolinesterase (AChE), que leva ? hiperestimula??o dos receptores colin?rgicos. O sistema colin?rgico ? caracterizado pela a??o da acetilcolina (ACh) nos receptores muscar?nicos e nicot?nicos. O n?vel dessa mol?cula ? regulado pela acetilcolinesterase (AChE), que catalisa a degrada??o da ACh em colina e acetato. O sistema purin?rgico ? caracterizado pela a??o do ATP e adenosina (ADO) nos purinoreceptores P2 e P1, respectivamente. Os n?veis dessas mol?culas s?o regulados pela a??o das ectonucleotidases que catalisam a hidr?lise do ATP a adenosina (ADO). A ADO pode ser desaminada a inosina (INO) pela a??o da adenosina desaminase (ADA). A dopamina (DA), sintetizada a partir da L-DOPA, ? o neurotransmissor do sistema dopamin?rgico e age atrav?s dos seus receptores D1-D5, controlando o in?cio e execu??o do movimento. Portanto, este estudo visa avaliar os efeitos da exposi??o ao mangan?s e agrot?xicos, tais como dicl?rvos e tebuconazol, sobre par?metros comportamentais em peixe-zebra nos est?gios larval e adulto, bem como os seus efeitos sobre a sinaliza??o purin?rgica, colin?rgica e dopamin?rgica. A exposi??o ao MnCl2 nos est?gios iniciais de desenvolvimento (1 hpf - 5 dpf) foi capaz de reduzir a dist?ncia percorrida, o ?ngulo de giro absoluto e o tempo de mobilidade das larvas aos 5, 7 e 10 dpf. Al?m disso, causou um aumento nos n?veis de TH aos 5 e 7 dpf, seguido por uma redu??o em 10 dpf. Tamb?m foi observado um aumento dos marcadores de morte celular. Em animais adultos, a exposi??o ao longo de 96 horas causou redu??o na dist?ncia percorrida, n?mero de cruzamentos e ?ngulo de giro absoluto, e ainda uma redu??o na aquisi??o de mem?ria atrav?s da tarefa de esquiva inibit?ria. Al?m disso, os animais adultos expostos a MnCl2 apresentaram aumento nos n?veis de marcadores de morte celular, al?m de TH e DOPAC (?cido 3,4-di-hidroxifenilac?tico) no enc?falo. A exposi??o por 96 horas a este metal tamb?m foi capaz de inibir a atividade das NTPDases e da ecto-ADA e reduzir os transcritos de RNAm do gene ADA2.1, al?m de aumentar os n?veis de ADP e reduzir a quantidade de AMP, ADO e INO no meio. O tebuconazol foi capaz de aumentar a dist?ncia ocular de larvas aos 5 dpf e reduzir a capacidade explorat?ria desses animais, e ainda diminuir a atividade locomotora de peixe-zebra adultos expostos por 96 horas. Al?m disso, este fungicida diminuiu a atividade da AChE tanto em larvas de 5 dpf, quanto em enc?falo de animais adultos. Al?m do mais, o tebuconacol aumentou a express?o relativa de mRNA da AChE em larvas, no entanto, n?o alterou em adultos. A exposi??o ao dicl?rvos, nas fases iniciais do desenvolvimento (1 hpf - 7 dpf), reduziu o tamanho corporal, a taxa de batimentos card?acos e a superf?cie dos olhos de larvas de 7 dpf, bem como afetou a capacidade de escape de larvas aos 7 e 14 dpf. Al?m disso, reduziu a capacidade explorat?ria aos 7 e 14 dpf, padr?o mantido quando os animais tornaram-se adultos jovens aos 30 dpf, e adultos aos 70 dpf. No entanto, as an?lises realizadas aos 120 dpf mostraram recupera??o da atividade comportamental similar ao grupo controle. A an?lise aos 120 dpf da exposi??o ao dicl?rvos nas fases iniciais do desenvolvimento sobre as enzimas do sistema purin?rgico mostrou que este inseticida ? capaz de elevar a atividade da ecto-5?-nucleotidase e reduzir a atividade da ecto-ADA. Os resultados mostram que os agentes neurot?xicos, tais como Mn, dicl?rvos e tebuconazol t?m atua??o significativa sobre o comportamento do peixe-zebra em est?gio larval e adulto, al?m de afetar diferentes sistemas de neurotransmiss?o. Tais altera??es comportamentais podem estar associadas aos d?ficits observados nos mecanismos neuroqu?micos e moleculares investigados.

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-04-04T12:33:24Z No. of bitstreams: 1 DARLAN_GUSSO_DIS.pdf: 1507228 bytes, checksum: e2389f511956dbb64bb7b767d8f73e40 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-04-19T16:26:33Z (GMT) No. of bitstreams: 1 DARLAN_GUSSO_DIS.pdf: 1507228 bytes, checksum: e2389f511956dbb64bb7b767d8f73e40 (MD5) / Made available in DSpace on 2018-04-19T16:43:13Z (GMT). No. of bitstreams: 1 DARLAN_GUSSO_DIS.pdf: 1507228 bytes, checksum: e2389f511956dbb64bb7b767d8f73e40 (MD5) Previous issue date: 2018-02-21 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Mosquitoes are responsible for the transmission of various pathogens. Over the past three years, the world has witnessed epidemics affecting all age groups, such as dengue fever, yellow fever, chikungunya and zika virus. To control and eliminate Aedes vectors responsible for the transmission of these diseases, it is necessary to use larvicides and insecticides such as pyriproxyfen. Pyriproxyfen is a larvicide used to control mosquitoes, mainly of the genus Aedes. This study evaluated the effects of pyriproxyfen on survival, morphology, behavior, and endocrine parameters at different stages of zebrafish development. We have demonstrated that pyriproxyfen can cause changes in the survival rate, behavior and morphology of exposed larvae, adults exposed at the larval stage, and adult offspring of exposed parents. Larvae exposed to pyriproxyfen for 1-96 hpf (hours post-fertilization) showed a decrease in survival rate, heart rate 3 dpf (days post-fertilization), and body length at 5 and 8 dpf. The distance traveled and the mean velocities of the exposed larvae were reduced in comparison to the vehicle group (1% DMSO). Therefore, pyriproxyfen changes the morphology and behavior of zebrafish in early stages of development and may affect the next generations. However, when the exposed larvae were maintained until the adult stage (6 months), we did not observe differences in locomotor parameters. Parental exposure to pyriproxyfen induced decrease in locomotion of zebrafish adult offspring as well as an anxiolytic-like behavior. We also tested pyriproxyfen exposure for 96 hours in adult zebrafish and the locomotion, anxiety, memory and endocrine parameters were analyzed as well as the gene expression of the glucocorticoid receptor (GR) and corticotropin-releasing factor (CRF). These results showed that there was no significant difference in locomotion, anxiety and endocrine parameters. In addition, there was an impairment of habituation memory in adult zebrafish exposed to pyriproxyfen. Therefore, it is important to control the use of larvicides due to their toxic effects on non-target species. Our findings demonstrated the importance of studies related to the use of larvicides, since they are potential causes of morphological and behavioral alterations in aquatic species, such as zebrafish. / Mosquitos s?o respons?veis pela transmiss?o de v?rios agentes patog?nicos. Nos ?ltimos tr?s anos, o mundo testemunhou epidemias que afetam todas as faixas et?rias, tais como dengue, febre amarela, chikungunya e zika v?rus. Para controlar e eliminar vetores do g?nero Aedes, respons?vel pela transmiss?o destas doen?as, ? necess?rio usar larvicidas e inseticidas como o piriproxifeno. O piriproxifeno ? um larvicida usado para eliminar mosquitos, principalmente do g?nero Aedes. Este estudo avaliou os efeitos do piriproxifeno sobre a sobreviv?ncia, morfologia, comportamento e par?metros end?crinos em diferentes est?gios de desenvolvimento de peixe-zebra. N?s demonstramos que o piriproxifeno pode causar altera??es na taxa de sobreviv?ncia, comportamento e morfologia de larvas expostas, adultos expostos no est?gio larval e descendentes adultos de pais expostos. As larvas expostas ao piriproxifeno durante 1-96 hpf (horas p?s-fertiliza??o) mostraram uma diminui??o na taxa de sobreviv?ncia, frequ?ncia card?aca 3 dpf e comprimento corporal nos 5 e 8 dpf (dias p?s- fertiliza??o). A dist?ncia percorrida e a velocidade m?dia das larvas expostas foram reduzidas em rela??o ao grupo ve?culo (1% de DMSO). Portanto, o piriproxifeno altera a morfologia e o comportamento do peixe-zebra em est?gios iniciais de desenvolvimento e pode afetar as pr?ximas gera??es. No entanto, quando as larvas expostas foram mantidas at? o est?gio adulto (6 meses), n?o observamos diferen?as nos par?metros locomotores. A exposi??o parental ao piriproxifeno induziu uma diminui??o na locomo??o da prole adulta, bem como um comportamento do tipo ansiol?tico. N?s tamb?m avaliamos o efeito da exposi??o ao piriproxifeno em peixe-zebra na fase adulta durante 96 horas sobre a locomo??o, ansiedade, mem?ria e par?metros end?crinos, como express?o g?nica do receptor de glicocorticoide (GR) e fator de libera??o de corticotrofina (CRF). Os resultados demonstraram que n?o houve diferen?a significativa na locomo??o, ansiedade e par?metros end?crinos. Al?m disso, houve um preju?zo da mem?ria de habitua??o do peixe-zebra adulto exposto ? piriproxifeno. Portanto, ? importante controlar o uso de larvicidas devido aos seus efeitos t?xicos em esp?cies n?o-alvo. Nossos achados demonstram a import?ncia de estudos relacionados com a utiliza??o de larvicidas, uma vez que s?o potenciais causadores de altera??es morfol?gicas e comportamentais em esp?cies aqu?ticas, como o peixe-zebra.