Thesis (Ph. D.)--Illinois State University, 1997. / Title from title page screen, viewed June 5, 2006. Dissertation Committee: H. Tak Cheung (chair), Sean Arkins, Herman E. Brockman, Paul A. Garris, Brian J. Wilkinson. Includes bibliographical references (leaves 113-121) and abstract. Also available in print.

De acordo com dados da Organização Mundial da Saúde (OMS) estimam-se que os agrotóxicos causam anualmente 70 mil intoxicações agudas e crônicas que evoluem para óbito. Dentre eles, o paraquat (PQ) é o que apresenta maior taxa de mortalidade, sendo responsável por cerca de 13% de todos os casos registrados, principalmente devido a falta de um tratamento efetivo. O principal mecanismo de toxicidade proposto está associado ao ciclo redox do PQ, onde ocorre a formação de espécies reativas (ERs) de oxigênio e nitrogênio, levando ao estresse oxidativo (EO). Na literatura há relatos do uso de antioxidantes para casos de intoxicação do PQ. Dessa maneira, nesse trabalho avaliou-se o uso de melatonina associada a nanocápsulas de núcleo lipídico (Mel-LNC) na proteção contra os danos causados pelo PQ, uma vez que o uso da nanotecnologia melhorou a atividade antioxidante dessa molécula. Para tal utilizou-se o sistema in vitro, linhagem celular de adenocarcinoma pulmonar (A549), e o modelo alternativo in vivo, Caenorhabditis elegans. Mel-LNC e nanocápsulas de núcleo lipídico (LNC) foram preparadas de acordo com o método de deposição do polímero pré-formado. Ambas as formulações foram caracterizadas avaliando tamanho de partícula, potencial zeta e pH, e para Mel-LNC foram determinadas a concentração de melatonina e porcentagem de encapsulação. Os resultados encontrados estão de acordo com os parâmetros já validados para essas formulações. Foi possível verificar que as formulações MEL-LNC e LNC se mantiveram estáveis nos meios de cultura utilizados nos ensaios in vitro e in vivo. No estudo in vitro foi observado que o tratamento com ambas as formulações não causaram diminuição da viabilidade nem dano de DNA na linhagem celular utilizada. Além disso, foi verificado a internalização da Mel-LNC utilizando-se a formulação marcada com rodamina B, sendo possível verificar uma intensa fluorescência vermelha ao redor do núcleo da célula. O pré-tratamento com Mel-LNC foi capaz de aumentar a viabilidade celular e diminuir o dano oxidativo de DNA causado pelo paraquat após 24 horas de exposição, porém isso não ocorreu quando as células foram pré-tratadas com melatonina livre. No estudo com o modelo alternativo C. elegans, foi utilizada uma formulação de Mel-LNC marcada com rodamina B (Mel-LNC-RoB), a fim de verificar a absorção dessa formulação pelo nematoide. Foi possível observar que a internalização da Mel-LNC no C. elegans ocorre principalmente pela via oral, uma vez que se verificou uma intensa fluorescência no intestino do nematoide após o tratamento com a Mel-LNC-RoB e após três horas, essa fluorescência se distribuiu pelo restante do corpo, apresentando inúmeros pontos de fluorescência fora do intestino. Com relação à avaliação do efeito protetor nesse modelo alternativo in vivo, pode-se inferir que o pré-tratamento com Mel-LNC aumentou a sobrevida, diminuiu a produção de espécies reativas (ERs) e manteve o desenvolvimento normal dos nematoides após a exposição ao PQ, sendo que isso não foi verificado quando os mesmos foram pré-tratados com melatonina livre. Além disso, verificou-se que as nanocápsulas de núcleo lipídico (LNC) são seguras para o uso no modelo C. elegans, uma vez que apresentou alto valor para a dose letal 50 (DL50), e alterações no desenvolvimento e produção de ERs somente ocorreram em doses mais elevadas que as utilizadas em nossos experimentos. Dessa maneira, a formulação de Mel-LNC mostrou-se um promissor candidato para estudos futuros nos casos de intoxicação por paraquat. / According to estimations by World Health Organization (WHO), pesticides are responsible for 70 thousand acute intoxication cases that lead to death per year. Among these compounds, paraquat (PQ) presents the highest mortality rate, about 13% of all registered cases, especially for the lack of effective treatment. The major mechanism of toxicity proposed is associated to its redox cycle, in which oxygen and nitrogen reactive species (RS) are generated culminating in oxidative stress (OS). Some reports in the literature support the use of antioxidants for PQ intoxication cases. The present study aimed to evaluate the use of melatonin-loaded lipid-core nanocapsules (Mel-LNC) in the protection against PQ-induced damages, considering that nanotechnology has improved the antioxidant activity of this molecule. For this purpose, an in vitro system composed by lung adenocarcinoma (A549) cell line, and the in vivo alternative model of Caenorhabditis elegans have been utilized. Mel-LNC and unloaded lipid-core nanocapsules were prepared by self-assembly and characterized by particle sizing, zeta potential and pH, and for Mel-LNC formulation it was determined the drug content and encapsulation efficiency. The results are in agreement with the parameters already validated for these formulations. It was possible verify that Mel-LNC and LNC formulations remained stable in the culture medium utilized in in vitro and in vivo experiments. Results from in vitro studies showed that none of the formulations induced reduction in cell viability or DNA damage in treated cells. Besides, it was observed the internalization of Mel-LNC marked with rhodamine B, showing an intense red fluorescence around the cell nucleus. Pretreatment with Mel-LNC was able to enhance cell viability and diminish DNA oxidative damage caused by paraquat after 24h exposure, which could not be observed when cells were pretreated with Mel. In the study with the alternative model C. elegans, a rhodamine (Ro)-linked Mel-LNC formulation was prepared in order to assess the absorption of the formulation by the nematode. Mel-LNC uptake in C. elegans was found to occur mainly by the oral route, once an intense fluorescence was observed in the intestine after treatment with Mel-LNC-RoB, which after 3h distributed to the rest of the body, presenting numerous fluorescence dots outside the intestine. In relation to the evaluation of protection with the in vivo alternative model, results indicate that pretreatment with Mel-LNC increased survival rate, reduced the production of reactive species and maintained the normal development of nematodes after paraquat exposure, while the same observations were not found after pretreatment with free melatonin. In addition, the lipid-core nanocapsules (LNC) were found to be safe in the C. elegans model, due to its high lethal dose (LD50) value, and development alterations and RS production only occurred in the higher doses than those utilized in our experiments. Therefore, the Mel-LNC formulation demonstrated to be a promising candidate for future studies aiming treatment of paraquat intoxication cases.

De acordo com dados da Organização Mundial da Saúde (OMS) estimam-se que os agrotóxicos causam anualmente 70 mil intoxicações agudas e crônicas que evoluem para óbito. Dentre eles, o paraquat (PQ) é o que apresenta maior taxa de mortalidade, sendo responsável por cerca de 13% de todos os casos registrados, principalmente devido a falta de um tratamento efetivo. O principal mecanismo de toxicidade proposto está associado ao ciclo redox do PQ, onde ocorre a formação de espécies reativas (ERs) de oxigênio e nitrogênio, levando ao estresse oxidativo (EO). Na literatura há relatos do uso de antioxidantes para casos de intoxicação do PQ. Dessa maneira, nesse trabalho avaliou-se o uso de melatonina associada a nanocápsulas de núcleo lipídico (Mel-LNC) na proteção contra os danos causados pelo PQ, uma vez que o uso da nanotecnologia melhorou a atividade antioxidante dessa molécula. Para tal utilizou-se o sistema in vitro, linhagem celular de adenocarcinoma pulmonar (A549), e o modelo alternativo in vivo, Caenorhabditis elegans. Mel-LNC e nanocápsulas de núcleo lipídico (LNC) foram preparadas de acordo com o método de deposição do polímero pré-formado. Ambas as formulações foram caracterizadas avaliando tamanho de partícula, potencial zeta e pH, e para Mel-LNC foram determinadas a concentração de melatonina e porcentagem de encapsulação. Os resultados encontrados estão de acordo com os parâmetros já validados para essas formulações. Foi possível verificar que as formulações MEL-LNC e LNC se mantiveram estáveis nos meios de cultura utilizados nos ensaios in vitro e in vivo. No estudo in vitro foi observado que o tratamento com ambas as formulações não causaram diminuição da viabilidade nem dano de DNA na linhagem celular utilizada. Além disso, foi verificado a internalização da Mel-LNC utilizando-se a formulação marcada com rodamina B, sendo possível verificar uma intensa fluorescência vermelha ao redor do núcleo da célula. O pré-tratamento com Mel-LNC foi capaz de aumentar a viabilidade celular e diminuir o dano oxidativo de DNA causado pelo paraquat após 24 horas de exposição, porém isso não ocorreu quando as células foram pré-tratadas com melatonina livre. No estudo com o modelo alternativo C. elegans, foi utilizada uma formulação de Mel-LNC marcada com rodamina B (Mel-LNC-RoB), a fim de verificar a absorção dessa formulação pelo nematoide. Foi possível observar que a internalização da Mel-LNC no C. elegans ocorre principalmente pela via oral, uma vez que se verificou uma intensa fluorescência no intestino do nematoide após o tratamento com a Mel-LNC-RoB e após três horas, essa fluorescência se distribuiu pelo restante do corpo, apresentando inúmeros pontos de fluorescência fora do intestino. Com relação à avaliação do efeito protetor nesse modelo alternativo in vivo, pode-se inferir que o pré-tratamento com Mel-LNC aumentou a sobrevida, diminuiu a produção de espécies reativas (ERs) e manteve o desenvolvimento normal dos nematoides após a exposição ao PQ, sendo que isso não foi verificado quando os mesmos foram pré-tratados com melatonina livre. Além disso, verificou-se que as nanocápsulas de núcleo lipídico (LNC) são seguras para o uso no modelo C. elegans, uma vez que apresentou alto valor para a dose letal 50 (DL50), e alterações no desenvolvimento e produção de ERs somente ocorreram em doses mais elevadas que as utilizadas em nossos experimentos. Dessa maneira, a formulação de Mel-LNC mostrou-se um promissor candidato para estudos futuros nos casos de intoxicação por paraquat. / According to estimations by World Health Organization (WHO), pesticides are responsible for 70 thousand acute intoxication cases that lead to death per year. Among these compounds, paraquat (PQ) presents the highest mortality rate, about 13% of all registered cases, especially for the lack of effective treatment. The major mechanism of toxicity proposed is associated to its redox cycle, in which oxygen and nitrogen reactive species (RS) are generated culminating in oxidative stress (OS). Some reports in the literature support the use of antioxidants for PQ intoxication cases. The present study aimed to evaluate the use of melatonin-loaded lipid-core nanocapsules (Mel-LNC) in the protection against PQ-induced damages, considering that nanotechnology has improved the antioxidant activity of this molecule. For this purpose, an in vitro system composed by lung adenocarcinoma (A549) cell line, and the in vivo alternative model of Caenorhabditis elegans have been utilized. Mel-LNC and unloaded lipid-core nanocapsules were prepared by self-assembly and characterized by particle sizing, zeta potential and pH, and for Mel-LNC formulation it was determined the drug content and encapsulation efficiency. The results are in agreement with the parameters already validated for these formulations. It was possible verify that Mel-LNC and LNC formulations remained stable in the culture medium utilized in in vitro and in vivo experiments. Results from in vitro studies showed that none of the formulations induced reduction in cell viability or DNA damage in treated cells. Besides, it was observed the internalization of Mel-LNC marked with rhodamine B, showing an intense red fluorescence around the cell nucleus. Pretreatment with Mel-LNC was able to enhance cell viability and diminish DNA oxidative damage caused by paraquat after 24h exposure, which could not be observed when cells were pretreated with Mel. In the study with the alternative model C. elegans, a rhodamine (Ro)-linked Mel-LNC formulation was prepared in order to assess the absorption of the formulation by the nematode. Mel-LNC uptake in C. elegans was found to occur mainly by the oral route, once an intense fluorescence was observed in the intestine after treatment with Mel-LNC-RoB, which after 3h distributed to the rest of the body, presenting numerous fluorescence dots outside the intestine. In relation to the evaluation of protection with the in vivo alternative model, results indicate that pretreatment with Mel-LNC increased survival rate, reduced the production of reactive species and maintained the normal development of nematodes after paraquat exposure, while the same observations were not found after pretreatment with free melatonin. In addition, the lipid-core nanocapsules (LNC) were found to be safe in the C. elegans model, due to its high lethal dose (LD50) value, and development alterations and RS production only occurred in the higher doses than those utilized in our experiments. Therefore, the Mel-LNC formulation demonstrated to be a promising candidate for future studies aiming treatment of paraquat intoxication cases.

Nuclear architecture and gene expression in Caenorhabditis elegans Mgr. Jitka Bolková ABSTRACT The parental genomes are initially separated in each pronucleus after fertilization. During the first mitosis this spatial distribution is being disintegrated. In my thesis we used green-to-red phoroconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their distribution in space in living Caenorhabditis elegans embryos starting shortly after fertilization. Both of the parental chromatin domains within the nucleus are separated in the zygote and at the 2-cell stage. Intermingling occurs first after chromatin decondensation at the beginning of the cell cycle at the 4-cell stage. To our knowledge, we report to the first live observation of the separation and subsequent mixing of parental chromatin during embryogenesis. Following of the photoconverted chromatin also allowed us to detect a reproducible 180ř rotation of the nuclei during cytokinesis of the zygote. Tracking of fluorescently-labelled P granules and polar bodies showed that the entire embryo rotates during the first cell division. In the second part of the thesis we used the C. elegans model to investigate relationship between nuclear architecture and gene expression. We focused on localization of...

Abstract Collagen prolyl 4-hydroxylases (C-P4Hs) and related enzymes catalyze the hydroxylation of certain proline residues in animal collagens and plant hydroxyproline-rich proteins, respectively. Animal C-P4Hs and their isoenzymes have been characterized to date from humans, rodents, insects and nematodes. Most of the animal C-P4Hs are α2β2 tetramers in which protein disulphide isomerase (PDI) serves as the β subunit, but the nematode C-P4Hs characterized so far have unique molecular compositions. Two P4Hs have been cloned from the plant Arabidopsis thaliana and one from the Paramecium bursaria Chlorella virus-1, these being monomeric enzymes. This thesis reports on the identification of a large P4H family in the green alga Chlamydomonas reinhardtii and the cloning and characterization of one member, Cr-P4H-1. This is a soluble monomer that hydroxylates in vitro several peptides representing sequences found in C. reinhardtii cell wall proteins. Lack of its activity led to a defective cell wall structure, indicating that Cr-P4H-1 is essential for proper cell wall assembly and that the other P4Hs cannot compensate for the lack of its activity. Two C. elegans genes, Y43F8B.4 and C14E2.4, predicted to code for C-P4H α subunit-like polypeptides were analyzed. Three transcripts were generated from Y43F8B.4, one of them coding for a functional C-P4H α subunit named PHY-4.1. C14E2.4 turned out not to be a C-P4H α subunit gene, as a frame-shift led to the omission of codons for two catalytically critical residues. PHY-4.1 formed active tetramers and dimers with PDI-2 and had unique substrate requirements in that it hydroxylated certain other proline-rich sequences besides collagen-like peptides. Inactivation of the Y43F8B.4 gene led to no obvious morphological abnormalities. Spatial expression of the phy-4.1 transcript and PHY-4.1 polypeptide was localized to the pharynx and the excretory duct. Taken together, these data indicate that PHY-4.1 is not involved in the hydroxylation of cuticular collagens but is likely to have other substrates in vivo. Cloning and characterization of the PHY-1 and PHY-2 subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in assembly properties between the C. elegans and C. briggsae PHY-2 subunits in spite of their high amino acid sequence identity. Genetic disruption of C. briggsae phy-1 resulted in a less severe phenotype than that observed in C. elegans, evidently on account of its more efficient assembly of the C. briggsae PHY-2 to an active C-P4H explaining the milder phenotype. Rescue of C. elegans and C. briggsae phy-1 mutants was achieved by injection of a wild-type phy-1 gene from either species.

No description available.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, late-onset, progressive neurodegenerative disease. A familial form of ALS, autosomal dominant ALS8, is characterized by a mutation in an ER membrane protein, VAPB. To characterize the role of VAPB in motor neurons, two C. elegans models were generated: one expressing human VAPB-P56S and another with the knockdown of C. elegans VAPB ortholog, VPR-1. Overexpression of human VAPB in DA neurons caused backward locomotion defects, enhanced vulnerability to oxidative stress and premature neuronal death. Knockdown of vpr-1 in C. elegans recapitulated the loss of protein function believed to be associated with human cases of ALS8. It caused backward locomotion defects, such as uncoordination and slowed rates of movement, as well as age-dependent motor neuronal death. In both models, DA6 and DA7 were the most vulnerable motor neurons. Because of the unexpected developmental defects associated with the VAPB transgenic model, the knockdown of vpr-1 may be a better model to recapitulate the human disease. This model provides further support that ALS8 pathogenesis is due to a loss of VAPB protein function and can also be used to test drugs or treatments that may delay the onset of neuronal death.

The regulation and evolution of the inverted repeat transposable element Tel, found in the nematode Caenorhabditis elegans, was studied. The stability of Tel elements in the N2 strain genome was investigated by cloning seventeen N2 Tel elements. To examine their structural integrity, sixteen cloned N2 Tel elements were restriction mapped and, in the case of some variants, their DNA was partially sequenced. Two restriction site variants, Tcl(Eco).12 and Tcl(Hpa-).9, were found. Tel(1.5).10b had lost 89 bp from one end, while Tcl(1.7).28 contained a 55 bp insertion. Two additional elements, Tcl(0.9).2 and Tcl(0.9).14, had different internal deletions. Each element was about 900 bp in length. The majority of Tel elements cloned from the N2 strain were found to have identical restriction maps. Somatic excision of Tel elements in the N2 genome was demonstrated. Tel elements in N2 are apparently both structurally and functionally intact. Nevertheless, mobilization of Tel elements in the N2 germline is restricted. Two new transposable element families, Barney (also known as TCbl) and TCb2, were discovered in a closely related nematode, Caenorhabditis briggsae due to Tel identity. These two families, distinguished through differential inter-element hybridization, showed multiple banding differences between strains. The open reading frames (ORFs) of Tel and Barney share 71% DNA sequence and 74% amino acid sequence identity. The putative terminus of Barney exhibits 68% identity with the 54 bp terminal repeat of Tel. Partial sequencing of TCb2 revealed that its ORF is equally diverged from Barney and Tel. The basis of the sequence heterogeneity observed in the C. briggsae transposons and not in the C. elegans transposons could be due to either horizontal transfer or alternate paths of divergence. Significant sequence identity was found between Tel, Barney, and HB1 (a transposable element from Drosophila melanogaster) within their coding regions and terminal repeats. These sequence similarities define a subclass of inverted repeat transposable elements inhabiting two different phylla, Arthropoda and Nematoda. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.