Thesis (Ph. D.)--University of Chicago. / Reprinted from Journal of morphology, Nov. 1898, v. 15, no. 2. "Literature referred to": p. 202-207.

Thesis (Ph. D.)--University of Chicago. / Reprinted from Journal of morphology, Nov. 1898, v. 15, no. 2. "Literature referred to": p. 202-207.

Graduation date: 1997

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: juvenile hormone; Limulus; horseshoe crab. Includes bibliographical references (p. 68-79).

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Nancy M. Targett and Timothy E. Targett, College of Marine and Earth Studies; Pamela J. Green, Dept. of Plant & Soil Science. Includes bibliographical references.

"Lipid-based hormones known as the juvenile hormones (JH) are ubiquitous among the arthropods, but their presence, functions, and sites of production in the horseshoe crab, Limulus polyphemus, remain unknown. Large size and lack of secondary sex characteristics in adult female horseshoe crabs may indicate continuous growth and molting throughout life, which is the outcome of high JH levels in insects and crustaceans. Here a study was undertaken to detect and localize lipid-based hormones in horseshoe crab hemolymph and tissue. Capillary electrophoresis and RP-HPLC analyses indicate the presence of a JH-like compound in subadult horseshoe crab hemolymph. The compound is present only in much lower amounts in the hemolymph of adult male and adult female horseshoe crabs. Identification of this compound was based on its similar retention time to standard JH, co-migration with added JH, and cross-reactivity with a polyclonal antibody to JH III. In addition, immunohistochemistry was used to localize the production site of this compound. Analysis of neural tissue, the assumed site of production, yielded no reactivity with labeled anti-JH III antiserum. In larval animals, however, reactivity was noted in yolk contained within the digestive tract. Since the larvae are lecithotrophic and feeding only on their yolk reserves, JH in the gut may be maternal, deposited in the egg before laying. Based on these results, we conclude that horseshoe crabs produce a lipid-based, JH-like hormone, with functional similarity to JH III in insects (i.e., maintenance of the juvenile form during growth and molting.) This paper is the first substantiation of such a hormone in horseshoe crabs. Our findings suggest that JH will be found in other chelicerates as well."

Endotoxin is a part of the cell wall of gram-negative bacteria, consisting of serotype-specific polysaccharide, core oligosaccharide, and lipid A. Lipid A is responsible for an array of pathophysiologic reactions in animal hosts. Amebocyte lysate originated from Limulus polyphemus (horseshoe crab) has been used extensively in various assay systems to detect endotoxin. One of the assays, a chromogenic Limulus amebocyte lysate (CLAL) test was developed in 1978 and has been used extensively in human clinical fields for its high sensitivity and ease in quantitation. The use of the CLAL test in veterinary fields has been limited to dogs, horses and cattle. The objective of the thesis research was to determine the level of endotoxin by the CLAL assay in broiler chickens. Since gram-negative septicemia is common in broiler chickens, the detection of endotoxemia would help in understanding the pathogenesis and in developing a new treatment or prophylactic mean. By the use of a kinetic method, the CLAL assay detected the standard endotoxin (phenol-water extract from Escherichia coli, 055:B5 strain) in the range between 100 ng and 10 pg/ml. The intra-assay variation was 1.2% and interassay variation was 18.8% based on 1.0 ng standard. The control showed spontaneous release of the chromophore starting around 40 min. after the start of the reaction. This spontaneous release was found not due to contamination of pyrogen-free water (PFW) or substrate by endotoxin. With chicken plasma, various non-specific reactions were detected. Plasma alone released the chromophore in a slow, steady manner, but this reaction was virtually eliminated by heating at 100 C. Chicken plasma contained both inhibitor(s) and enhancer(s) for the test. Endotoxin-free plasma samples were prepared by absorption and reconstituted with 1.0 ng/ml of endotoxin. After 1:10 dilution in PFW, heating (10 min.) at 100 C was found most adequate to inactivate these factors as compared with heating to 70 or 85 C. With plasma samples which had been diluted and heated at 100 C, however, still some nonspecific reaction was detected; the lysate, in the absence of substrate, caused some precipitates with chicken plasma in a nonspecific manner, making it difficult to interpret the OD readings. Because of these nonspecific reactions largely inherent to the state of lysate, sensitivity was judged only to 100 pg endotoxin/ml of chicken plasma. A commercial test kit also showed 100 pg/ml sensitivity with the end point method, but found unreliable since proper controls cannot be evaluated in a similar manner as the kinetic method. Thirty chicken plasma were collected from 3 local broiler farms and was judged to contain less than 100 pg/ml in 29 birds, while one bird showed 12.0 ng/ml of endotoxin. Twenty-three per cent of the chickens showed gram-negative bacteremia without detectable endotoxemia, and the bird with endotoxemia did not have bacteremia. One microgram of the standard endotoxin was injected intravenously to 20 broiler chickens raised in the laboratory, and 5 were sacrificed at 2, 30, 60, and 90 minutes after the injection. The endotoxin was found to be cleared from the blood at the rate of 152 pg/min. To increase the sensitivity and to decrease the cost of the CLAL test, future efforts should be made; 1) to significantly decrease the nonspecific reaction between the lysate and substrate; and 2) to block the precipitation or clotting reaction between the lysate and chicken plasma. If these nonspecific reactions be controlled, the CLAL test could be run in a simple end-point method and/or in an automated manner with chicken plasma. / Graduation date: 1997

No description available.

O objetivo do presente trabalho foi quantificar, in vitro e in vivo, a endotoxina bacteriana (LPS) aderida a ligaduras ortodônticas elastoméricas estéticas de poliuretano e de silicone, empregando o teste Limulus Amebocyte Lysate (LAL). Para o estudo in vitro foram utilizadas quatro tipos de ligaduras elastoméricas estéticas: Sani-Ties (poliuretano) e Sili-Ties (silicone), da GAC, e Mini Single Case Ligature Stick (poliuretano) e Synergy&reg; Low-friction ligatures (silicone), da Rock Mountain, sendo 5 contaminadas com solução de endotoxina (controle positivo) e 5 não-contaminadas (controle negativo). Réplicas feitas de fio de amarrilho torcido e de aço inox fundido, de mesmo tamanho e formato das ligaduras elastoméricas, contaminadas ou não com endotoxina, foram usadas como controle. A quantificação de endotoxina foi realizada por meio do teste LAL (Kit QCL-1000&trade;), sendo os resultados expressos em unidades de endotoxina (UE/mL). No estudo in vivo, 20 pacientes de ambos os gêneros, com faixa etária entre 15 e 30 anos, que iniciaram tratamento com aparelho ortodôntico fixo na Faculdade de Odontologia de Ribeirão Preto - Universidade de São Paulo receberam, randomicamente, em quadrantes alternados, os mesmos quatro tipos de ligaduras elastoméricas utilizadas no estudo in vitro, sendo uma ligadura de cada marca inserida nos caninos superiores e inferiores (13, 23, 33 e 43), aleatoriamente. Vinte e um dias após, as ligaduras foram removidas e processadas para quantificação da endotoxina bacteriana, utilizando também o Kit QCL-1000&trade;. Todos os dados obtidos foram submetidos à análise estatística apropriada de acordo com a distribuição dos dados, por meio dos testes de Kruskal-Wallis e pós-teste de Dunn (estudo in vitro) e ANOVA de medidas repetidas e pós-teste de Tukey (estudo in vivo). Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. De acordo com os resultados obtidos, observou-se que a endotoxina bacteriana aderiu-se a todos os materiais testados. No estudo in vitro, o grupo GAC silicone foi o que apresentou menor mediana de contaminação (1,15 UE/mL), com relação aos outros grupos (p<0,0001), os quais não apresentaram diferença estatisticamente significante quando comparados entre si (p>0,05). No estudo in vivo, de maneira semelhante ao observado no estudo in vitro, o grupo GAC silicone foi o que apresentou menor média de contaminação (0,577±0,017 UE/mL), com diferença estatisticamente significante (p<0,001) em comparação aos demais grupos. Pôde-se concluir, que a endotoxina bacteriana apresentou afinidade pelas ligaduras elastoméricas estéticas à base de silicone e poliuretano testadas. As ligaduras de silicone da marca GAC foram as que apresentaram menor quantidade de endotoxina aderida às suas superfícies / The objective of this study was to quantify, in vitro and in vivo, bacterial endotoxin (LPS) attached to esthetic elastomeric orthodontic ligatures made of polyurethane and silicone using the Limulus Amebocyte Lysate test (LAL). For the in vitro study, were used four types of aesthetic elastomeric ligatures: Sani-Ties (polyurethane) and Sili-Ties (silicone) - GAC, and Mini Single Case Ligature Stick (polyurethane) and Synergy&reg; Low-friction ligatures (silicone) - Rock Mountain; 5 of each were contaminated with endotoxin solution (positive control) and 5 non-contaminated (negative control). Replicas made of twisted wire ligatures and of cast stainless steel, of the same size and shape than the elastomeric ligatures, contaminated or not with endotoxin, were used as control. Endotoxin quantification was performed using the LAL test (QCL-1000&trade; kit), the results being expressed in EU/mL. In the in vivo study, 20 patients of both genders, with ages ranging from 15 to 30 years old, who started treatment with a fixed orthodontic appliance at the School of Dentistry of Ribeirão Preto - University of São Paulo, received randomly the same four types of elastomeric ligatures used in the in vitro study, being a ligature of each brand inserted in the upper and lower canines (UR3, UL3, LR3, LL3), randomly. Twenty-one days later, ligatures were removed and processed for quantification of bacterial endotoxin, using the same test as the in vitro study. All data were submitted to appropriate statistical analysis according to the data distribution, using KruskalWallis tests and Dunn post-test (in vitro study) and ANOVA of repeated measurements and Tukey\'s post-test (in vivo study). All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. According to the results obtained, it was observed that the bacterial endotoxin attached to all materials tested. In the in vitro study, the GAC silicone group had the lowest median contamination (1.15 EU/mL), in relation to the other groups (p<0.0001), which did not present a statistically significant difference when compared to each other (p>0.05). In the in vivo study, similar to that observed in the in vitro study, the GAC silicone group had the lowest mean contamination (0.577 ± 0.017 EU/mL), with a statistically significant difference (p< 0.001) compared to the others groups. It could be concluded that bacterial endotoxin exhibited affinity for the tested silicone and polyurethane aesthetic elastomeric ligatures. The silicone ligatures of GAC brand were the ones that presented less amount of endotoxin attached to their surfaces

ABSTRACT This thesis was divided into two parts. The purpose of part I was to clone and sequence the full-length ependymin gene from the invertebrate Limulus polyphemus, or portions of the gene, and to use RT-PCR to determine whether expression of this gene increases during leg regeneration. PCR was chosen as the method for obtaining the gene due to the success our lab had previously characterizing several ependymin genes using this approach. Three sets of primers were designed based on the conserved domains between teleost fish and three invertebrate ependymin sequences. “Sea primers" were designed based on the nucleotide sequence of the sea cucumber H. glaberrima for each conserved domain, and these primers produced all four of the expected size amplicons with Limulus DNA, but surprisingly only one such band with the sea cucumber Sclerodactyla briareus. The consensus primers (con-primers) were designed based on the most conserved nucleotide among all known ependymin species at each particular position in the conserved domains. Primers designated“5-11 primers" were designed based on the absolutely conserved domains among the three known invertebrate ependymins. Neither con-primers nor 5-11 primers produced any bands of the expected size; this was true for both species of DNA. One very strong band was produced using“5-11" primer pair 6/10 with both species. One of the bands from this reaction from Limulus was cloned and sequenced, and showed a very strong homology (88% over 292 bp) with mouse FGF-14, a neurotrophic factor involved in mouse neurogenesis. The expression of this gene during leg regeneration will be tested in future experiments. Limulus GAPDH was also cloned and sequenced, and a genomic intron was identified for the first time in this study. This Limulus housekeeping gene will be used in future studies for gene expression comparisons. The purpose of part two of this thesis was to study the up-regulation of growth-related genes induced by treatment of a human neuroblastoma SH-SY5Y cell line with a human ependymin peptide mimetic (hEPN-1), in an attempt to help provide a basis for using human EPN mimetics as therapeutics in stroke and neurodegenerative diseases. The sequence of this mimetic is derived from an area of human MERP-1 analogous to goldfish mimetic CMX-8933. The human mimetic was previously found to up-regulate growth related genes L-19, EF-2 and ATP Synthase in the mouse neuroblastoma cell line Nb2a (Saif, 2004). The expression levels of genes encoding ribosomal proteins and ribosomal RNA were studied using RT-PCR as hallmarks of proliferating cells. hEPN-1 was found to increase the expression of the nuclear-encoded ribosomal proteins S-19 and S-12, an average of 2.76 fold and 1.74 fold, with statistically significant p-values of 0.031 and 0.015 (<0.05), respectively. The expression levels of nuclear-encoded 5.8S ribosomal RNA (p = 0.018) and the mitochondrial-encoded 16S RNA (p = 0.046) were found to be increased an average of 14.04 fold and 3.91 fold, respectively. Thus, human ependymin mimetic hEPN-1 appears to stimulate growth-related genes, a property which can be useful to regenerate neuronal tissue after injury.