Among the genetic mutations involved in carcinogenesis, TP53 mutation is a frequent event in many types of cancer. P53 is a transcription factor that regulates activities such as cell cycle arrest, apoptosis, DNA repair and angiogenesis. The majority of TP53 mutations are missense mutations that accumulate in cancer and are often retained in distant metastases. The effects of the mutant p53 proteins include loss of function, dominant-negative effects over wild-type (WT) p53 and possible acquisition of new properties (gain-of-function). However, some of these properties may differ from one mutant p53 protein to another. These differences could have implications for the in vivo behaviour of tumours carrying particular mutations and hence patient prognosis. The aim of this project was to investigate the phenotypic variation between cells transformed with different p53 mutants. This was achieved by constructing a range of TP53 mutants (R175H, G245S, R248W, R248Q, R273H, R282W) using PCR-based mega-primer site directed mutagenesis. These mutants were cloned into a mammalian bi-cistronic expression vector (designed for the co-expression of WT and mutant TP53 from a single plasmid) to allow transient expression in NCI-H358 cells (p53 null). Regard to the method for PCR site directed mutagenesis, the main technical difficulty with conventional methods was the insufficiency of the mutant TP53 product yield (75%). This thesis has modified these methods by carrying over the start template to a second round of PCR and increasing the MgCl2 concentration. This modified PCR-based site directed mutagenesis method has demonstrated an increased mutant TP53 product yield (100%). The tetracycline expression system is the most widely used for conditional inducible systems in mammalian cells, although high background expression has been a main problem. The ecdysone inducible system potentially allows for the study of the conditional expression of the exogenous reporter gene even though it may be cell lethal or alter the phenotype during the selection of transfectants. This system relies on two independent transfections of two plasmids namely pVgRXR and pIND. However, disruption of the regulatory element within the plasmid during stable integration can result in silence or high background expression of the exogenous reporter gene. A previous study reported a transient luciferase reporter assay to screen the cell line stably transfected with pVgRXR plasmid. However, there is no suitable method to screen the subsequent pIND transfection. This thesis has demonstrated a real time RT-PCR strategy to screen for the background expression problem associated with the ecdysone expression system. However, due to the project’s time limitations, a transient expression system rather than a stable expression system was used. The metastasis related cellular activity of WT/mutant TP53 transfected NCI-H358 cells was examined using a range of in vitro functional assays including a proliferation assay, a p21 promoter binding activity assay, a colony formation assay, and a migration assay. To extend the study, this thesis also employed real-time RT-PCR to examine the mRNA expression level of three metastatic related genes, VEGF, HER-2, and E-cadherin, in the WT/mutant TP53 transfected NCI-H358 cells. The results showed that different WT/mutant TP53 transfected cell linse could contribute to markedly different cellular activity. Among these mutants, R175H produced the highest cellular proliferation activity, the strongest dominant-negative activity over the WT on the p21 promoter binding activity and apoptosis activity, and the greatest effect on cellular migration. Furthermore, the real-time PCR results showed that the WT p53 inhibited transcription of key metastasis-related genes such as VEGF and HER-2. Considered with recent literature, this led me to postulate a feedback amplification cycle involving defective p53 and HER-2 mRNA expression. In conclusion, cancer cells with the R175H mutant could contribute to aggressive tumours. This conclusion, based on the in vitro data, is consistent with some clinical observations and animal model experiments. In the past few years it has become apparent that epigenetic changes also play a vitally important role in the cancer developmental process. Recent studies have reported the p53 protein can contribute in methylation which is one of the processes involved in epigenetic modification. This thesis employed a very new PCR-based AMP technique to examine the change of the global genome methylation pattern as a result of knocked-out p53 protein. The results showed defective p53 protein expression may associate with the global genome methylation pattern changes. However, it is important to note that antibiotic reagents, which were used for stable transfectant selection, could also contribute to the global genome methylation changes. In conclusion, this thesis has successfully developed two new methods. One allows the generation of a genetic mutant construct using PCR-based site directed mutagenesis while the other screens the tightly regulated ecdysone reporter system. In terms of effect of p53 in in vitro cell activity, this thesis has postulated that the R175H mutation is associated with much more aggressive metastatic cellular activity. Finally, this thesis also reported that loss of p53 expression could also result in changes in the global genome methylation pattern.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Also available online.

Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has revealed the stage and tissue specificity of NR activation for each of the fly NRs. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production are also a prominent feature that has been revealed using this approach. In addition, I went on to characterize the ligand regulated function of a single Drosophila nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75, reversing its ability to block the activity of its heterodimer partner Drosophila Hormone Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.

Nuclear receptors (NR’s) comprise a large, ancient, superfamily of eukaryotic transcription factors that govern a wide range of metabolic, homeostatic, and developmental pathways, and which have been implicated in disease states including cancer, inflammation, and diabetes. The ability of NRs to activate or repress gene transcription is modulated through direct binding of small lipophilic ligands which induce conformational changes in their cognate receptor. These changes are structural in nature and lead to the recruitment of coactivator or corepressor complexes, ultimately regulating the expression of target genes to whose response elements NRs are bound. In Drosophila 18 NRs have been identified which have representative members belonging to each of the six major NR subfamilies, and which show a high degree of homology to their vertebrate counterparts. This fact, in addition to the power and ease of genetic manipulation, make Drosophila an excellent model system in which to study NR function. When I began my project, 17 of the 18 NRs in Drosophila were ‘orphan’ receptors for which no cognate ligand had been identified. As a first step in an effort to identify potential ligands for these 17 receptors I first set out to determine how, where and when nuclear receptors are regulated by small chemical ligands and/or their protein partners. In order to do so I contributed to developing a ‘ligand sensor’ system to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live, developing animals. This system is based upon transgenic lines that express the ligand binding domain of each Drosophila NR fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, these fusion proteins show tissue- and stage-specific patterns of activation. Analysis using this system has revealed the stage and tissue specificity of NR activation for each of the fly NRs. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation that are indicative of sweeping changes in ligand and/or co-factor production are also a prominent feature that has been revealed using this approach. In addition, I went on to characterize the ligand regulated function of a single Drosophila nuclear receptor, Ecdysone inducible protein 75 (E75). Previous work from our lab has demonstrated that E75 binds to heme, and that its function as a transcriptional repressor is regulated in vitro by binding of the small diatomic gases nitric oxide (NO) and carbon monoxide (CO) to its heme moiety. In an effort to validate and to further understand the in vivo relevance of E75 regulation by NO I used gain and loss of function transgenes, as well as tissues manipulated in culture to show that NO acts directly on the Drosophila nuclear receptor E75, reversing its ability to block the activity of its heterodimer partner Drosophila Hormone Receptor 3 (DHR3). By specifically focusing on the Drosophila larval ring gland, the principal endocrine organ responsible for the production of the metamorphosis-inducing hormone, ecdysone, I have shown that failure to produce NO and to inactivate E75 results in failure to recognize the signals that normally trigger metamorphosis.

<p>A key question in developmental biology is how organisms attain a final size. Deviations in growth patterns can produce different/new phenotypes and these changes can play fundamental roles in ecology and evolution. The size of an organism and of its constitutive organs is determined by the growth rate and the duration of the growing period. In insects, peptide hormones such as insulin-like growth factors have been shown to be involved in determining the growth rates by coordinating metabolism, cell proliferation and cell size. In contrast, steroid hormones, such as ecdysone, are involved in determining life stage transitions, and thus the termination of the growing period. Although it is clear that insulin and steroid hormones are both involved in the regulation of growth, the ways in which these two regulators interact is yet to be determined. Furthermore, it is not clear how organs and body growth are coordinated during development to arrive to their correct proportions. In this study, using the tobacco hornworm Manduca sexta and its wings as a model system, I examine the developmental mechanisms involved in the regulation of organ growth and how developmental processes can drive morphological evolution. First, I examine how the hormonal events that take place during the termination of the body growth period affect wing disk growth. Second, by using gene expression assays and in vitro cultures, I examine the interaction between bombyxin, the Lepidopteran insulin-like growth factor, and ecdysone, the molting hormone, and their contributions to wing imaginal disk growth. Finally, by using three different size strains of M. sexta, I examine the developmental basis of the allometric relationship between the wings and the body. My results show that during the final instar of M. sexta larval development, wing imaginal disks are sensitive to the hormonal events that terminate the growth period. Furthermore, I show that the bombyxin requirement for wing disk growth is restricted to the early days of the final instar unlike the constitutive effects seen in other species. After the larva has passed a particular critical weight, bombyxin is not necessary for wing disk growth, although its absence does decrease the growth rate. In contrast, ecdysone is required for promoting the growth of wing imaginal disks primarily through its stimulation of cell proliferation. Finally, I show how selection on body size has unpredictable consequence for the response of wing size. These results demonstrate how specific allometries have a developmental basis in the cross-talk of the various signals that regulate growth itself. Therefore, direct selection on allometric relationships may not need to be strong in order to hold scaling relationships constant, at least over short evolutionary periods.</p> / Dissertation

Thesis (M.S.)--University of Missouri-Columbia, 2003. / RGL after WOC in title is in superscript. Typescript. Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--University of Missouri-Columbia, 2003. / RGL after WOC in title is in superscript. Typescript. Includes bibliographical references. Also available on the Internet.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on September 22, 2009). Thesis advisor: Dr. Bruce Barrett. Includes bibliographical references.

Doctor of Philosophy / Entomology / David C. Margolies / Yoonseong Park / Insect metamorphosis is driven by two major hormones, juvenile hormone (JH) and ecdysone (Ec). The presence of JH with an Ec peak in each stadium results in larval-larval molting whereas in the last larval instar a decline of JH to undetectable level combined with pulses of Ec leads to larval-pupal metamorphosis. Larval-pupal metamorphosis normally occurs after a certain number of larval instar and upon reaching a certain size (critical weight). However, in the flour beetle, Tribolium freemani, under crowded conditions larva continue larval-larval molting (LLC) without pupation for longer than 14 instars (6 months). Previous studies have implicated high JH titer as preventing the metamorphosis leading to supernumerary molts. My investigation of JH roles in LLC started by asking whether suppression of JH would rescue the LLC phenotype and allow pupal metamorphosis. Using RNA interference (RNAi), I found that under crowded conditions RNAi of T. freemani methyltransferase3 (TfMT3), which encodes a crucial enzyme for the final methylation step in the JH biosynthesis, or RNAi of T. freemani Krüppel homolog1 (TfKr-h1), the JH downstream gene, did not rescue the larvae but resulted in prepupal lethality. Surprisingly, under crowded conditions prepupal lethality was rescued by RNAi of both TfMT3 and TfKr-h1 administered together, although developmental arrest occurred at the pharate adult stage; this is also the phenotype of TfKr-h1 RNAi-treated larvae under isolated conditions. In investigations of the role of Ec titer in LLC, lethality of the larvae with RNAi of TfMT3 under crowded conditions was associated with the loss of the major ecdysteroid peak, while TfKr-h1 RNAi-treated larvae under crowded conditions showed a delayed, but normal, Ec peak occurring at prepupal arrest. The pattern of Ec peak in RNAi of both TfMT3 and TfKr-h1 together was similar to that with TfKr-h1 RNAi alone. I suggest that a hormonal imbalance, high JH and high Ec in the prepupal arrest of TfKr-h1 RNAi, was rescued by RNAi of both TfMT3 and TfKr-h1 for low JH and high Ec. These results demonstrate that the signaling pathways for LLC are through at least two independent pathways; JH biosynthesis and TfKr-h1-mediated JH response.

O consumo de produtos marinhos vem aumentando nos últimos anos. A aquicultura foi responsável por 47 % de todo alimento de origem marinha em 2010. A ecdise é importante para a aquicultura, pois os cultivos de Siri-mole (Callinectes sapidus) utilizam o animal no estágio pós-muda, para agregar valor ao produto. O evento da ecdise é a troca do exoesqueleto antigo para permitir o crescimento nos artrópodes, sendo coordenado por uma interação neuro-humoral entre dois órgãos, o complexo órgão X/glândula do seio (que produz o hormônio inibidor da muda HIM) e o órgão Y (que produz a ecdisona). As fitoecdisonas são metabólitos secundários dos vegetais e esses compostos são análogos aos hormônios da muda dos Artrópodes. Neste trabalho, a fitoecdisona de Pfaffia paniculata foi extraída, contendo no final 32% de fitoecdisona. A fitoecdisona e a ecdisona sintética a 30% foram testadas como indutores de muda, utilizando náuplios de Artemia salina como modelo de estudo (N=60/ concentração de 0,01 - 0,3 mg/mL). Os náuplios expostos a fitoecdisona foram fotografados e mensurados para comparação no incremento de tamanho após a primeira muda. Os resultados foram negativos para antecipação ou atraso da ecdise nos náuplios experimentados com fitoecdisona. Para os experimentos com ecdisona sintética, os resultados mostraram, por outro lado, um efeito deterrente ou de atraso da ecdise em relação aos animais controle. A morfometria mostra que apesar da fitoecdisona não estimular a ecdise, os animais expostos a este tiveram incremento de tamanho após a primeira ecdise em relação aos animais controle / The consumption of marine products is increasing in recent years. Aquaculture was responsible for 47% of all food from marine origin in 2010. Ecdysis is important for aquaculture because soft crab culture (Callinectes sapidus) uses the animals in posmolt stage to increase value to the final product. The event of ecdysis is the change of the old exoskeleton, so that arthropods increase in growth. This event is regulated by a neurohumoral interaction by two endocrine organs: X organ complex (producing the molt inhibiting hormone MIH) and Y organ (producing the ecdysone). Phytoecdysones are secondary metabolites of plants and are analogues to moult hormones of arthropods. In this work, phytoecdysone was extracted from Pfaffia paniculata showing a final content of 32% phytoecdysone. This phytoecdysone and a synthetic ecdysone at 30% were utilized as molting inducers using Artemia salina nauplii as a study model (N=60/ concentration range 0.01mg/mL - 0.3mg/mL). The nauplii exposed to phytoecdysone was photographed and measured to compare growth after first moulting. The results showed no stimulation of ecdyses in the nauplii exposed to phytoecdysone. The experiment with synthetic ecdysone, on the other hand, showed a deterrent effect and/or ecdyses delay. The morphometry showed that the nauplii exposed to phytoecdysone had increased growth increment after first ecdysis when compared with control nauplii