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Aberrant cell proliferation is a fundamental feature of cancer. It is thus not unexpected that many studies have been devoted to the exploration of tumour cell proliferation as a potential indicator of outcome. Indeed, in most malignancies, high expression of proliferation markers and more accurately, increased expression of proliferation-related genes have been strongly associated with poor outcome. In colorectal cancer (CRC), however, discordant results have been reported and the prognostic significance of cell proliferation has not been demonstrated in this type of cancer. As these results were mostly based on the subjective assessment of a single proliferation marker, this work set out to evaluate the association between the proliferative activity of CRCs and their malignant potential using an objective microarray-based multi-gene proliferation signature. In the first step, a gene proliferation signature (GPS) was derived from analysis of a CRC cell line model. Ten CRC cell lines were harvested under semi- and fully-confluent conditions to obtain RNA from two stages that differed in their proliferative activity. Gene expression profiles of the two growth stages were analyzed on oligonucleotide arrays and a GPS was identified by gene ontology analysis of differentially expressed genes. In the second step, the performance of the signature to classify patients into prognostic groups was examined using two independent cohorts of primary CRC tumours (cohort A: 73 tumours in stages I-IV, cohort B: 55 tumours in stages I-II). Further, the signature was applied to a population of liver metastases to establish its association with the malignant potential of CRC. Finally, the capacity of the signature to detect clinically distinct CRC populations was compared with that of the proliferation marker Ki-67 in a classic immunohistochemical approach. The GPS consisted of 38 mitotic cell cycle genes, which were over-expressed in actively cycling cells relative to growth-inhibited cells in vitro. Intriguingly, a reduced GPS expression was associated with (i) the presence of lymph node or distant metastasis in cohort A, (ii) an increased risk of recurrence, and (iii) shorter overall and recurrence-free survival in both cohorts of primary CRCs (p<0.05). While the association between the GPS and clinical outcome was not independent of the disease stage in cohort A, reduced GPS expression was an independent predictor of outcome in cohort B. Importantly, adjuvant therapy had no impact on this association. Furthermore, GPS expression was reduced in CRC liver metastases, confirming that decreased proliferation is an indicator of the malignant potential in CRC. While reduced proliferation in liver metastases was also observed by Ki-67 immunostaining, the classic proliferation marker was not able to stratify primary CRCs into different prognostic groups. To the best of our knowledge, this study is the first to report an association between the reduced expression of a multi-gene proliferation signature and poor outcome in cancer. This finding contradicts the long-held belief that increased proliferation is an indicator of tumour malignancy. In contrast to many other cancers, reduced proliferation appears to be a significant component of a biological signature associated with malignant potential of CRC tumours. Investigating the reasons why CRC differs from other cancer types may provide insights into important underlying biological mechanisms. If this association can be verified in larger cohorts, the GPS may have important clinical implication for identification of high-risk early stage CRC patients.

武器在第三世界的擴散基本上是東西德冷戰下的產物，但並未隨冷戰結束 而告落幕，反而構成當今國際情勢中一股潛在的不穩定因子。一九九一年 海灣戰爭更凸顯了此一問題在國際安全事務中的重要性。 供應國籍接 受國基於各自利益及需求輸出及引進武器，加上第三世界若干國家本國武 器工業的發展，都是造成第三世界武器擴散愈演愈烈的原因。而除了傳統 武器之外，核生化武器及彈道飛彈等大規模毀滅性武器的逐漸擴散，更在 海灣戰爭之後成為國際間關切的焦點。因此在戰後要求加強對第三世界武 器擴散管制的呼籲及實際措施愈來愈多，惟其真正效果如何仍有待評估。 另外，第三世界武器擴散之勢在未來數年將更更為嚴重或趨於收歛，目前 亦難論斷。然而，如何避免使第三世界武器擴散成為影響國際局勢的不安 因素，並在同時得以兼顧第三世界國家的安全，則將是後冷戰時期世界各 國致力的目標。 本文架構係分為六章。第一章導論。第二章為第三世 界武器擴散的 現狀、特色及因素之分析。第三章為第三世界武器擴散與 區域情勢的關係，並以海灣戰爭為例加以說明。第四章為第三世界武器擴 散之管制，以供應國之輸出管制，接受國的區域性管制，及國際間的全球 性管制詳 述之。地五章係針對第三世界武器擴散的晚進發展進行分析， 指出近來助長及抑制第三世界武器擴散的成因及現象，並探討武器擴散與 第三世界安全及發展的相互關聯。地六章則為結論及簡評。文中並引用美 國武器管制暨裁軍總署、瑞典斯德哥爾摩國際和平研究所、及美國國會圖 書館國會研究處對全球及第三世界武器轉移的統計資料，來加強說明。

Includes summary / Includes bibliography / [586] leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Anatomy and Histology, 1988

Limb-Bud and Heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of functional TCF/LEF binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a non-canonical pathway involving Lmx1b. Furthermore, we show that Lbh is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice and in aggressive basal-subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human breast cancer appears to be Wnt/beta-catenin dependent as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. RNAi mediated knockdown of LBH in basal breast cancer cell lines resulted in loss of CD44high/CD24low tumor cells, luminal differentiation, reduced cell growth, reduced colony forming ability, and increased apoptosis, suggesting a novel pro-survival and stem cell maintenance function of LBH in breast cancer. Reciprocal overexpression studies in the basal breast carcinoma line BT549 resulted in increased tumorigenicity in vitro, suggesting that LBH overexpression is indeed oncogenic. Finally, we further characterized LBH protein expression patterns and post-transcriptional regulation. Collectively, this thesis demonstrates that LBH is a direct Wnt target gene in both development and basal breast cancer that promotes the undifferentiated phenotype and survival of basal breast tumor cells.

Organochlorine pesticides (OCs) are environmental toxicants with important links to human health. They have been found to activate signalling pathways within cells and thereby affect cell survival and proliferation. Receptor Activator of Nuclear Factor kB (RANK) ligand and its receptor RANK are crucial for mammary epithelial proliferation in pregnancy and have recently been linked to hormone induced breast cancers. The objectives of this study were to confirm the proliferative effects of an OC (endosulfan) on human MCF-7 breast cancer cells, identify activated intracellular signaling pathways and investigate changes in RANK and RANKL gene expression. This study showed that endosulfan has a stimulatory effect on human MCF-7 cell proliferation, which may be invoked through activated intracellular signaling pathways (JNK, ERK1/2 and p38). In addition, there was a down regulation of RANK and upregulation of RANKL gene expression suggesting endosulfan is capable of modulating both cellular behavior and gene expression. / UOIT

Pebble-fueled high temperature gas-cooled reactor (HTGR) technology was first developed by the Federal Republic of Germany in the 1950s. More recently, the design has been embraced by the People's Republic of China and the Republic of South Africa. Unlike light water reactors that generate heat from fuel assemblies comprised of fuel rods, pebble-fueled HTGRs utilize thousands of 60-mm diameter fuel spheres (pebbles) comprised of thousands of TRISO particles. As this reactor type is deployed across the world, adequate methods for safeguarding the reactor must be developed. Current safeguards methods for the pebble-fueled HTGR focus on extensive, redundant containment and surveillance (C/S) measures or a combination of item-type and bulk-type material safeguards measures to deter and detect the diversion of fuel pebbles. The disadvantages to these approaches are the loss of continuity of knowledge (CoK) when C/S systems fail, or are compromised, and the introduction of material unaccounted for (MUF). Either vulnerability can be exploited by an adversary to divert fuel pebbles from the reactor system. It was determined that a solution to maintaining CoK is to develop a system to identify each fuel pebble that is inserted and removed from the reactor. Work was performed to develop and evaluate the use of inert microspheres placed in each fuel pebble, whose random placement could be used as a fingerprint to identify the fuel pebble. Ultrasound imaging of 1 mm zirconium oxide microspheres was identified as a possible imaging system and microsphere material for the new safeguards system concept. The system concept was evaluated, and it was found that a minimum of three microspheres are necessary to create enough random fingerprints for 10,000,000 pebbles. It was also found that, over the lifetime of the reactor, less than 0.01% of fuel pebbles can be expected to have randomly the same microsphere fingerprint. From an MCNP 5.1 model, it was determined that less than fifty microspheres in each pebble will have no impact on the reactivity or temperature coefficient of reactivity of the reactor system. Finally, using an ultrasound system it was found that ultrasound waves can penetrate thin layers of graphite to image the microsphere fingerprint.

Las fosfolipasas D (PLDs) son enzimas regulables que catalizan la hidrólisis de la fosfatidilcolina, un fosfofolípido mayoritario de las membranas de células eucariotas, para dar lugar a fosfatidato (PtdOH), molécula capaz de funcionar como segundo mensajero y que se ha implicado en procesos celulares como el tráfico vesicular, la reorganización del citoesqueleto y la proliferación celular. Los alcoholes primarios, como el 1-butanol, pueden derivar la formación de PtdOH por la PLD hacia la formación de los correspondientes fosfatidilalcoholes (moléculas inactivas). Es por ello que los alcoholes primarios son ampliamente usados en el estudio de las funciones celulares en las que se hayan implicados enzimas PLD. Esta tesis se centra en el estudio de procesos celulares en los que intervienen enzimas PLD, y en particular, el estudio de su implicación en el ondulamiento de la membrana plasmática (membrane ruffling) y en la proliferación celular.El ondulamiento de la membrana plasmática es un proceso dinámico que requiere del reordenamiento del citesqueleto de actina. Es fácilmente observable en células especializadas en secreción e indicativo de que la célula está exocitando activamente. Tanto mastocitos como línias celulares relacionadas responden a la estimulación por antígenos con una reorganización profunda de su citoesqueleto cortical y con la exocitosis de sus gránulos de secreción. Nosotros hemos observado que la inhibición de la formación de PtdOH por la PLD mediante la incubación con 1-butanol se traduce en la inhibición de la ondulación de la membrana plasmática en células RBL-2H3 (una línea celular mastocitaria). Dicha inhibición por 1-butanol es totalmente reversible: si se elimina el 1-butanol del medio las células recuperan la capacidad de ondular su membrana plasmática en respuesta a la estimulación por antígeno. Medidas de la actividad PLD en respuesta a antígeno indican que la ondulación de la membrana requiere de la producción continua de PtdOH por la PLD. Tanto PLD1 como PLD2 son expresadas por células RBL. En estudios de transfección con PLD1 y PLD2 marcadas con GFP (green fluorescent protein) se observa la colocalización de PLD2 en las ondulaciones de membrana de células RBL estimuladas con antígeno. Por contra, GFP-PLD1 muestra una distribución intracelular localizada en vesículas citoplasmáticas. Por ello asumimos que la isoforma PLD2 es esencial en los cambios a nivel de citoesquelto que se observan en respuesta a la estimulación por antígeno.En el estudio de la implicación de la PLD en la proliferación celular se emplearon astrocitos en cultivo como modelo celular. Se determinó la activación de la PLD en respuesta a una batería de estímulos incluyendo el promotor de tumores PMA, factores de crecimiento y agonistas de receptores metabotrópicos. Asimismo, se deteminó la estimulación de la incorporación de timidina al DNA como reflejo de la actividad mitogénica en respuesta a los mismos estímulos. Observamos una clara ausencia de correlación entre la activación de la PLD y la magitud de la respuesta mitogénica. De este dato concluimos que la formación de PtdOH por la PLD no parece ser un mecanismo comunmente implicado en la transducción de señales proliferativas. Por otra parte, la inhibición de la formación de PtdOH (por incubación en presencia de alcoholes primarios) no se traduce en la reducción de la actividad proliferativa en respuesta a diversos mitógenos, apoyando la conclusión anterior.Tras concluir que la activación de la PLD no era un requerimiento necesario en la transducción de señales mitogénicas, nos propusimos explorar mecanismos moleculares implicados en la proliferación de astrocitos. Observamos que concentraciones de litio en el rango milimolar inducen un bloqueo en el ciclo celular a nivel de G2/M en astrocitos proliferantes. Asimismo, el litio retrasa la progresión a través del ciclo celular en astrocitos deprivados de suero y estimulados por el mitógeno endotelina-1. La adición exógena de myo-inositol no es capaz de revertir los efectos del litio sobre el ciclo celular y estos no se reproducen por la inhibición selectiva de la glucógeno sintasa quinasa-3 (GSK3). Estos resultados sugieren que los efectos del litio no son mediados ni por inhibición de inositol monofosfatasas ni por inhibición de GSK3, dos de sus dianas farmacológicas reconocidas. Por contra, el pretratamiento con litio de astrocitos proliferantes reduce la activación de ERK1/2 (extracellular regulated kinase 1/2) y de su quinasa reguladora MEK 1/2. El litio priviene también la activación de dichas quinasas en respuesta a endotelina-1 en astrocitos deprivados de suero. En células granulares de cerebelo concentraciones milimolares de litio promueven la activación de MEK y ERK. Los efectos opuestos del litio en neuronas y astrocitos nos llevan a proponer su uso terapeútico en situaciones de daño traumático en el sistema nervioso central. / Phospholipase Ds (PLDs) are regulated enzymes that catalyse the hydrolysis of phosphatydylcholine, a major lipid contituent of eukaryotic cell membranes, and generate phosphatidic acid (PtdOH), a putative second messenger molecule implicated in the regulation of vesicular trafficking, cytoskeletal reorganisation and cell proliferation. Primary alcohols such as butan-1-ol can divert the production of PtdOH generated by PLD to the correspondent phosphatydylalcohol (inactive molecule) and therefore constitute useful tools in the study of cellular functions of PLD enzymes. The present thesis aims to explore the role of PLD in different cellular events, in particular in membrane ruffling and in cell proliferation.Membrane ruffling is a dynamic phenomenon taking place at the plasma membrane that requires the rearrangement of the actin cytoskeleton. It is very prominent in cells specialised in secretion and is indicative of ongoing exocytosis. Mast cells and related cell lines, when stimulated with antigen, show a dramatic alteration of their cytoskeleton required for the release of secretory granules by exocytosis. We have observed that inhibition of PLD-derived PtdOH fomation by butan-1-ol results in the halt of antigen-stimulated membrane ruffling in a mast cell line (RBL-2H3 cells). Inhibition by butan-1-ol was completely reversible because its removal restored membrane ruffling. Measurements of PLD activation by antigen indicate a requirement for continual PtdOH formation during ruffling. PLD1 and PLD2 are both expressed in RBL cells and green fluorescent-tagged (GFP) proteins were used to identify PLD2 localizing to membrane ruffles in antigen-stimulated RBL cells. In contrast, GFP-PLD1 localised in intracellular vesicles and remained in this location after stimulation with antigen. We therefore assume that PLD2 is essential for actin cytoskeletal rearrangements triggered by antigen. The role of PLD in cell proliferation was studied in cultured astrocytes. We monitored PLD activation elicited by a set of different stimuli including the tumour promoter PMA, growth factors and G protein-coupled receptor agonists. As an index of the mitogenic response, thymidine incorporation into DNA in response to these stimuli was also monitored. We observed a lack of correlation between PLD activation and the extent of the mitogenic response, suggesting that PtdOH formation was not a common mechanism involved in mitogenic signal transduction. In agreement with this conclusion, primary alcohol inhibition of PtdOH formation by PLD does not result in the inhibition of the mitogen-stimulated incorporation of thymidine into DNA. Having observed that PLD may not be part of the mitogenic signal transduction machinery, we further explored molecular mechanims involved in astrocyte proliferation. We observed that millimolar lithium concentrations induced a G2/M cell cycle arrest in proliferating astrocytes and also delays cell cycle reentry by the mitogen endothelin-1. Myo-inositol supplementation of the culture medium could not overcome lithium-induced cell cycle arrest and selective inhibition of glycogen synthase kinase-3 (GSK3) by compound SB216763 could not reproduce lithium effects on cell cycle distribution. This results indicate that lithium effects are unlikely mediated by inhibition of inositol monophosphate phosphatases nor GSK3 inhibition, two pharmacological targets mediating lithium actions in other settings. Interestingly, lithium pretreatment of proliferating astrocytes results in reduced phosphorylaton of extracellular regulated kinase 1/2 (ERK1/2) and the upstream kinase MEK1/2. Lithium also prevents endothelin-1-stimulated phosphorylation of ERK1/2 and MEK1/2 in serum-deprived astrocytes. In cerebellar granule cells millimolar lithium enhanced ERK and MEK phosphorylation. We propose that he opposing effects of lithium in neurons and astrocytes make lithium treatment a promising strategy to favour neuronal repair and reduce reactive gliosis after traumatic injury.

Optic nerve section (ONS) is an experimental model for damage of the optic nerve associated with diseases such as glaucoma and optic neuritis. Damage to the optic nerve causes loss of retinal ganglion cells that are attached, once the cells are damaged, they are not typically replaced. Recently, Fischer and Reh (2003) found that Müller glia have the potential to adopt phenotypes and functional capabilities similar to those of retinal progenitors, a potential source of retinal regeneration. In the chick, when the specific retinal cells are targeted for damage by chemotoxins, there is widespread apoptosis but also mitotically active cells that label with retinal progenitor markers. Fischer and Reh (2002) also discovered that the combination of growth factors FGF2 and insulin is capable of stimulating the regenerative response of the Müller glia to retinal progenitor cells in chick eyes. This study was conducted to analyse damage to the ganglion cells by optic nerve section in chicks to determine the effect of age on the cell death timeline, the proliferative qualities of the retina and to see if injections of growth factors had the ability to increase the proliferation. Histological methods were used to analyse cellular changes and ultrasound to monitor eye growth. Apoptotic activity preceded retinal thinning and ganglion cell loss, indicating that ONS-related cell death is mediated at least in part by apoptotic mechanisms and age did not affect the time course, although, age did affect the eye growth changes, which may be attributed to the plasticity of the younger eyes. ONS damage elicited proliferative activity in the retina as did growth factor injections alone. The combination of ONS damage and growth factor injections increased the proliferative activity and the overall total number of cells in the ganglion cell layer. These findings can potentially lead to the development of therapeutic strategies for the preservation or restoration of retinal cells in diseased eyes.

Little is known about how the size of a tissue is established during development and maintained subsequently. Proliferation-inhibiting signals secreted by cells within a tissue that act specifically on cells within that tissue can provide negative feedback on cell number, thus regulating tissue size. A better understanding of tissue-specific inhibitors of proliferation could be useful for designing therapies for cancer and other diseases. However, few signals of this sort have been identified, and little is known about how these signals function. Two examples of such signals are the proteins AprA and CfaD, which are secreted by the social amoeba Dictyostelium discoideum and inhibit cell proliferation in a concentration-dependent manner. Cells lacking either AprA or CfaD proliferate rapidly, and adding recombinant AprA or CfaD to cells reduces proliferation. However, little is known about the signal transduction pathways downstream of AprA and CfaD. I identified three proteins that are required for the normal function of AprA and CfaD: the kinase QkgA, the putative transcription factor BzpN, and the putative kinase PakD. Cells lacking any one of these proteins proliferate rapidly, and adding AprA or CfaD to cells lacking these proteins does not cause reduced proliferation, indicating that these proteins are involved in AprA/CfaD signal transduction. I also found that, in addition to its proliferation-inhibiting activity, AprA also functions as an autocrine chemorepellant. Colonies of cells lacking AprA expand less rapidly than wild-type colonies, despite the fact that individual cells lacking AprA show a random motility like that of wild-type cells. Further, two independent assays demonstrate that cells show a biased movement away from a source of AprA. The chemorepellant activity of AprA requires CfaD, QkgA, and PakD, but not BzpN, indicating that AprA affects proliferation and chemorepulsion through distinct but overlapping pathways. These results suggest that AprA functions as a readout of local cell density, to which cells respond by slowing proliferation and chemotaxing to regions of lower cell density, where nutrients are more likely to be present. The study of human AprA, CfaD, QkgA, BzpN, and PakD orthologs may serve to guide therapeutic approaches that modulate chemorepulsive or antiproliferative processes.