Preeclampsia is an increasingly prevalent disorder of placentation that has very limited options for treatment. The disease is characterized by aberrant invasion of placental trophoblasts into the decidualized maternal endometrium. In order to identify pathways of therapeutic interest during placentation, we are focusing on the pathway of the neuropeptide kisspeptin and its receptor KISS1R, both highly expressed in the human placenta. Early functional studies of the ligand-receptor system identified a role for kisspeptin in the inhibition of cancer metastasis. Parallels exist between cancer and placentation, suggesting the possibility of an inhibitory role for kisspeptin during pregnancy as well. Existing functional data supports kisspeptin's inhibitory influence on cellular invasion, but the mechanism remains unknown. Evidence for the localization of the KISS1R receptor in the current literature was established via a nonspecific antibody and requires further investigation. Current literature suggests involvement of the ERK (extracellular signal-regulated kinase) pathway as well. Our work aims to solidify the localization of kisspeptin and KISS1R, avoiding the use of KISS1R antibodies. Using immunohistochemistry for protein localization of kisspeptin and placental fractionation followed by quantitative PCR analysis for gene expression, we provide evidence of kisspeptin's restriction to the syncytiotrophoblast layer of the placenta, and KISS1R gene expression limited to the villous cytotrophoblast layer. This distribution of ligand and receptor suggests a paracrine mechanism for kisspeptin action, with syncytiotrophoblasts secreting kisspeptin to act on its receptor on the villous cytotrophoblast layer, and thus restricting cytotrophoblast invasion. We further attempt to support these data with the use of laser capture microdissection of placental tissue to isolate the different layers, followed by quantitative PCR. This technique introduced a particularly challenging aspect of working with the placenta: maintaining tissue morphology while also preserving RNA integrity. This thesis outlines our troubleshooting process for that technique and introduces alternatives for future work. We also employed Western blot analysis of ERK activation to establish the mechanism of kisspeptin's inhibitory effect on fractionated trophoblasts.

Cancer cell metastasis is induced by actin-dependent cell migration and is affected by cytoskeletal remodelling proteins. FMNL2 is one such protein which promotes colorectal cancer (CRC) cell metastasis and amoeboid style invasion of melanoma cells. FMNL2 mRNA is subject to alternative splicing and studies suggest that the resulting encoded proteins are likely to differ in their regulation, subcellular localization and activity. We identified four FMNL2 isoforms (ITM, YHY, PMR and TQS) expressed in non-invasive (SW480) and invasive (SW620) CRC cells, as well as in highly invasive A375 amoeboid melanoma cells. qPCR data suggests that an “invasive” isoform (TQS) may be preferentially expressed in highly invasive and amoeboid cell lines. Boyden chamber invasion assay results show that FMNL2 knockdown inhibits amoeboid style invasion in two melanoma cell lines and that TQS is the most efficient isoform at rescuing the invasive phenotype. This study provides a further understanding of FMNL2’s role in invasion and metastasis and identifies specific targets for the development of future antimetastatic therapies.

Homeostasis of trace metal ions is essential for a variety of cellular processes and virulence mechanisms, including resistance to oxidative stress, DNA replication, and regulation of cell adhesion/invasion. Understanding how pathogenic microorganisms overcome metal starvation and intoxication provides insight into how these mechanisms could be targeted by novel antimicrobial therapies. Streptococcus pneumoniae, or pneumococcus, is a Gram-positive, commensal of the human nasopharynx and upper respiratory tract. Though this organism is primarily an asymptomatic colonizer, it is also the causative agent of infections ranging in severity from reoccurring acute otitis media to life-threatening community acquired pneumonia or bacterial sepsis. This study aims to characterize aspects of pneumococcal physiology and infection that are responsive to changes in micronutrient zinc availability. Two zinc-binding lipoproteins of S. pneumoniae, AdcA and AdcAII, were characterized as playing redundant roles in zinc acquisition; however, this study shows that these proteins are not equally sensitive to zinc starvation and have additional functionality in adhesion and invasion. Mutant strains lacking AdcAII but not AdcA suffer decreased fitness when exposed to a zinc-chelator; and following chelation adcAII was upregulated 42old whereas adcA was only upregulated 4old. Zinc-deficient mutants lacking AdcA and AdcAII show increased invasion at levels reaching 200-300% compared to parental strains. Additionally, AdcAII-deficient strains show decreased ability to adhere to epithelial cells and colonize nasal tissues during murine challenge, suggesting a role for AdcAII or zinc homeostasis in biofilm formation. Analysis of biofilms grown in varying concentrations of metals revealed that increased zinc, specifically, resulted in the formation of larger, more architecturally complex biofilms. The increase in biofilm size was determined to be due to the formation of cell-to-cell aggregates. In addition to encountering high concentrations of metals, pathogens are competing for micronutrients from the host, and are thus adept to surviving metal starvation. A previously uncharacterized operon, SP1434-1438, was found to be sensitive to zinc-starvation and proteomics strongly suggest an importance for these genes in cellular metabolism. These results have identified roles for zinc homeostasis in cell adhesion, colonization, cell and bloodstream invasion, biofilm formation, and the maintenance of cellular metabolism.

No description available.

No description available.

The ability of cancer cells to adopt various invasive modes (the plasticity of cancer cell invasiveness) represents a significant obstacle in the treatment of cancer metastasis. Cancer invasiveness involves various modes of migration. Cells can move together (with the preserved intercellular junctions; collective invasiveness) or individually. Within individual invasiveness, we distinguish two principal invasive modes - mesenchymal and amoeboid. The mesenchymal mode of migration is characterized by an elongated shape, proteolytic degradation of the fibres of the extracellular matrix, and the formation of strong contacts with the extracellular matrix. The amoeboid mode of migration is not dependent on proteolytic activity, the cells are characterized by a round shape and increased contractility, which they use to squeeze themselves through the pores of the extracellular matrix. This thesis deals with the analysis of the plasticity of cancer cell invasiveness, specifically the transitions between individual amoeboid and mesenchymal migration modes, in the 3D environment of the collagen gel as a model of extracellular matrix. The work presents models of mesenchymal-to-amoeboid transition (MAT), which include BLM, HT1080 and MDA-MB-231 cell lines, in which MAT is induced by the expression of...

BACKGROUND:The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.RESULTS:We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.CONCLUSIONS:Our functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

This thesis presents a new analysis of British naval policy before and during the First World War which challenges both orthodox and revisionist interpretations of the period and highlights a highly significant yet much neglected facet of Admiralty planning. It argues that safeguarding the British Isles from invasion was one of the Admiralty’s prime concerns between 1888 and 1918 and that these defensive considerations played a hitherto unappreciated role in shaping British naval strategy. By exploiting source material generally overlooked by previous writers, it demonstrates that, contrary to popular historical belief, Britain’s naval leadership planned extensively to ensure the inviolability of the British coastline during this period. Before 1900, these plans were characterized by relying upon an extensive flotilla of small vessels, supported by a small number of old armoured warships, to secure the position in the Channel and North Sea, while the Navy’s most modern warships focused upon the main French Fleet in the Mediterranean. The Admiralty’s willingness to rely primarily upon flotilla craft for home defence ended after 1900, however, when German displaced France as Britain’s primary naval rival. Germany posed a very different threat to Britain than had previously been the case with France, since it possessed a merchant marine large enough to transport a significant military force without major disruption to the normal operation of its commerce and had her naval forces concentrated in northern waters. Despite the paucity of German planning for the invasion of the United Kingdom, the Admiralty became haunted by the possibility of a ‘surprise’ German invasion attempt, launched before the outset of war and escorted by a strong German Fleet. The Admiralty identified the danger of a surprise German raid or invasion by early 1907 and formed a series of highly secretive plans to deploy the Navy’s most modern armoured warships into the North Sea at the outset of war to meet this danger. These plans were updated constantly between 1910 and 1918 as perceptions of the German threat developed. The nature and extent of these plans has highly significant implications for our understanding of naval policy throughout the period, and for our appreciation of the role of sea power during the First World War.

Thesis (PhD)--Stellenbosch University, 2013. / Australian Acacia species are an important group of invaders and are known to form dense monospecific cultures in invaded habitats. Despite the ecological and economic importance of invasive acacias, little is known about their invasive biology both from an ecological and evolutionary perspective. Molecular genetic methods have increasingly become important in identifying source populations for invasive species and determining the population genetic structure of these populations. This thesis applied molecular tools to understand the invasion ecology of Acacia pycnantha and its rhizobial symbionts as a model system of Australian Acacia introductions. Specific objectives were to: reconstruct the molecular phylogeny of invasive and native populations of populations of Acacia pycnantha and identify the native provenance of A. pycnantha; identify microsatellite markers for Acacia pycnantha and other invasive Australian acacias based on transferring microsatellite markers developed for A. mangium, A. saligna, Paraserianthes lophantha and universal chloroplast microsatellites developed from tobacco; assess the introduction dynamics of Acacia pycnantha in South Africa and identify the source populations in the species’ native range ; and determine which nitrogen fixing symbionts nodulate A. pycnantha and determine whether A. pycnantha brought its symbionts along from its native range or acquired them in the invasive range. Nuclear and chloroplast DNA sequence data were used to reconstruct phylogeographic relationships between native and invasive A. pycnantha populations. The chloroplast phylogeny showed that Australian populations of A. pycnantha are geographically structured into two previously informally recognized lineages (representing wetland and dry land forms). Habitat fragmentation is probably the result of cycles of aridity and abundant rainfall during the Pleistocene0. The invasive population in Portugal was found to be the wetland form while South African populations were found to be predominantly wetland form although some dryland forms were identified. Thirty microsatellites out of the forty nine tested microsatellites successfully amplified across all species tested (A. implexa, A. longifolia, A. melanoxylon, A. pycnantha and A. podalyriifolia). High Transfer rates varied between 85% for microsatellites developed for A. mangium to 50% for those developed in A. saligna. Although transfer rates were high only twelve microsatellites (24%) out of the fifty tested were polymorphic while the chloroplast microsatellites showed no polymorphism. The low level of polymorphic loci calls for development of more microsatellites in this genus especially for species that have high commodity value. Nuclear microsatellites revealed three genetic groupings with substantial admixture in the native range (1. wetland Victoria and South Australia populations; 2. dryland Victoria and Flinders Range population; and 3. New South Wales). Admixture in the native range may have occurred as a result of reforestation exercises. Acacia pycnantha has been widely used in rea forestation projects in Australia because of its fast growth rate and ease of germination. Admixed populations were most - likely introduced to South Africa thus establishment of A. pycnantha may have been facilitated by already admixed propagules in the invasive range. Extensive admixture in the native range made it difficult to identify source populations of invasive A. pycnantha found in South Africa. The rhizobial symbionts of A. pycnantha were identified, showing that this species utilizes a wider suite of symbionts in its invasive range than its native range and there is support for both the co-introduction and host jumping hypotheses. This creates substantial opportunities for horizontal gene transfer between previously allopatric bacterial lineages, with as yet unknown consequences for plant and bacterial invasions.

No description available.