Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

Improving oncolytic adenoviral therapies for gastrointestinal cancers and tumor initiating cells

Although the treatment of most cancers has improved steadily, only few metastatic solid tumors can be cured. Despite responses, refractory clones often emerge and the disease becomes refractory to available treatment modalities. Furthermore, resistance factors are shared between different treatment regimens and therefore loss of response typically occurs rapidly, and there is a tendency for cross-resistance between agents. Therefore, new agents with novel mechanisms of action and lacking cross-resistance to currently available approaches are needed. Modified oncolytic adenoviruses, featuring cancer-celective cell lysis and spread, constitute an interesting drug platform towards the goals of tumor specificity and the implementation of potent multimodal treatment regimens. In this work, we demonstrate the applicability of capsid-modified, transcriptionally targeted oncolytic adenoviruses in targeting gastric, pancreatic and breast cancer. A variety of capsid modified adenoviruses were tested for transductional specificity first in gastric and pancreatic cancer cells and patient tissues and then in mice. Then, oncolytic viruses featuring the same capsid modifications were tested to confirm that successful transductional targeting translates into enhanced oncolytic potential. Capsid modified oncolytic viruses also prolonged the survival of tumor bearing orthotopic models of gastric and pancreatic cancer. Taken together, oncolytic adenoviral gene therapy could be a potent drug for gastric and pancreatic cancer, and its specificity, potency and safety can be modulated by means of capsid modification. We also characterized a new intraperitoneal virus delivery method in benefit for the persistence of gene delivery to intraperitoneal gastric and pancreatic cancer tumors. With a silica implant a steady and sustained virus release to the vicinity of the tumor improved the survival of the orthotopic tumor bearing mice. Furthermore, silica gel-based virus delivery lowered the toxicity mediating proimflammatory cytokine response and production of total and anti-adenovirus neutralizing antibodies (NAbs). On the other hand, silica shielded the virus against pre-excisting NAbs, resulting in a more favourable biodistribution in the preimmunized mice. The silica implant might therefore be of interest in treating intraperitoneally disseminated disease. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in cancer relapse and the formation of metastasis. Therefore, we examined if transcriptionally modified oncolytic adenoviruses are able to kill these cells. Complete eradication of CD44+CD24-/low putative breast cancer stem cells was seen in vitro, and significant antitumor activity was detected in CD44+CD24-/low –derived tumor bearing mice. Thus, genetically engineered oncolytic adenoviruses have potential in destroying cancer initiating cells, which may have relevance for the elimination of cancer stem cells in humans.

Invasion impacts on biodiversity: responses of ant communities to infestation by cat’s claw creeper, Macfadyena unguis-cati (Bignoniaceae) in subtropical Australia

Ants are the dominant soil faunal group in many if not most terrestrial ecosystems, and play a key role in soil structure and function. This study documents the impacts of invasion by the exotic cat’s claw creeper vine, Macfadyena unguis-cati (L.) Gentry (Bignoniaceae) on surface-situated (epigaeic) and subterranean (hypogaeic) ant communities in subtropical SE Queensland Australia where it is a major environmental weed of riparian areas, rainforest communities and remnant natural vegetation, smothering standing vegetation and causing canopy collapse. Soil ants were sampled in infested and uninfested areas at eight sites spanning both riparian and non-riparian habitats in subtropical SE Queensland. Patterns of ant species composition and functional grouping in response to patch invasion status, landscape type and habitat stratum were investigated using ANOVA and non-metric multidimensional scaling ordination. The epigaeic and subterranean strata supported markedly different ant assemblages, and ant communities also differed between riparian and non-riparian habitats. However, M. unguis-cati invasion had a surprisingly limited impact. There was a tendency for ant abundance and species richness to be lower in infested patches, and overall species composition was different between infested and uninfested patches, but these differences were relatively small, and did not occur consistently across sites. There were changes in functional group composition that conformed to known functional group responses to environmental change, but these were similarly limited and inconsistent across sites. Our study has shown that ant communities are surprisingly resilient to invasion by M. unguis-cati, and serves as a warning against making assumptions about invasion impacts based on visual appearances.

Regulation of tumor suppressor protein p53 in cellular stress and tumorigenesis

Functional loss of tumor suppressor protein p53 is a common feature in diverse human cancers. The ability of this protein to sense cellular damage and halt the progression of the cell cycle or direct the cells to apoptosis is essential in preventing tumorigenesis. Tumors having wild-type p53 also respond better to current chemotherapies. The loss of p53 function may arise from TP53 mutations or dysregulation of factors controlling its levels and activity. Probably the most significant inhibitor of p53 function is Mdm2, a protein mediating its degradation and inactivation. Clearly, the maintenance of a strictly controlled p53-Mdm2 route is of great importance in preventing neoplastic transformation. Moreover, impairing Mdm2 function could be a nongenotoxic way to increase p53 levels and activity. Understanding the precise molecular mechanisms behind p53-Mdm2 relationship is thus essential from a therapeutic point of view. The aim of this thesis study was to discover factors affecting the negative regulation of p53 by Mdm2, causing activation of p53 in stressed cells. As a model of cellular damage, we used UVC radiation, inducing a complex cellular stress pathway. Exposure to UVC, as well as to several chemotherapeutic drugs, causes robust transcriptional stress in the cells and leads to activation of p53. By using this model of cellular stress, our goal was to understand how and by which proteins p53 is regulated. Furthermore, we wanted to address whether these pathways affecting p53 function could be altered in human cancers. In the study, two different p53 pathway proteins, nucleophosmin (NPM) and promyelocytic leukemia protein (PML), were found to participate in the p53 stress response following UV stress. Subcellular translocations of these proteins were discovered rapidly after exposure to UV. The alterations in the cellular localizations were connected to transient interactions with p53 and Mdm2, implicating their significance in the regulation of p53 stress response. NPM was shown to control Mdm2-p53 interface and mediate p53 stabilization by blocking the ability of Mdm2 to promote p53 degradation. Furthermore, NPM mediated p53 stabilization upon viral insult. We further detected a connection between cellular pathways of NPM and PML, as PML was found to associate with NPM in UV-radiated cells. The observed temporal UV-induced interactions strongly imply existence of a multiprotein complex participating in the p53 response. In addition, PML controlled the UV response of NPM, its localization and complex formation with chromatin associated factors. The relevance of the UV-promoted interactions was demonstrated in studies in a human leukemia cell line, being under abnormal transcriptional repression due to expression of oncogenic PML-RARa fusion protein. Reversing the leukemic phenotype with a therapeutically significant drug was associated with similar complex formation between p53 and its partners as following UV. In conclusion, this thesis study identifies novel p53 pathway interactions associated with the recovery from UV-promoted as well as oncogenic transcriptional repression.

Comparative studies on the invasion of cattle ticks (Rhipicephalus (Boophilus) microplus) and sheep blowflies (Lucilia cuprina) by Metarhizium anisopliae (Sorokin).

Microscopic investigations over time were carried out to study and compare the pathogenesis of invasion of ticks and blowflies by Metarhizium anisopliae. The scanning electron microscope and stereo light microscope were used to observe and record processes on the arthropods' surfaces and the compound light microscope was used to observe and record processes within the body cavities. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle, particularly the outer endocuticle. Although large numbers of conidia adhered to the surface of blowflies, no conidia were seen to germinate on external surfaces. A single germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggests that fungal invasion of the blowfly is primarily through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on tick cuticles, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16.

Cat's claw creeper vine, Macfadyena unguis-cati (Bignoniaceae), invasion impacts: comparative leaf nutrient content and effects on soil physicochemical properties

Macfadyena unguis-cati (L.) Gentry (Bignoniaceae) is a major environmental weed in coastal Queensland, Australia. There is a lack of quantitative data on its leaf chemistry and its impact on soil properties. Soils from infested vs uninfested areas, and leaves of M. unguis-cati and three co-occurring vine species (one exotic, two native) were collected at six sites (riparian and non-riparian) in south-eastern Queensland. Effects of invasion status, species, site and habitat type were examined using univariate and multivariate analyses. Habitat type had a greater effect on soil nutrients than on leaf chemistry. Invasion effect of M. unguis-cati on soil chemistry was more pronounced in non-riparian than in riparian habitat. Significantly higher values were obtained in M. unguis-cati infested (vs. uninfested) soils for ~50% of traits. Leaf ion concentrations differed significantly between exotic and native vines. Observed higher leaf-nutrient load (especially nitrogen, phosphorus and potassium) in exotic plants aligns with the preference of invasive plant species for disturbed habitats with higher nutrient input. Higher load of trace elements (aluminium, boron, cadmium and iron) in its leaves suggests that cycling of heavy-metal ions, many of which are potentially toxic at excess level, could be accelerated in soils of M. unguis-cati-invaded landscape. Although inferences from the present study are based on correlative data, the consistency of the patterns across many sites suggests that M. unguis-cati may improve soil fertility and influence nutrient cycling, perhaps through legacy effects of its own litter input.

Circulating tumor cells: Isolation, expansion, characterization and translation to clinic

Circulating tumor cells (CTCs) are the seeds for cancer metastases development, which is responsible for >90% of cancer-related deaths. Accurate quantification of CTCs in human fluids could be an invaluable tool for understanding cancer prognosis, delivering personalized medicine to prevent metastasis and finding cancer therapy effectiveness. Although CTCs were first discovered more than 200 years ago, until now it has been a nightmare for clinical practitioners to capture and diagnose CTCs in clinical settings. Our society needs rapid, sensitive, and reliable assays to identify the CTCs from blood in order to help save millions of lives. Due to the phenotypic EMT transition, CTCs are undetected for more than one-third of metastatic breast cancer patients in clinics. To tackle the above challenges, the first volume in “Circulating Tumor Cells (CTCs): Detection Methods, Health Impact and Emerging Clinical Challenges discusses recent developments of different technologies, which have the capability to target and elucidate the phenotype heterogenity of CTCS. It contains seven chapters written by world leaders in this area, covering basic science to possible device design which can have beneficial applications in society. This book is unique in its design and content, providing an in-depth analysis to elucidate biological mechanisms of cancer disease progression, CTC detection challenges, possible health effects and the latest research on evolving technologies which have the capability to tackle the above challenges. It describes the broad range of coverage on understanding CTCs biology from early predictors of the metastatic spread of cancer, new promising technology for CTC separation and detection in clinical environment and monitoring therapy efficacy via finding the heterogeneous nature of CTCs. (Imprint: Nova Biomedical)

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-d-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.