Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is extremely prevalent in people from Laos and Thailand whose staple diet is uncooked fish which harbour the liver fluke, Opisthorchis viverrini. There is no stronger link between a parasite and cancer than that between O. viverrini and CCA. Indeed WHO data suggests that one in six infected people contract liver cancer derived from the fluke. In vitro and in vivo studies have indicated that the fluke’s excretory/secretory (ES) proteins are mitogenic and likely make significant contributions to the initiation of CCA. To identify these carcinogenic components I undertook two distinct yet related approaches - (1) traditional protein purification methods to separate ES products, specifically targeting mitogenic proteins, and, (2) bioinformatic screening of 5,000 expressed sequences tags (ESTs) and ES proteins characterized by shotgun proteomic approaches, searching for homologues of molecules that are associated with human cancers. The protein purification approach utilized a cell proliferation assay that I developed for measuring cell replication rates in NIH-3T3 fibroblast and human CCA (KKU-100) cell lines stimulated with ES products. ES products were separated by a combination of ion exchange, hydrophobic interaction, size exclusion and a final ion exchange polishing chromatography steps. ES products and chromatographically separated ES proteins were added to cultured cells to observe mitogenic activity. A four-step purification process resulted in the isolation of 23 and 31 kDa proteins that stimulated cell proliferation at just picomolar quantities. These proteins account for a very small proportion of the total protein biomass (6 ppm and 39 ppm respectively) secreted by the parasite. Their identities are currently being explored using alternate proteomic approaches. Some growth factors bind to heparin, so an alternative purification process was developed using a heparin affinity column to purify ES mitogens. In combination with ion exchange chromatography a 20 kDa heparin-binding protein was identified using tandem mass spectrometry as a member of the sperm-coating protein 65 (SCP)-like extracellular proteins, also called SCP/Tpx-1/Ag5/PR-1/Sc766 (SCP/TAPS; Pfam accession number no. PF00188). The O. viverrini heparin-binding SCP/TAPs protein shared similarity with secreted proteins from other parasitic helminths including the hookworm activation-associated protein family, some of which are known to bind to host cells. In silico screening of the O. viverrini ESTs and ES peptides generated by mass spectrometry for proteins associated with cell proliferation and cancer revealed numerous secreted proteins of interest. One of these proteins shared identity with granulin, a vertebrate growth factor. The cDNA corresponding to this protein was termed Ov-grn-1. The predicted molecular characteristics of Ov-GRN-1 (isoelectric point and molecular weight) corresponded with the biochemical properties of the semi-purified mitogen that was chromatographically purified from ES products. Recombinant Ov-GRN-1 was expressed in E. coli in inclusion bodies and the purified denatured protein was refolded to produce a soluble protein. Refolded Ov-GRN-1 stimulated proliferation of NIH-3T3 fibroblasts at nanomolar concentrations and induced shape changes in affected cells. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of fibroblasts and the KKU-100 CCA cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumourigenic environment that may ultimately manifest as CCA.

Hohenheim, Univ., Diss., 2005. / Aus: 1) Yeast 21:1375-89, 2004 2) .Agric. Food Chem. 53:8230-38, 2005. 3) Food Additives and Contaminants 22:397-405, 2005.

No description available.

CCCTC-binding factor (CTCF) binds DNA, thereby helping to partition the mammalian genome into discrete structural and regulatory domains. In doing so, it insulates chromatin and fine-tunes gene activation, repression, and silencing. Complete removal of CTCF from mammalian cells causes catastrophic genomic dysregulation, most likely due to widespread collapse of 3D chromatin looping within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction in CTCF expression are viable but have an increased incidence of spontaneous multi-lineage malignancies. In addition, CTCF is mutated in many human cancers and is thus implicated as a tumour suppressor gene. This study aimed to interrogate the genome-wide consequences of a reduced genomic concentration of Ctcf and its implications for carcinogenesis. In a genetically engineered mouse model, Ctcf hemizygous cells showed modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these were enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, several hundred genes concentrated in cancer-related pathways were dysregulated due to changes in transcriptional regulation. Global chromatin structure was preserved but some loop interactions were destabilised, often around differentially expressed genes and their enhancers. Importantly, these transcriptional alterations were also seen in human cancers. These findings were then examined in a hepatocyte-specific mouse model of Ctcf hemizygosity with diethylnitrosamine-induced liver tumours. Ctcf hemizygous mice had a subtle liver-specific phenotype, although the overall tumour burden in Ctcf hemizygous and wild-type mice was the same. Using whole genome sequencing, the highly reproducible mutational signature caused by DEN exposure was characterised, revealing that Braf(V637E), orthologous to BRAF(V600E) in humans, was the predominant oncogenic driver in these liver tumours. Taken together, while Ctcf loss is partially physiologically compensated, chronic CTCF depletion dysregulates gene expression by subtly altering transcriptional regulation. This study also represents the first comprehensive genome-wide and histopathological characterisation of this commonly used liver cancer model.

In this thesis, it is shown how aflatoxins can be determined in biological material. The thesis is a summary of five papers. Aflatoxins are carcinogenic mycotoxins produced by Aspergillus moulds. Methods were developed for the determination of aflatoxins in samples of airborne dust and human urine collected at feed factories. For the dust samples from such agricultural products as copra, cotton seed and maize, methods were developed for the determination of aflatoxins B1, B2, G1 and G2. For urine samples, methods were developed for analysing the four aflatoxins above that naturally occur in dust, and the metabolites aflatoxins M1 and Q1. Sample preparation of dust samples included solvent extraction, filtration and immunoaffinity column extraction. Urine samples were cleaned up using immunoaffinity column extraction or solid-phase extraction using ethyl bonded-phase columns. All extractions with these columns were automated by means of a laboratory robot. Reversed-phase liquid chromatography was used to separate the aflatoxins in the cleaned-up extracts. Detection was performed by fluorescence after post-column derivatization by addition of bromine. Parameters for the derivatization were studied using factorial designs. To confirm the identity of aflatoxins in naturally contaminated airborne dust samples and spiked urine, liquid chromatography was combined with electrospray mass spectrometry. The detection limits of the aflatoxins in dust samples were in the range 1.8-3.1 ng/g in 10-mg dust samples using fluorescence detection. Aflatoxins were determined in spiked urine down to the 6.8-18 pg/ml level. In naturally contaminated dust of copra and cotton seed, aflatoxins were detected with a content of 9-50 pg/mg of aflatoxin Bi. No aflatoxins could be detected in any urine sample obtained from feed factory workers that were less than 6.8 pg/ml of aflatoxins B1, B2, G1 and G2 and less than 18 pg/ml of aflatoxins M1 and Q1. / <p>Diss. (sammanfattning) Umeå : Univ., härtill 5 uppsatser</p> / digitalisering@umu

Diagnosis plays a very crucial role in medicine and health care, which makes biosensors extremely important in modern technological context. Till date, various types of biosensors have been developed that are capable of detecting a wide range of biologically important species with great sensitivity and selectivity. However, most of these sensing units require highly sophisticated instrumentation and often lack the desired portability. Liquid crystal (LC) droplets, on the other hand, are a new type of functional material that are finding increasing research attention as a new sensing unit due to their tunable optical property, high surface area, portability and cost-effectiveness. In this dissertation, functionalized LC droplets for biosensing at aqueous-LC interface are highlighted. Chemically functionalized LC droplets dispersed in aqueous solution were prepared by the self-assembly of amphiphilic molecules at the aqueous/LC interface. These functionalized LC droplets showed a well-defined director of configuration and a specific optical pattern when observed with a polarizing light microscope. It was discovered that the interaction of chemically functionalized LC droplets with an analyte triggers transition of the director of configuration of the LC within the droplets, providing a simple and unique optical sign for the detection of the analyte. Moreover, the director of configuration transition happened in a concentration dependent manner, allowing both qualitative and quantitative detection of the analyte. The sensitivity of chemically functionalized LC droplets depends not only on the nature of amphiphilic molecules but also the size and number of the droplets. iv The dissertation essentially deals with the application of these chemically functionalized LC droplets in detecting several biologically important species. It was observed that the adsorption of charged macromolecules (dendrimers, proteins, and viruses) on polyelectrolyte functionalized LC droplets triggered a bipolar-to-radial configuration transition based on the polar verses nonpolar interaction. By using a simple optical microscope, microgram per milliliter concentrations of bovine serum albumin, cowpea mosaic virus, and tobacco mosaic virus could be detected in aqueous solution. The detection limit of Mastoparan X polypeptide decorated LC droplets in detecting E. coli could reach to approximately 10 bacteria per milliliter. In this case, the high affinity of the polypeptide towards the bacterial causes the former to detach from the LC droplets, triggering the director of configuration transition of the LC inside the droplets. Finally, surfactant decorated LC droplets were used to detect lithocholic acid (LCA), a toxic bile acid used as a specific biomarker for colon cancers. In this case, the director of configuration transition of the LC inside the droplets is a result of the replacement of the surfactant from the aqueous/LC interface by LCA. The microgram per milliliter concentration of LCA, a clinically significant concentration, could be easily detected by changing the length of surfactants. These studies highlight the novel use of surface functionalized LC droplets to detect biologically important species. Due to their tunable optical property, coupled with high surface area and portability, surface functionalized LC droplets have great potentials in the design of next generation biosensors

A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program.