Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide. Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL). CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity. In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.

Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.

The antitumoural cyanoguanidine CHS 828 has shown promising activity in a number of preclinical and clinical studies. However, the mechanisms underlying the cell death induced by CHS 828 has not been clarified. This thesis describes in vitro studies of the cellular pharmacology of CHS 828. CHS 828 induced cell death with necrosis like features in the lymphoma cell line U-937 GTB. Addition of 3-aminobenzamide, an inhibitor of ADP-ribosylation, resulted in a decreased sensitivity to CHS 828 and a shift in the mode of cell death towards apoptosis. Mouse fibroblasts lacking the enzyme PARP-1 were more sensitive to CHS 828 compared to normal fibroblasts. CHS 828 was able to induce p53 in normal fibroblasts but this effect does not seem to be necessary to induce cell death. Characterization of two CHS 828 resistant cell lines indicated that they were selectively resistant to cyanoguanidines. Known mechanisms of anticancer drug resistance did not seem to account for the cyanoguanidine resistance. One possible resistance mediating protein, which was upregulated in the resistant cells, was epidermal fatty acid binding protein. A novel high content screening assay was also developed. The assay was shown to be suitable both for screening of potential novel antitumoural substances as well for mechanistic studies. In the assay, CHS 828 induced caspase-3 activity and reduction in mitochondrial membrane potential, both signs of apoptosis, in U-937 GTB cells. However, nuclei in exposed cells did not show nuclear fragmentation, one of the hallmarks of apoptosis. CHS 828 was also shown to indirectly inhibit the proteasome activity in U-937 GTB cells. In conclusion, the results presented provide new insights into the metabolic and molecular events involved in cell death induced by CHS 828.

The novel cyanoguanidine CHS 828 has shown promising antitumor activity in many in vitro and in vivo studies. The long-term 14 days in vitro hollow fiber cultures, where tumor cells from different tumor cell lines were cultured inside semipermeable fibers, were more resistant to CHS 828 and other cytotoxic drugs than the shorter-term 3 days cultures. CHS 828 was generally more effective against haematological than solid tumor cells from both cell lines and patients samples. In vivo, the hollow fibers were implanted into immunocompetent rats and the pharmacokinetics, tumor response and/or toxicity (pharmacodynamics) of CHS 828 were successfully assayed. CHS 828 showed higher activity in this model when a more protracted schedule was used. The quantitative relationships between dose, plasma concentration and response (PK/PD model) developed for CHS 828 explained this phenomenon partly by dose-dependent fraction absorbed and partly by a schedule-dependent pharmacodynamic effect. Modelling of the in vitro CHS 828 and standard cytotoxic drugs concentration-time effect data in different tumor cell types and characterization of pattern of change of the potency and the slope of the concentration-time effect curves were performed. The results suggest two different mechanisms of action for CHS 828 and that CHS 828 cytotoxicity may depend on the schedule used. The NF-kB pathway that regulates the transcription of anti-apoptotic genes proved to be inhibited by CHS 828 in different tumor cell lines and the inhibition was correlated to the cell death induced by this agent. CHS 828 did not seem to induce the NF-kB inhibition by affecting the proteasome activity. The in vitro and in vivo hollow fiber methods were also used successfully to evaluate the new paclitaxel formulation, Pacliex. Pacliex had a similar activity to that of the clinically used formulation Taxol®.

CHS 828 is a cyanoguanidine with anti-tumour properties which has shown promising effects in several preclinical models. This thesis describes both preclinical and clinical studies aiming to investigate disease specific activity, clinical tolerability and efficacy of CHS 828. In paper I we investigated CHS 828 activity in a cell line panel with human myeloma cells, three of these cell-lines were also tested in vivo using a hollow fibre rat-model. In paper II we investigated CHS 828 activity in primary human tumour samples from patients. CHS 828 showed an effect on all tumour cell types tested both the primary human tumour samples and the myeloma cell lines. Notably, CHS 828 showed a high relative in vitro activity against tumour cells from chronic lymphocytic leukaemia and high-grade lymphoma. In a phase I trial we determined the maximum tolerated dose (MTD) of CHS 828. Haematological toxicity was generally mild and dominated by transient thrombocytopenia and lymphocytopenia. Non-haematological toxicity was mostly of gastrointestinal origin. The recommended phase two dose (RPTD) of CHS 828 was estimated to be 20 mg once daily for five days in cycles of 28 days duration. In a phase II trial we investigated the effect of CHS 828 on patients diagnosed with B-CLL. In total 12 patients were enrolled. CHS 828 was found to be well tolerated and the most common haematological toxicity was thrombocytopenia. Non-haematological toxicities were generally mild. Transient decreases in lymphocyte counts could be discerned coinciding with drug dosing, but no sustained clinical responses could be achieved. In conclusion, CHS 828 demonstrated marked effects in the preclinical investigations suggesting haematological malignancies as the main target. The clinical phase I study established a safe dose and the subsequent phase II trial in B-CLL patients showed biological effect but with no clinical disease response.

Ultraviolet –B (UV-B) radiation is an abiotic stress that has significant effects on plant growth, development, and gene regulation. Due to the depletion of the stratospheric ozone layer over the past several decades, the amount of UV-B light that is reaching the earth’s surface has significantly increased. As a result, research over the past few decades on the effects of UV-B light on plant growth, development, and the mechanisms that regulate a plant’s protection and survival against UV-B light has grown greatly. Brachypodium distachyon is a relatively new model system and one that has not been extensively studied. The aim of this study was to determine the UV-B dose time required to elicit a significant increase in phenolic content, while subsequently assessing protein production to qualitatively implicate whether or not the experimental dosage of UV-B administered was initiating a UV-B specific or non-specific response. In addition, this research annotated the genes that encode the protein sequences for UVR8 and CHS proteins to see if B. distachyon possessed the necessary proteins to undergo a UV-B specific response similar to that of Arabidopsis. The results of the study show that in response to artificial UV-B light, the dose time of UV-B required to elicit a significant increase in total phenolic content is 2 hours. The data also shows an increase in total protein content after 4 hours of UV-B exposure. In addition to the metabolic data, computational analysis of chalcone synthase (CHS) and UV-RESISTANCE LOCUS 8 (UVR8) revealed that there are seven genes in B. distachyon that encode the protein transcripts for CHS and CHS-like proteins, and two genes that code for UVR8 proteins. The results of this study suggest that the UV-B dose regimen used in this study may be initiating the non-specific UV-B signaling pathway. In addition, the presence of UVR8 and CHS protein sequences suggest that B. distachyon has the capacity to work through the UV-B specific signaling pathway.

This paper presents the behaviour and design of axially loaded concrete filled stainless steel circular and square hollow sections. The experimental investigation was conducted using different concrete cube strengths varied from 30 to 100 MPa. The column strengths and load-axial shortening curves were evaluated. The study is limited to cross-section capacity and has not been validated at member level. Comparisons of the tests results together with other available results from the literature have been made with existing design methods for composite carbon steel sections ¿ Eurocode 4 and ACI. It was found that existing design guidance for carbon steel may generally be safely applied to concrete filled stainless steel tubes, though it tends to be over-conservative. A continuous strength method is proposed and it is found to provide the most accurate and consistent prediction of the axial capacity of the composite concrete filled stainless steel hollow sections due largely to the more precise assessment of the contribution of the stainless steel tube to the composite resistance.

<p>This thesis describes the use of a new technology, the Cytosensor^® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. </p><p>The Cytosensor^® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure.</p><p>In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development.</p><p>In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. </p><p>It is concluded that measurement in the Cytosensor^® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents. </p>

This thesis describes the use of a new technology, the Cytosensor® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. The Cytosensor® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure. In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development. In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. It is concluded that measurement in the Cytosensor® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents.

This thesis presents the results of a research project to develop methods to carry out fire safety design of welded steel tubular trusses at elevated temperatures due to fire exposure. It deals with three subjects: resistance of welded tubular joints at elevated temperatures, effects of large truss deflection in fire on member design and effects of localised heating. The objectives of the project are achieved through numerical finite element modelling at elevated temperatures using the commercial Finite Element software ABAQUS v6.10-1 (2011). Validation of the simulation model for joints is based on comparison against the test results of Nguyen et al. (2010) and Kurobane et al. (1986). Validation of the simulation model for trusses is through checking against the test results of Edwards (2004) and Liu et al. (2010).For welded tubular joints, extensive numerical simulations have been conducted on T-, Y-, X-, N- and non-overlapped K-joints subjected to brace axial compression or tension, considering a wide range of geometrical parameters. Uniform temperature distribution was assumed for both the chord and brace members. Results of the numerical simulations indicate for gap K- and N-joints (two brace members, one in tension and the other in compression) and for T-, Y- and X-joints with the brace member under axial tensile load (one brace member only, in tension), it is suitable to use the same ambient temperature calculation equation as in the CIDECT (2010) or EN 1993-1-8 (CEN, 2005a) design guides and simply replace the ambient temperature strength of steel with the elevated temperature value. However, for T-, Y- and X-joints under brace compression load (one brace member only, in compression), the effect of large chord deformation should be considered. Large chord deformation changes the chord geometry and invalidates the assumed yield line mechanism at ambient temperature. For approximation, the results of this research indicate that it is acceptable to modify the ambient temperature joint strength by a reduction factor for the elastic modulus of steel at elevated temperatures. In the current fire safety design method for steel truss, a member based approach is used. In this approach, the truss member forces are calculated at ambient temperature based on linear elastic analysis. These forces are then used to calculate the truss member limiting temperatures. An extensive parametric study has been carried out to investigate whether this method is appropriate. The parametric study encompasses different design parameters over a wide range of values, including truss type, joint type, truss span-to-depth ratio, critical member slenderness, applied load ratio, number of brace members, initial imperfection and thermal elongation. The results of this research show that due to a truss undergoing large displacements at elevated temperatures, some truss members (compression brace members near the truss centre) experience large increases in member forces. Therefore, using the ambient temperature member force, as in the current truss fire safety design method, may overestimate the truss member critical temperature by 100 °C. A method has been proposed to analytically calculate the increase in brace compressive force due to large truss deformation. In this method, the maximum truss displacement is assumed to be span/30. A comparison of the results calculated using the proposed method against the truss parametric study results has shown good agreement with the two sets of results, with the calculation results generally being slightly on the safe side. When different members of a truss are heated to different temperatures due to localised fire exposure, the brace members in compression experience increased compression due to restrained thermal expansion. To calculate the critical temperature of a brace member in a localised heated truss, it is necessary to consider this effect of restrained thermal expansion. It is also necessary to consider the beneficial effects of the adjacent members being heated, which tends to reduce the increase in compressive force in the critical member under consideration. Again, an extensive set of parametric studies have been conducted, for different load ratio, slenderness and axial restraint ratio. The results of this parametric study suggest that to calculate the critical temperature of a brace member, it is not necessary to consider the effects of the third or further adjacent members being heated. For the remainder of the heated members, this thesis has proposed a linear elastic, static analysis method at ambient temperature to calculate the additional compressive force (some negative, indicating tension) in the critical member caused by the heated members (including the critical member itself and the adjacent members). The additional compressive force is then used to calculate the limiting temperature of the critical member. For this purpose, the approximate analytical equation of Wang et al. (2010) has been demonstrated to be suitable.