This thesis reports a study of the chemical stability and coordination chemistry of several antitumour metallocenes Cp2MCl2 (Cp = h5-C5H5; M = Ti 1, V 2, Nb 3, Mo 4), as well as derivatives of Cp2TiCl2 1, with nucleic acids, nucleic acid constituents and proteins. These studies were carried out in order to identify the biologically active species and more fully understand the molecular level mechanism of action of the antitumour metallocenes, in particular Cp2TiCl2 1, which is currently undergoing phase II clinical trials. The interactions of Cp2MoCl2 4 with four oligonucleotides were studied by 1H and 31P NMR spectroscopy. In 50 mM salt solutions of Cp2MoCl2 4, hydrolysis of the halide ligands occurred to give a solution with pD -2, containing a species in which both Cp rings remain metal bound for 24 h. At pD -7, partial hydrolysis of the Cp rings (-30percent) occurred after 24 h. Addition of an aqueous solution of Cp2MoCl2 4 in 50 mM salt to the self-complementary sequence d(CGCATATGCG)2, maintaining the pD at 6.0-7.0, showed no evidence for the formation of a metallocene-oligonucleotide complex and only peaks arising from hydrolysis of Cp2MoCl2 4 were detected. A similar result was obtained in titration experiments with the single stranded sequence d(ATGGTA) at pD 6.5-7.0. However, at pD 3.0, new signals assigned to a molybdocene-oligonucleotide complex(es), which was stable for hours at pD 3.0, were detected; while at pD -7 the complex is destabilised and only peaks arising from hydrolysis of Cp2MoCl2 4 were detected. Titration experiments at low pD with Cp2MoCl2 4 and the dinucleotide dCG were consistent with formation of a complex arising due to coordination of molybdenum to guanine N7 and/or cytosine N3. The results obtained showed that stable oligonucleotide adducts were not formed in 50 mM salt at pD -7 and hence it is highly unlikely that formation of molybdocene-DNA adducts in vivo is the primary action that is responsible for the antitumour properties of Cp2MoCl2 4. The rate of hydrolysis of the aromatic rings of Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) and the dimethylsubstituted derivatives (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41), in aqueous solutions at pD 2-8 was studied by 1H NMR spectroscopy. Rapid hydrolysis of both the halide/glycine and Cp ligands in Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) occurred and predominantly gave a precipitate at pD -7. In contrast, under the same experimental conditions, the predominant species present in aqueous solutions of (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) at pH 2-8 contained both MeCp rings metal bound. At pD < 5, Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) and (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) formed similar complex(es) with purine nucleotides. However, at pD >5, stable adducts between nucleotides and Cp2TiX2 (X equals Cl 1, OCOCH2NH3Cl 27) were not formed. In contrast, (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) formed complex(es) with 5'-dAMP or 5'-dGMP, which were stable for 24 h. These results suggest that formation of stable chelates between (MeCp)2TiX2 (X equals Cl 34, OCOCH2NH3Cl 41) and nucleic acid constituents in vivo is possible. However, the methyl substituted derivatives 34 and 41 did not show any antitumour activity against EAT in mice when administered in either 10percentDMSO/90percentsaline or in water at pH 6.2-6.4, which suggests that the labile Cp-Ti bond present in Cp2TiCl2 1 is required for antitumour activity. The synthesis of a range of Cp substituted titanocene derivatives was investigated in an attempt to prepare derivatives with modified Cp stability in comparison to the methyl substituted derivatives. The synthesis of derivatives (CpCH2Y)2TiCl2 where Y equals ?CHO 43, ?CONMe2 44, ?NO2 45, (RCp)2TiCl2 where R equals ?COMe 46, ?COOMe 47 or ?CONMe2 48, (CpNMe2)2TiCl2 62 and (Cp(CH2)2NMe2)2TiCl2 63 was unsuccessful, due to the presence of coordinating substituents on the Cp rings and poor stability in polar, protic solvents. Hence, these derivatives were excluded from further studies. The rate of hydrolysis of the Cp rings of Cp2TiX2 (X equals Cl 1, OCOCCl3 22 and OCOCH2NH3Cl 27) in aqueous solutions, 10percentDMSO/90percentD2O and 100percent DMSO was monitored by 1H NMR spectroscopy. Rapid hydrolysis of both the carboxylate and Cp ligands of Cp2TiX2 (OCOCCl3 22 and OCOCH2NH3Cl 27) occurred in DMSO to give biologically inactive species. The rate of these reactions were concentration dependent as dilution of these samples with saline or water to give the therapeutic conditions of 10percentDMSO/90percentD2O slowed the hydrolysis chemistry. In contrast, samples of Cp2TiX2 (X equals Cl 1 and OCOCH2NH3Cl 27) dissolved in water, gave solutions containing the presumed antitumour active species in which the halide or glycine ligands have been hydrolysed but the Cp rings remain metal bound. Thus, charged X ligands may be incorporated into Cp2TiX2 and will give comparable activity to Cp2TiCl2 1 provided the samples are administered in water. The antitumour metallocenes Cp2MCl2 (M equals Ti 1, V 2, Nb 3, Mo 4) and the inactive derivative (MeCp)2TiCl2 34 were found to inhibit the relaxation of supercoiled plasmid DNA pBR322 by human topoisomerase II in vitro. These results implicated the inhibition of topoisomerase II in the mechanism of antitumour activity although there was no direct correlation between the in vitro results with biological activity against EAT in vivo. UV spectroscopy confirmed that the metallocenes Cp2MCl2 (M equals Ti 1, Mo 4) became associated with and were stabilised to hydrolysis by calf thymus DNA but not with human serum albumin. ICP-AES was used to measure the amount of metal associated with either DNA or human serum albumin after incubation with Cp2MCl2 (M equals Ti 1, Nb 3, Mo 4) and dialysis of these solution. The results confirmed that DNA stabilises or becomes associated with the metallocenes. However, errors associated with the ICP-AES measurements did not allow these results to be quantified. 1H NMR spectroscopy was used to show that the antitumour metallocene Cp2MoCl2 4 formed an adduct with glutathione 72 in the pH range 3-7 through the sulfur donor group. In comparison, the antitumour metallocenes Cp2MCl2 (M equals Ti 1, Nb 3) showed limited adduct formation with glutathione 72 at pH -3 and no adducts were detected at pH > 5.5.

Doctor of Philosophy (PhD) / The toxicity of platinum anticancer drugs presents a major obstacle in the effective treatment of tumours. Much of the toxicity stems from a lack of specificity of the drugs for the sites at which they are able to exert maximum anticancer activity. An improved understanding of the behaviour of the drugs in the tumour environment may assist in the rational design of future platinum anticancer agents with enhanced specificity and reduced toxicity. In the work presented herein, the specificity of two classes of platinum anticancer agents was assessed (platinum(IV) cisplatin analogues and platinum(II) anthraquinone complexes). The interaction of the platinum(IV) agents with DNA, believed to be their main cellular target, was examined using XANES spectroscopy. This experiment was designed to assess the ability of the drugs to interact with DNA and thus exert their anticancer activity. It was shown that the platinum(IV) complexes were not reduced by DNA during 48 hr incubation. It was not possible to conclusively determine whether the interaction of the complexes with DNA was direct or platinum(II) catalysed, or whether interaction had occurred at all. The distribution of platinum(II) anthraquinone complexes and their corresponding anthraquinone ligands in tumour cells (A2780 ovarian and DLD-1 colon cancer cell lines) was investigated. The cytotoxicity of the compounds in DLD-1 cells was also assessed. It was found that the compounds were efficiently taken up into the cells and entered the lysosomal compartments almost exclusively. This suggested that the cytotoxicity of the drugs was caused by lysosomal disruption, or that the platinum complexes were degraded, leaving a platinum species to enter the cell nuclei and interact with DNA. Alternatively, the complexes may bind to proteins and transport into the nuclei of the cells, though with their fluorescence quenched by the protein. The penetration and distribution of platinum(IV) complexes was assessed in DLD-1 multicellular tumour spheroids (established models of solid tumours) using a number of synchrotron techniques, including micro-tomography, micro-SRIXE, and micro-XANES. The complexes were found to be capable of penetrating throughout the entire volume of the spheroids. Micro-XANES indicated that in central and peripheral spheroidal regions, bound platinum species were present largely as platinum(II).

No description available.

The pathology of a disfiguring and debilitating fatal disease affecting a high proportion of the wild population of Tasmanian Devils (Sarcophilus harrisii) that was discovered is described. The disease, named devil facial tumour disease (DFTD), has been identified in devils found across 60% of the Tasmanian landscape. The prevalence of this disease was extremely variable, possibly reflecting seasonal trapping success. Between 2001 and 2004, 91 DFTD cases were obtained for pathological description. Grossly, the tumours presented as large, solid, soft tissue masses usually with flattened, centrally ulcerated and exudative surfaces. They were typically multi-centric, appearing first in the oral, face or neck regions. Histologically, the tumours were composed of circumscribed to infiltrative nodular aggregates of round to spindle-shaped cells often within a pseudocapsule and divided into lobules by delicate fibrous septae. They were locally aggressive and metastasised in 65% of cases. There was minimal cytological differentiation amongst the tumour cell population under light and electron microscopy. The diagnostic values of a number of immunohistochemical stains were employed to further characterise up to 50 representative cases. They were negative for cytokeratin, epithelial membrane antigen, von Willebrand factor, desmin, glial fibrillary acid protein, CD16, CD57, CD3 and LSP1. DFTD cells were positive for vimentin, S-100, melan A, neuron specific enolase, chromogranin A and synaptophysin. In conclusion, the morphological and immunohistochemical characteristics together with the primary distribution of the neoplasms indicate that DFTD is an undifferentiated neoplasm of neuroendocrine histogenesis.

Tumour development has long been recognised as an evolutionary process during which cells accumulate mutations and evolve into a mix of genetically distinct cell subpopulations. The resulting genetic intra-tumour heterogeneity poses a major challenge to cancer therapy, as it increases the chance of drug resistance. To study tumour evolution in more detail, reliable approaches to infer the life histories of tumours are needed. This dissertation focuses on computational methods for inferring trees of tumour evolution from single-cell and multi-sample sequencing data. Recent advances in single-cell sequencing technologies have promised to reveal tumour heterogeneity at a much higher resolution, but single-cell sequencing data is inherently noisy, making it unsuitable for analysis with classic phylogenetic methods. The first part of the dissertation describes OncoNEM, a novel probabilistic method to infer clonal lineage trees from noisy single nucleotide variants of single cells. Simulation studies are used to validate the method and to compare its performance to that of other methods. Finally, OncoNEM is applied in two case studies. In the second part of the dissertation, a comprehensive collection of existing multi-sample approaches is used to infer the phylogenies of metastatic breast cancers from ten patients. In particular, shallow whole-genome, whole exome and targeted deep sequencing data are analysed. The inference methods comprise copy number and point mutation based approaches, as well as a method that utilises a combination of the two. To improve the copy number based inference, a novel allele-specific multi-sample segmentation algorithm is presented. The results are compared across methods and data types to assess the reliability of the different methods. In summary, this thesis presents substantial methodological advances to understand tumour evolution from genomic profiles of single cells or related bulk samples.

The incidence of colorectal cancer (CRC) is increasing and the prognosis for patients with advanced or metastatic disease is relatively poor. Immunotherapies hold great promise, but deploying them effectively in CRC patients will require further knowledge of the complex cellular and molecular interactions that occur between intestinal tumours and the host immune system. The objective of this study is to understand the mechanisms by which lack of immune cell regulation in the gut can drive the formation of colon adenocarcinomas. In addition, this work aims to identify new mechanisms involved in progression to metastatic disease. Using mouse model systems, we found that aberrant activity of Treg cells deficient in IL-10 can promote inflammation-driven CRC. IL-10 deficient Tregs have increased capacity to drive tumourigenesis compared to their CD4⁺ effector T cell counterparts. RNA sequencing revealed specific upregulation of several genes, including a newly-described cytokine, in tumour-promoting Tregs. We explored cytokine regulation and the tumour microenvironment, and show that the inflammatory cytokine IL-6 and TGFÎ2 are necessary for tumour formation in this model. Moreover, disease is associated with a marked stromal cell signature that is induced as a consequence of Treg deficiency in IL-10 production. Gp38⁺ stromal cells are dominant producers of IL-6, and potent ECM modellers. Furthermore, tumours driven by IL-10 deficient Tregs express high amounts of the pro-mesenchymal transcription factor Sox4. Using combined in vitro and in vivo analyses, we confirm that Sox4 is involved in tumour growth and characterise its expression in CRC patients. Collectively, our findings suggest that Tregs and stromal cells act together to foster a microenvironment that promotes disease progression, notably through the expression of Sox4 in tumour cells. These findings open an exciting avenue to explore the phenotype of tumour-promoting Tregs and to study Sox4 function in metastatic disease.

Orientadores: Ubirajara Ferreira, Fernandes Denardi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T15:47:05Z (GMT). No. of bitstreams: 1 Matheus_WagnerEduardo_D.pdf: 1544170 bytes, checksum: b8c2ff3d48db298c2b90e9243e4fad82 (MD5) Previous issue date: 2007 / Resumo: O melhor tratamento para o carcinoma urotelial invasivo de bexiga é a cirurgia de cistectomia radical. O objetivo principal desse estudo foi de comparar a taxa de sobrevida global dos tumores músculo invasivos primários dos tumores invasivos progressivos. O objetivo secundário foi comparar a taxa de sobrevida global dos subgrupos pT3/4, acometimento linfonodal e presença de metástases, dos tumores primários e invasivos. Nesse estudo multicêntrico retrospectivo, foram avaliados 242 pacientes submetidos à cistectomia radical, no período de 1992 a 2005, para tratamento de carcinoma urotelial invasivo de bexiga. Os pacientes foram divididos em dois grupos: Grupo I ¿ 185 pacientes com tumor invasivo primário e Grupo II - 57 pacientes com carcinoma urotelial invasivo progressivo. Além disso, conforme achados histopatológicos, ambos os grupos foram divididos em subgrupos: pT2 (invasão de musculatura vesical), pT3/4 (invasão de gordura perivesical e órgãos ou tecidos adjacentes), N+ (acometimento de linfonodos) e M+ (presença de metástases). Para análise estatística foram aplicados os testes de qui-quadrado, Mann-Whitney, Kaplan-Meier e Wilcoxon (Breslow). A média e mediana de seguimento foram de 98 e 90 meses para o Grupo I, e 96 e 88 meses para o Grupo II, respectivamente, e sem diferença estatística significativa (p = 0.0734). No seguimento, foram observadas as seguintes taxas de sobrevida global: no primeiro ano, 77% para o Grupo I e 84% para o Grupo II; no terceiro ano, 59% e 74% e, no quinto ano, 52% e 58% para os grupos I e II, respectivamente, sem diferença estatística significativa. Quando analisados separadamente, os três subgrupos: tumores PT3/T4, acometimento linfonodal e presença de metástases, também não foram observadas diferenças estatísticas significativas nos grupos I e II. No presente estudo, não houve diferença significativa de sobrevida global dos pacientes portadores de tumores vesicais invasivos primários e progressivos, no seguimento de cinco anos. Também não houve diferença significativa na sobrevida global, quando analisados separadamente os subgrupos: PT3/4, com acometimento linfonodal e presença de metástases / Abstract: The best treatment for all-invasive bladder cancer is radical cystectomy. The main purpose of this study was to compare the overall survival rate of primary muscle-invasive urothelialbladder carcinoma (UC) to the progressive muscle-invasive bladder carcinoma. A secondary aim was to compare the survival rate of the subgroups pT3/4, lymph nodes involvement and the presence of metastasis in primary and invasive bladder carcinomas. A retrospective multicentric analysis was performed studying a total of 242 patients who underwent radical cystectomy for invasive TCCB from 1992 to 2005. The patients were divided into two groups. There were 185 patients in Group I with progressive invasive TCCB, while Group II had 57 patients with primary invasive TCCB. Both groups were further divided according to the pathological findings in pT2 (muscle invasion), pT3/4 (perivesical fat and/or adjacent organs/structure invasion), N+ (positive lymphaticnodes) and M+ (distant organ metastasis). Several tests were employed for the statistical analysis: qui-square, Mann-Whitney, Kaplan-Meier method and Wilcoxon (Breslow). The average and median follow-ups were, respectively, 98 and 90 months in Group I and 96 and 88 months in Group II, without a significant statistical difference (p = 0.0734). The 1-year survival rate was 77% in Group I and 84% in Group II. After 3 years of follow-up the survival rate fell to 59% in Group I and 74% in Group II. Finally, the 5-year survival rate was 52% in Group I and 58% in Group II, without a significant statistical difference. When the three subgroups were analyzed separately for tumors pT3/T4, invasivelymphatic nodes and the presence of metastasis, no significant statistical differences were found in either Group I or Group II. In the present study, patients with primary invasive and progressive invasive TCCB showed a similar 5-year global survival rate. Pathological stage (PT, N and M) and patient demography did not interfere with the results / Doutorado / Cirurgia / Doutor em Cirurgia

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Positron emission tomography (PET) imaging is an emerging medical imaging modality. Due to its high sensitivity and ability to model physiological function, it is effective in identifying active regions that may be associated with different types of tumour. Increasing numbers of patient scans have led to an urgent need for the development of new efficient data analysis system to aid clinicians in the diagnosis of disease and save decent amount of the processing time, as well as the automatic detection of small lesions. In this research, an automated intelligent system for oncological PET volume analysis has been developed. Experimental NEMA (national electrical manufacturers association) IEC (International Electrotechnical Commission) body phantom data set, Zubal anthropomorphic phantom data set with simulated tumours, clinical data set from patient with histologically proven non-small cell lung cancer, and clinical data sets from seven patients with laryngeal squamous cell carcinoma have been utilised in this research. The initial stage of the developed system involves different thresholding approaches, and transforming the processed volumes into the wavelet domain at different levels of decomposition by deploying Haar wavelet transform. K-means approach is also deployed to classify the processed volume into a distinct number of classes. The optimal number of classes for each processed data set has been obtained automatically based on Bayesian information criterion. The second stage of the system involves artificial intelligence approaches including feedforward neural network, adaptive neuro-fuzzy inference system, self organising map, and fuzzy C-means. The best neural network design for PET application has been thoroughly investigated. All the proposed classifiers have been evaluated and tested on the experimental, simulated and clinical data sets. The final stage of the developed system includes the development of new optimised committee machine for PET application and tumour classification. Objective and subjective evaluations have been carried out for all the systems outputs, they show promising results for classifying patient lesions. The new approach results have been compared with all of the results obtained from the investigated classifiers and the developed committee machines. Superior results have been achieved using the new approach. An accuracy of 99.95% is achieved for clinical data set of patient with histologically proven lung tumour, and an average accuracy of 98.11% is achieved for clinical data set of seven patients with laryngeal tumour.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that localises to focal adhesions. FAK is crucial for many cellular processes that are disturbed in malignancy, including proliferation, cell cycle, cell survival, adhesion, and migration. Mouse models have shown that FAK is involved in tumour formation and progression. Other studies demonstrated a functional correlation between FAK expression, tumour progression and malignancy in human cancer, making FAK a potentially important therapeutic target. Several FAK inhibitors have been developed most of which target the FAK kinase function. However, FAK may predominantly act as a scaffolding molecule rather than as a kinase, therefore, disruption of FAK’s interaction with protein binding partners could be a good strategy to inhibit some cancer processes. The identification and characterisation of novel FAK interactions may help to uncover important molecular mechanisms that, in turn, regulate key cellular processes involved in tumour formation and/or progression. Disruption of their function, or inhibition of their binding to FAK, will define their roles and identify whether they are good anti-cancer targets. In this thesis work, I set out to identify novel binding partners of FAK, and study the role of a sub-set of these in tumour biology by impairing them in squamous cell carcinoma cancer cells in vitro. To do this I employed protein microarray and phage display methodologies using FAKΔ375 and FAK-FERM recombinant proteins as bait, respectively. I identified a number of novel proteins that interact directly with FAK. Then I set out to characterise some of these proteins. The first of these, Axl, is a protein receptor tyrosine kinase that has previously been linked with tumour progression and metastasis in number of human cancers. I confirmed the interaction between FAK and Axl in SCC cells and showed that the FAK-Axl interaction is predominantly a scaffolding function of FAK, which seems to be unregulated, at least by any of the major phosphorylation events characterised for FAK. I also found that Axl controls cell spreading, cell polarisation and invasive migration in this cancer cell lines. The second protein I characterise is the autophagy protein Ambra1. I found that Ambra1 is required for selective targeting of active Src to the autophagy pathway – a process that SCC cancer cells use when they are under adhesion stress, such as when FAK is deleted. Thus, Axl and Ambra1 are potentially important proteins in SCC biology. They bind to FAK and function at cell adhesions to promote cancer-associated cellular processes. Analysis of FAK binding proteins may be a useful strategy to discover proteins that function in various aspects of cancer cell behaviour.