Cervical cancer still represents the fourth most common cause of cancer deaths in women worldwide. Human papilloma virus (HPV) infection plays a role in cervical carcinoma initiation, but other genomic changes are needed for pre-malignant abnormalities to fully develop to cancer. This often happens through genomic instability caused by the virus oncoproteins. Several integrative genomic analysis studies have found that one of the most common imbalances in cervical squamous cell carcinoma (SCC) is copy number gain and amplification of chromosome 5p. In this region, copy number gain of the OSMR gene was found to correlate significantly with adverse outcome independent of the tumour stage (p=0.046). Furthermore, this copy number gain correlated with Oncostatin M receptor (OSMR) overexpression and sensitised these cells to Oncostatin M (OSM) leading to increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation, cell migration, invasion and proangiogenic signalling. The aim of this PhD project was to study the role of OSMR overexpression in the SCC tumour microenvironment (TME) and tumour growth in vivo and to study the role of hypoxia inducible factor driven hypoxia signalling in OSMR overexpressing SCC cells and their tumour microenvironment. OSMR overexpression was found to sensitise tumour cells to induce Hypoxia inducible factor 1a and 2a (HIF1a, HIF2a) signalling in normoxic conditions, to promote pro-angiogenic signalling. Furthermore, hypoxic conditions were found to enhance OSM signalling in OSMR overexpressing cells leading to increased expression of markers of epithelial to mesenchymal transition, angiogenesis and migration. In the SCC tumour microenvironment, OSMR overexpression was found to sensitise tumour cells to OSM secreted from macrophages and other immune cells leading to improved tumour growth, angiogenesis and STAT3 activation at the tumour site. Removal of OSMR from either tumour cells or tumour microenvironment led to reduced tumour growth and angiogenesis, along with increased tumour necrosis. I conclude that OSMR overexpression is an important driver of SCC tumour progression and malignancy via STAT3- and HIF-driven signalling and removal of it from either tumour cells or tumour microenvironment drastically hampers tumour growth in vivo. Based on the results of this study, OSMR blockade is a potential novel therapeutic option in advanced SCC.

Natural products have long been known for their broad range of useful therapeutic properties, and have been widely utilised in the field of medicine. This dissertation describes work towards the total synthesis of natural products possessing biological activity in two important areas. The first section concerns the total synthesis of six 4-quinolone natural products, four of which had never been synthesised before. These compounds were originally isolated from a soil bacterium of the genus Pseudonocardia, and bear intriguing structural resemblance to the Pseudomonas Quinolone Signal. This signalling molecule is vital to the quorum sensing activity of the human pathogen Pseudomonas aeruginosa, which is a phenomenon by which it regulates many of its virulence factors. These natural products possess the potential to disrupt this system, hence attenuating the pathogenicity of the bacteria. The routes that were developed are highly divergent, efficiently giving access to multiple natural products from mutual late stage intermediates. Key steps included regioselective epoxidation, palladium-catalysed heterocylisation and acid catalysed 1,3-transposition of an allylic alcohol. In the second section, attention turns towards the total synthesis of the complex sesquiterpene lactone 1β-Hydroxyalantolactone. The compound possesses five stereogenic centres, one of which is quaternary, alongside a challenging tricyclic core scaffold. Previous biological studies have revealed a range of intriguing properties, including anti-inflammatory and anti-tumour activity. The chosen route utilises as its key step the catalytic desymmetrisation of a diene which was itself accessed by Birch reduction chemistry. Whilst the synthesis is as yet incomplete, access was granted to a key intermediate encompassing around half of the stereocentres present in the natural product.

Neurofibromatosis type 2 (NF2) is an inherited, multiple tumour disease caused by loss of the tumour suppressor protein, Merlin. There are several tumours associated with NF2 including; ependymomas, meningiomas and schwannomas. Merlin loss can also occur sporadically in all of these tumours and is associated with upregulation of various growth factor receptors and their relevant signalling pathways. At present the only treatment options for NF2 are surgery or radiosurgery, both of which incur serious morbidity and are unable to prevent recurrence of tumours. Either new drug treatments, or re-profiling of other drugs already commercially available, are urgently needed to improve outcome for NF2 patients. Cellular prion protein (PrPC), encoded by PRNP gene, is involved in tumour development by altering proliferation, adhesion, and survival in some cancers via focal adhesion kinase (FAK) /Src/ NFκB, cyclin D1 and p53 -proteins. Our group previously showed a strong elevation of PRNP gene activity in schwannoma. I hypothesise that PrPC may contribute to schwannoma development. To study the role of PrPC in schwannoma development I have used the well-established in vitro model of schwannoma that comprises primary human Schwann and schwannoma cells. I show that PrPC is upregulated in schwannoma as well as in Merlin-deficient meningiomas and human malignant mesotheliomas. In schwannoma PrPC is released both via exosomes and by α-cleavage which forms biologically active N- and C-terminal portions of the protein. PrPC contributes to pathological proliferation, adhesion and survival of schwannoma cells by activating ERK1/2, PI3K/AKT, cyclin D1, FAK, p53 pathways via the 37/67kDa non-integrin laminin receptor (LR/37/67kDa) and CD44. Furthermore, schwannoma cells appear to be intrinsically drug-resistant due to upregulation of MDR1 protein p-glycoprotein (p-gp) expression. P-gp expression is dependent on PrPC thus, inhibiting PrPC may be a good potential new therapeutic option for schwannoma patients, either alone or in combination with Sorafenib and p-gp inhibitor Valspodar (PSC833). An inhibitor of LR/37/67kDa/PrP interaction, NSC47924, or Bortezomib, a proteasome/NFκB inhibitor which has been approved for the treatment of multiple myeloma, could also be of beneficial therapeutic effect and is something to investigate in future work. I conclude that PrPC is an interesting new therapeutic target through its involvement with schwannoma patholgenesis and resistance to drug treatments PrPC may prove to be a good therapeutic target in other NF2-related tumours like meningiomas and schwannomas.

Orientador: Maria Cristina Cintra Gomes Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-19T11:39:14Z (GMT). No. of bitstreams: 1 Oliveira_AndreGustavode_M.pdf: 2220744 bytes, checksum: ad8fc9b9eb42ef7af7f4733b165e19b0 (MD5) Previous issue date: 2011 / Resumo: O crescimento do câncer promove o desenvolvimento de caquexia em função de intensa espoliação de nutrientes, principalmente, de gordura e proteína corpórea total. A via de sinalização da mTOR controla o crescimento celular e alguns estudos apontam que a inibição dessa via por metformina (M) pode diminuir a taxa de desenvolvimento tumoral. Desse modo, o presente estudo avaliou os efeitos da administração de metformina associada à dieta rica em leucina (L), em animais com tumor de Walker 256 (W), sobre o metabolismo protéico muscular, na hipótese de melhorar o estado caquético. Ratos Wistar jovens foram distribuídos em oito grupos, de acordo com a presença ou não de tumor, tratamento com metformina (36 mg x Kg-1) e/ou dieta rica em leucina (dieta com excesso de 3%). Foram analisados parâmetros somáticos e bioquímicos bem como vias de sinalização celular no músculo gastrocnêmico. No grupo W, o crescimento tumoral induziu perda de 20% da massa corpórea; proporcionou redução de 70% na massa gorda, alem da diminuição da concentração sérica de glicose, de proteínas totais e albumina. As concentrações de GH e IGF-1 foram reduzidas, porém a concentração de ACTH foi elevada em todos os grupos com tumor. Os grupos portadores de tumor tiveram maiores taxas de degradação protéica muscular, embora as taxas de síntese não tenham sido alteradas. A suplementação com leucina estimulou a síntese protéica nos animais não portadores de tumor, e os resultados sugerem que esse efeito tenha sido via Akt ou Erk, enquanto que a metformina estimulou a sinalização via IRS-1, levando à ativação de Erk. A evolução tumoral promoveu espoliação da massa protéica e modulação dos processos de síntese protéica, através da inibição da Erk e IRS-1. A suplementação com leucina foi capaz de estimular a síntese protéica nos animais não portadores de tumor, porém o tratamento com metformina não foi eficaz em diminuir o crescimento das células tumorais, provavelmente em função da baixa concentração que foi administrada aos animais / Abstract: Tumour growth induces cachexia by intense nutrient waste, characterized by involuntary host weight loss, mainly depleting the total body protein and fat. mTOR signaling pathway controls the cell growth regulating translation of mRNA, and the inhibition of this pathway by compounds such as metformin (M) could decrease tumour growth rate. Knowing these facts, the aim of this study was to evaluate metformin effects associated to leucine-rich diet in Walker 256 (W) tumour-bearing animals, on muscle protein metabolism, trying to improve the cachectic state. Young male Wistar rats were distributed into 8 groups, according to the tumour implant, the treatment with metformin (36 mg x kg-1) and/or leucine-rich diet (3% leucine). After the sacrifice of the animals we evaluated some somatic and biochemical parameters as well as the muscle cell signaling pathways.. Tumour growth promotes 20%reduction of body mass and 70% of fat mass. Glucose serum was 50% decreased and also the total serum proteins (20% less) and albumin (25% reduced). GH and IGF-1 concentrations were decreased in all tumour-bearing groups, while ACTH concentration was increased. Tumour-bearing groups had higher protein degradation rates while protein synthesis rates were not changed, showing decreased protein turnover. Leucine rich-diet stimulated protein synthesis in non-tumour-bearing groups and these results suggest that this effect was through Akt or Erk pathways and metformin stimulated signaling through IRS-1, leading to Erk activation. Tumour growth promoted lean body mass spoliation and modulated protein synthesis through Erk and IRS-1 inhibition. Leucine-rich diet was able to stimulate protein synthesis in non tumour-bearing animals, although the treatment with metformina was not thus effective in decreasing tumour's cells growth, probably by low dose concentration / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Les fibroblastes activés qui composent les stromas tumoraux sont des acteurs majeurs des interactions tumeur-stroma impliquées dans la croissance et la dissémination des cellules tumorales. Ce processus d'activation des fibroblastes est caractérisé par l'expression de marqueurs protéiques spécifiques parmi lesquels figure l'alphaSMA et FAPalpha;. Le TGFbeta;, cytokine secrétée massivement par les cellules tumorales, est un des éléments impliqués dans l'activation des fibroblastes et la formation du stroma tumoral qui en résulte. L'EMMPRIN, glycoprotéine transmembranaire surexprimée dans les cellules tumorales est également un médiateur des interactions tumeur-stroma puisqu'il a la capacité d'induire la synthèse des MMPs par les fibroblastes péri-tumoraux accroissant ainsi la propagation des cellules tumorales à travers l'organisme. Nos travaux identifièrent que le TGFbeta secrété par les cellules tumorales induisait la synthèse du marqueur FAPalpha par les fibroblastes. L'EMMPRIN stromal apparaît comme récepteur de ces signaux tumoraux et est nécessaire à la synthèse du marqueur FAPalpha; par les fibroblastes. L'EMMPRIN participe donc à l'activation TGFbeta; dépendante des fibroblastes. Son inhibition dans ces cellules conduit à un dysfonctionnement de la signalisation médiée par les protéines Smad2/Smad3 aboutissant à une diminution de la synthèse du marqueur alphaSMA ainsi que de certaines protéines matricielles induites par le TGFbeta. L'étude du mécanisme d'action de l'EMMPRIN dans ce processus a permis d'identifier l'EMMPRIN comme nouvelle protéine chaperonne du récepteur de type I au TGFbeta;. / Tumor stroma activated fibroblasts are major actors of tumor stroma interactions taking to tumor growth and spreading. Activated fibroblasts are characterized by the expression of specific markers including alphaSMA and FAPalpha;. The TGFbeta;, a cytokine highly secreted by tumor cells, is one of the key factors involved in fibroblast activation and tumor stroma formation. EMMPRIN, a transmembrane glycoprotein overexpressed in tumor cells, is also a mediator of tumor-stroma interactions by its ability to induce the synthesis of MMPs by peri-tumor fibroblasts enhancing then tumor cells dissemination across the organism.Here, we demonstrate that TGFbeta; secreted by tumor cells is the tumor factor involved in the synthesis of FAPalpha; by fibroblasts. Stromal EMMPRIN appeared to be the receptor of these tumor-stroma interactions and is required for the synthesis of FAPalpha; by fibroblasts. EMMPRIN was also evidenced to take part in TGFbeta;-dependent fibroblast activation. Its inhibition in these cells correlate to a dysfunction in Smad2/Smad3 signaling leading to a decrease in the expression of alphaSMA and matrix proteins induced by TGFbeta;. The study of the mechanism used by EMMPRIN in this process evidenced this protein as a new chaperone for the type I TGFbeta; receptor.

No description available.

Epidemiology of head and neck cancers is currently intensively studied topic. Recent shift in the age of incidence towards younger population is generally attributed to growing proportion of head and neck cancers caused by human papillomavirus - sexually transmitted virus which causes asymptomatic, but sometimes persisting infection that can lead to malignant transformation of the infected tissue. Significance of the topic lies mainly in the prognostic ad- vantage of patients with virally induced head and cancers and preventabil- ity of infections with human papillomavirus. First aim of this thesis is to demonstrate epidemiological trends of head and neck cancers in the Czech Republic, with the focus on change of age-specific incidence and mortality in the last three decades. Second aim of this thesis is to identify risk factors of oral infections with human papillomaviruses and head and neck cancer in a case-control and cross-sectional study of a hospital-based cohort. 1

Many tumours contain regions of hypoxia which are difficult to treat by conventional radiotherapy. There is much interest in the ability of nitric oxide (^•NO) to radiosensitize hypoxic mammalian cells as a possible adjunct to radiotherapy but mechanisms for its action are unclear. It has been proposed that ^•NO may radiosensitize cells by ‘fixing’ radiation-induced DNA free radicals, and elevated radiation response by ^•NO in cells has been partly attributed to increased formation of DNA double strand breaks. In the work carried out for this thesis it is shown that reaction of ^•NO with radiation-induced nucleobase radicals produces some novel products. New pathways for the reactions of radiation-induced hydroxyl radicals with purine radicals are proposed. In addition, the effects of ^•NO on the yields of radiation-induced single strand breaks in anoxic plasmid DNA, and on anoxic mammalian cell radiosensitivity are investigated. Kinetics of formation and repair of radiation-induced double strand breaks indicate different effects of ^•NO on radiation-induced clustered and non-clustered DNA damage involving replication-induced DNA breaks. As ^•NO is an inhibitor of ribonucleotide reductase, some of the radiosensitizing properties of ^•NO may be due to reduction in the availability of 2-deoxyribonucleotides. Through studying reactions of ^•NO with tyrosine radicals, essential components of ribonucleotide reductase, this work has enhanced understanding into how ^•NO may inhibit the enzyme, which may offer new insights into the development of ^•NO-releasing anti-cancer agents. The potential for delivery of ^•NO to hypoxic tissue for radiotherapy has also been investigated in this work, through the development of bioreductively-activated pro-drugs. These novel agents are stable until reduced by one-electron reductants, when a ^•NO-releasing pro-drug is rapidly evolved, only in those regions which are sufficiently hypoxic. By increasing our understanding into the mechanisms involved in the ability of ^•NO to radiosensitize hypoxic cells, especially the reactivity of ^•NO with DNA radicals, knowledge has been gained into the identification, development and repair of radiation-induced DNA damage in cells, including clustered damage, in the presence of ^•NO. These studies contribute to further development of novel anti-cancer therapies based upon the release of ^•NO in hypoxic cells.

Glioblastoma Multiforme (GBM) is a primary malignant brain cancer, affecting children and adults and has a very poor prognosis. Up to 30% of the tumour mass consists of host derived immune cells, and a better understanding of how these cells affect tumour behaviour is required. These cells, called ‘Tumour Associated Macrophages’ (TAM) have been shown to occur in peripheral solid organ cancers, where they can cause local immune suppression, increase invasiveness and aid tumour growth. In the brain however, TAMs can consist of centrally derived microglia and peripherally derived macrophages, and although these cells could be exerting different effects on the tumour, there is currently no reliable way of distinguishing between the two. Cancer Stem Cells (CSC) are a subpopulation of cells within the tumour mass with stem-like features, are capable of self-renewal, and can recapitulate a tumour in an immunocompromised mouse host. It is thought that these cells play a role in disease recurrence and hence represent a potential target for anti-GBM therapies. In the first project we investigate the interaction between Cancer Stem Cells and TAMs. We first describe a method of culturing CSCs and TAMs from a single human patient sample, followed by an investigation into the functional properties of these cell types. We found functional differences between established cell line pairings of U87-MG and CHME3 versus primary patient derived CSCs and TAM cell line pairings. Polarisation of microglia/TAMs with lipopolysaccharide caused significant decrease in proliferative capacity of the GBM cell lines. Next we used a Non-Myeloablative Conditioning System (NMCS) to selectively replace the peripheral bone marrow compartment of wild type animals with labelled bone marrow cells, without disturbing brain homeostasis. We demonstrate that peripheral cells home exclusively to the orthotopically implanted tumour, and that some of these cells are CD11b+ and Gr1+, suggestive of myeloid derived suppressor cells (MDSC). We evaluate current CD45 based gating strategies for distinguishing peripheral and central cells and show them to be inadequate. Finally we compared the chemosensitivity profiles of different patient derived CSC lines using high throughput content screening (HTCS), against currently approved chemotherapeutic drugs and rank these drugs in a response space, based on HTCS parameters including 2D and 3D culture with and without irradiation. Differential chemosensitivities were noted between different patient derived cell lines. Drugs not currently used in the treatment of GBM such as Actinomycin D and Mitoxantrone were also identified using HTCS, suggesting the potential utility of such an approach to personalised treatments in GBM.

Philosophiae Doctor - PhD / Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.