Magnetic Resonance Elastography is a method of imaging the elasticity of soft tissues through measurement of small motions induced into a sample. It shows great promise in the detection of a wide variety of pathologies, especially tumours. An imaging protocol was developed to acquire MR elastography data for use in a clinical setting. A 3D gradient echo sequence was modified to allow for the detection of harmonic motion and tested on silicone phantoms and ex-vivo muscle and brain samples. The time for acquiring a high resolution, quantitative dataset of 3D motions was about 45 minutes. Our imaging method included motion encoding along all three coordinate axes and at several time points along the motion cycle. This time could be easily be reduced by more than half for future clinical use, while still retaining full quantitative data. A modified EPI sequence shows promise for even faster acquisition. The ability to detect the mechanical anisotropy of brain and muscle tissue in ex-vivo samples was also investigated. Initial results from the muscle data indicate a change in shear wavelength is observed for actuation along orthogonal axes. This is a strong indicator of anisotropy detection. Further work needs to be done to improve results from the brain sample as preliminary results are inconclusive.

Doctor of Philosophy(PhD) / Hyperparathyroidism, a disease of the parathyroid glands, is one of the most common endocrinopathies, having a prevalence of 1 – 3 per 1000 individuals. It is characterised by calcium insensitive hypersecretion of parathyroid hormone, and increased cell proliferation. While the treatment for familial as well as many sporadic tumours associated with hyperparathyroidism includes parathyroidectomy, the extent of surgery and the follow-up monitoring regime, are dependent on accurate clinical and histopathological classification of the lesion. However, overlaps in histopathological and morphological features confound distinctions between the three main classifications of adenoma, hyperplasia and carcinoma and differential diagnosis of these lesions remains challenging. At the start of this candidature in January 2002, the genes associated with two familial syndromes in which hyperparathyroidism may feature, Multiple Endocrine Neoplasia (MEN) 1 and 2 had been identified, respectively MEN1 and RET. In addition, overexpression or translocation of cyclin D1 had been identified in both benign and malignant sporadic lesions, indicating a role for cyclin D1 in parathyroid tumorigenesis. However, the underlying events leading either directly, or indirectly, to the development of a large proportion of parathyroid lesions are still largely unknown. The work described in this thesis has contributed to the understanding of parathyroid lesions and the diagnosis and prognosis of affected individuals. During this candidature, constitutive mutation of HRPT2 was associated with Hyperparathyroidism–Jaw Tumour syndrome (HPT-JT). HRPT2 mutation analysis and loss of heterozygosity studies at 1q24-32 in parathyroid tumours presented in this thesis identified the strong association of HRPT2 mutation with sporadic parathyroid malignancy. In addition, 2-hits affecting HRPT2 were identified in several tumours suggestive of a role for HRPT2 as a tumour suppressor gene in sporadic parathyroid tumorigenesis. Microarray analysis of parathyroid tumours presented in this thesis identified three broad clusters of tumours. Cluster 1 comprised predominantly hyperplastic specimens and also included the normal tissue. Cluster 2, the most robust of the clusters, consisted of tumours harbouring HRPT2 mutations. The HPT-JT-associated tumours, both benign and malignant, and sporadic carcinomas, comprised this cluster. Cluster 3 contained the majority of the sporadic adenoma specimens, some hyperplasia, as well as all of the MEN 1-associated tumours. The cluster data is strongly suggestive that parathyroid tumours with somatic HRPT2 mutation, or tumours developing on a background of germline HRPT2 mutation, follow pathways distinct from those involved in mutant MEN 1-related parathyroid tumours. The results of this work provide strong evidence for an adenoma to carcinoma progression model for parathyroid tumorigenesis in the presence of a germline HRPT2 mutation. With the knowledge that both HRPT2 and MEN1 have significant roles in familial as well as sporadic parathyroid tumorigenesis, assays for mutation screening of these two genes have been developed as part of this thesis. These assays will facilitate a rapid molecular diagnosis for patients with one of these familial syndromes. Furthermore, novel putative biomarkers for different parathyroid tumour subtypes have also been identified. VCAM1 and UCHL1 (PGP9.5) were found to be significantly overexpressed in tumours harbouring an HRPT2 mutation at both the transcript and protein level. These two molecules are suggested as putative biomarkers for the discrimination of sporadic carcinoma or HPT-JT-associated tumours. RALDH2 transcript and protein were highly significantly overexpressed in the hyperplasia class relative to the adenoma class, and this molecule is suggested as a putative biomarker for discrimination of these classes of parathyroid tumours. These biomarkers may assist in the accurate diagnosis and prognosis of hyperparathyroidism. Large cohort studies of these putative biomarkers will be required to determine their robustness in discriminating parathyroid tumour subtypes. Further studies of their putative role in parathyroid tumorigenesis may identify them as novel molecular targets for future therapeutics to treat both hyperplastic and neoplastic parathyroid lesions.

Radiotherapy is the use of ionising radiation to induce localised DNA damage to cancerous tissues, leading to cell death and disease control. In order to maximise tumour growth control and to limit damage of the healthy surrounding tissues and the consequent side effects for the patient, molecular determinants of tumour radioresistance are investigated as potential clinical targets. A high-throughput siRNA colony formation assay screen in HeLa cervical carcinoma cells previously published by our laboratory identified modulators of radiosensitivity. From the list CSF1R, EPHB2, GAK and TOPK, were selected and validated. TOPK (T-LAK cell-originated protein kinase, also known as PDZ-binding kinase, PBK) was selected for further investigation because it is overexpressed in most malignancies but not in normal tissues, apart from testis and placenta. Knockdown of TOPK was shown to induce radiosensitisation in a panel of cancer cell lines with no significant effects on normal cells. A role for TOPK in the cell cycle response to ionising radiation (IR) was discovered in HCT116 colorectal cancer cells, with alterations in the G₁/S and G₂/M checkpoints. Furthermore, immunoprecipitation experiments identified a physical interaction between TOPK and CDKN1A (p21) at 8 hours after IR. Apoptosis and the number of multinucleated cells were significantly increased in TOPK depleted cells exposed to IR, suggesting the possibility of aberrant mitosis and mitotic catastrophe in these cells. High TOPK expression in early breast cancer patients was shown to be associated with poor recurrence-free survival. In addition, immunohistochemistry (IHC) analysis on samples from prostate cancer patients identified a strong correlation between high levels of TOPK and poor clinical response to radiotherapy. In order to facilitate future in vivo experiments, an HCT116 shRNA stable knockdown cell line was developed and two commercially available TOPK inhibitors were tested and optimised. Taken together, these data suggest that TOPK is a molecular determinant of radiosensitivity with a great potential for future clinical applications.

Radiation therapy (RT) plays a crucial role in the management of cancer, however, current standards of care have yet to account for patient specific radiation sensitivity. Raman spectroscopy (RS) is a promising technique for radiobiological studies as a way to measure radiation responses in biological samples and could provide a method for monitoring and predicting radiation response in patients. The work in this dissertation gives way to significant advances in the implementation of RS for applications in radiation oncology. Specifically, instrumentation improvements for clinical implementation of RS were achieved through the investigation and development of Raman microfluidic systems. Unique magnesium fluoride based microfluidic systems were engineered and evaluated for applications in radiobiological studies. These systems were found to yield superior spectral quality over traditional microfluidic designs. Furthermore, in order to assert RS as a key technique for clinical monitoring and prediction of radiation responses, human non-small cell lung cancer (NSCLC) and breast adenocarcinoma tumour xenograft models were investigated for Raman signatures of radiation response. These studies found that RS can identify unique and distinct signatures of radiation response in tumours, that can be tracked over time. In particular, NSCLC tumours were found to have key radiation induced modulations in cell cycle and metabolic linked spectral features- including glycogen. Breast adenocarcinoma tumours were found to exhibit distinct fluctuations in spectral features linked to cell cycle as well as protein content. In the case of NSCLC, radiation response signatures were found to be linked to tumour regression and hypoxic status of the tumour- a key factor that dictates radiation resistance in the disease. This work provides the first application of RS to measure radiation response signatures of tumours irradiated \textit{in vivo}. These results show that RS is a versatile technique that can offer insight into radiation induced molecular changes that are unique to the type of cancer and can be monitored over several days following radiation exposure. Together with improved instrumentation for radiobiological studies using microfluidics, the work presented in this dissertation further emphasizes the key role RS can have in radiation oncology and personalization of RT. / Graduate / 2019-08-21

Shock waves have been used in medicine for more than 30 year. At the beginning was mainly use for lithotripsy, but today is also applied in other fields of medicine, such as orthopedics, rheumatology and others. Single shock wave is one shock that usually is repeated every 1-1.5 seconds. By contrast tandem shock waves are two shocks consecutively (ideal interval between shocks is from 8 to 15 microseconds), that are repeated. In this work we investigated the clinical use of single and tandem shock waves that are generated entirely new source. It is based on the principle of multichannel discharge. It was found that a single shock wave can destroy the union between bone and bone cement, this effect could be used in orthopedics. Single and tandem shock wave can damage the tumor in vivo, but the principle damage is different. Tandem shockwave is able to cause damage in a depth of acoustically homogeneous medium and enhances the effect of chemotherapy. It would therefore be possible to used single and tandem shock waves in oncology either alone, or their combination with other chemicals. Functional sample of clinically usable applicator of shock waves with a new source was made for these applications. Powered by TCPDF (www.tcpdf.org)

Splicing é o processo de remoção de introns e ligação de exons em eucariotos. É realizado pelo spliceossomo, um complexo macromolecular composto por RNAs e mais de cem proteínas. Alguns introns possuem microRNAs, os quais devem ser processados para gerar moléculas maduras. O cluster intrônico miR-17-92 é composto por sete miRNAs que têm sido associados ao desenvolvimento de diferentes tumores em vários tecidos. Neste trabalho o splicing de dois miRNAs deste cluster foi analisado em células HeLa, BCPAP e TPC-I. Os resultados mostraram que introns com miR19a tem o splicing mais eficiente do que aqueles com miR18a em todas as três células analisadas. Além disso, a composição dos spliceossomos foi analisada por espectrometria de massas. Entre as principais proteínas encontradas, destaca-se a presença das hnRNPs, como hnRNP_A1 e hnRNP_A2/B1. Estes resultados são importantes para entender como esses miRNAs são processados, e quais são os principais componentes recrutados em diferentes tipos celulares. / Pre-mRNA splicing is the process of intron removal and exon ligation in eukaryotes. It is performed by the spliceosome, a multi-megadalton machinery composed of RNAs and more than a hundred proteins. Intronic miRNAs must be processed from the host gene to generate mature molecules. miR-17-92 is an intronic cluster composed of seven miRNAs which have been associated to the development of different tumors, in several different cells. In this work, we analyzed the splicing of two miRNAs belonging to this cluster in HeLa, BCPAP and TPC-I cells. Interestingly, we observed miR19a is more efficiently spliced than miR18a in all three cells. We also searched for specific proteins that can be involved in their respective splicing process. We observed hnRNP proteins are especially concentrated in spliceosomes assembled in introns containing these miRNAs, based on mass spectrometry data. These results are important to understand how these miRNAs are spliced and matured and also can explain their different expression levels in different cells.

T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer. / Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen.

Cancer related mortality is almost always due to metastatic dissemination of the primary disease. While research into the biological mechanisms that drive the metastatic cascade continues to unravel its molecular underpinnings, progress in our understanding of biological phenomena such as tumour heterogeneity and its relevance to the origins of distant recurrence or the emergence of resistance to therapy has been limited.In parallel to major breakthroughs in the development of high throughput molecular techniques, researchers have begun to utilise next generation sequencing to explore the relationship between primary and matched metastatic tumours in diverse types of neoplasia. Despite small cohort sizes and often, a limited number of matched metastases for each patient, pioneering studies have uncovered hitherto unknown biological processes such as the occurrence of organ specific metastatic lineages, polyclonal seeding and homing of metastatic cells to the primary tumour bed. While yet other studies continue to highlight the potential of genomic analyses, at the time this thesis was started, an in-depth knowledge of disease progression and metastatic dissemination was currently lacking in breast cancers.Herein, we employed phylogenetic inference methods to investigate intra-tumour heterogeneity and evolution of breast cancers. A combination of whole exome sequencing, custom ultra-deep resequencing and copy number profiling were applied to primary tumours and their associated metastases from ten autopsied breast cancer patients. Two modes of metastatic progression were observed. In the majority of cases, all distant metastases clustered on a branch separate from their primary lesion. Clonal frequency analysis of somatic mutations showed that the metastases had a monoclonal origin and descended from a common metastatic precursor. Alternatively, the primary tumour was clustered alongside metastases with early branches leading to distant organs. This dichotomy coincided with the clinical history of the patients whereby multiple seeding events from the primary tumour alongside cascading metastasis-to-metastasis disseminations occurred in treatment naïve de novo metastatic patients, whereas descent from a common metastatic precursor was observed in patients who underwent primary surgery followed by systemic treatment. The data also showed that a distant metastasis can be horizontally cross-seeded and finally revealed a correlation between the extent of somatic point mutations private to the distant lesions and patient overall survival. In an unrelated dataset of relapsed breast cancer patients with matched primary and distant lesions profiled using whole genome sequencing, the landscape of somatic alterations confirmed the time dependency of copy number aberrations implying that cancer phylogenies can be dated using a molecular clock.The work presented here harnesses the strength of high throughput genomic techniques and state of the art phylogenetic tools to tell the evolutionary history of breast cancers. Our results show that the linear and parallel models of metastatic dissemination which have been held as near doctrines for many years are overstated point of views of cancer progression. Beyond the biological insights, these results suggest that surgical excision of the primary tumour in de novo metastatic breast cancers might reduce dissemination in selected cases hence providing a potential biological rationale for this practice. Similarly, there is no strong evidence of benefit in overall survival from surgical resection of oligo-metastases in breast cancer. From our analyses, metastatic lesions constitute an additional source of seeding and heterogeneity in advanced breast cancer. The data presented here is too small to derive practice-changing evidence, but supports the concept that resecting isolated metastases may be of clinical benefit in oligo-metastatic breast cancer patients. In both cases, results from larger prospective studies are warranted. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Abstract The goal of this study was to explore preoperative evaluation of patients with intracranial tumours using magnetic resonance imaging (MRI) methods: oedema-attenuated inversion recovery (EDAIR) sequence in low-field MRI, and diffusion-weighted imaging (DWI) and resting-state functional MRI (fMRI) in high-field MRI. The aim was also to increase our knowledge about the effects of brain surgery on eloquent brain cortices using new MRI techniques. The total number of patients in these studies was 50 (24 women). Enhancement of the tumour in ten patients after intravenous administration of gadolinium-based contrast agent in low-field MRI was examined with a new sequence, EDAIR, and compared with more conventionally used partial saturation spin echo sequences. EDAIR may facilitate the perception of small enhancing lesions and is valuable in low-field imaging, where T1-based contrast is inferior to high-field imaging. DWI was performed on 25 patients in order to evaluate the potential of this imaging method to assist in differential diagnosis of intracranial tumours. It was shown that apparent diffusion coefficient values of the tumour and peritumoural oedema produced by DWI were different in benign and malignant tumours. Resting-state blood oxygen level-dependent (BOLD) fMRI was performed on eight patients and ten healthy volunteers to examine if functional sensorimotor areas in the brain could be determined without any task-related activations. It was shown that intracranial tumours do not appear to hamper visualization of the sensorimotor area in resting-state BOLD fMRI when independent component analysis is performed, and this method may be used in preoperative imaging when activation studies cannot be performed. Conventional BOLD fMRI with motor and auditory stimuli was used with seven patients as the effect of brain surgery was studied. The results suggest that resection of a tumour with preoperative oedema probably decreases pressure on the brain and makes the functional cortex transiently more easily detectable in BOLD fMRI. In conclusion, the MRI imaging methods used in this study can give valuable additional information about the tumour, specifically for preoperative imaging and planning for surgery.

Abstract Aptosis denotes a biochemically and morphologically distinct chain of events leading to self-destruction of cell. It is pivotal in the maintenance of tissue homeostasis and also plays a role in neoplasm. In this work, the extent of apoptosis and apoptosis regulating proteins and factors was studied in a total of 94 patients operated for lung carcinoma, including 56 small cell lung carcinomas (SCLC) and 38 large cell lung carcinomas (LCLC). The extent of apoptosis was determined by detecting and counting the relative and absolute numbers of apoptotic cells and bodies using 3'- end labelling of the apoptotic DNA. The extent of apoptosis in SCLC was compared with the cell proliferation activity as determined by Ki-67 immunohistochemistry, with the volume density of necrosis and with the occurrence of immunohistochemically detectable p53 and bcl-2 proteins. In order to test the hypothesis that increased apoptotic activity is connected with neuroendocrine differentiation and with low differentiation degree in LCLC and that it is regulated by bcl-2 family proteins, the extent of apoptosis and tumour necrosis was analysed in relation to the expression of bcl-2 family proteins bcl-2, mcl-1, bax and bak. Apoptosis, tumour infiltrating lymphocytes (TILs), and angiogenesis are important factors that contribute to tumour growth. In the present study immunohistochemical methods were used to investigate the relationships of these factors and their role in the prognosis of the patients with LCLC and SCLC. A remarkably high apoptotic activity was detected in both SCLC and LCLC. The mean apoptotic index in SCLC was 2.70 % and in LCLC 2.49 %. Exceptionally high proliferation activity and high percentage of tumour necrosis was seen in SCLC. 58 % of SCLC showed more than 40 % of Ki-67 positive nuclei, and tumour necrosis was seen in 83 % of the cases. P53 protein accumulation was detected in 38 % and bcl-2 expression in 50 % of SCLC. The extent of apoptosis in SCLC was inversely related to tumour necrosis and p53 protein accumulation. In LCLC, bcl-2 expression was detected in 40 % of the cases. It was associated with neuroendocrine differentiation and predicted favourable prognosis of the patients. A high number of T cells and macrophages with a small number of B cells was detected in both SCLC and LCLC. The occurrence of intratumoural cytotoxic CD8 cells was associated with the occurrence of apoptotic bodies in SCLC. The increased number of intratumoural T cells, CD8-positive cells and macrophages predicted favourable prognosis of the patients with SCLC. In LCLC, an increased number of B cells and macrophages, but not T cells, was associated with better survival. Iaddition to tumour cells, numerous apoptotic bodies could also be found within alveolar macrophages within and close to tumour tissue. In order to test whether such cells could be found in sputum smears and if their presence could be utilised as a marker of malignancy in tumour diagnosis, the occurrence of alveolar macrophages with apoptotic bodies (AMWABs) was analysed in 84 sputum samples and 13 broncho-alveolar lavage (BAL) specimens from patients with and without lung carcinoma. AMWABs could be found in cytological samples of the patients with lung carcinoma. In sputum and BAL specimens, enhanced apoptosis, as measured by an increased number of AMWABs reflected and was indicative of malignancy. This was also true for cytological specimens of the patients even when the actual malignant cells were not found. Therefore the AMWABs served as a marker of pulmonary malignancy.