Background: Genetic and environmental factors have been associated with colorectal cancer (CRC) risk. However, their association with prognosis has been less studied. Methods: Path analysis was employed to examine causal pathways from education to environmental (diet, alcohol, smoking, physical activity) and personal factors (screening), and then to obesity and ultimately to tumour characteristics (stage, grade, microsatellite instability (MSI), and site) that are associated with CRC prognosis. Data came from the Ontario Familial Colon Cancer Registry. Pathways were evaluated for effect modification by sex and two indicators of CRC genetic susceptibility (Bethesda criteria and newly identified familial cancer clusters). Results: Four food patterns (healthy foods, high-fat foods, sweet and processed foods, and oriental foods) and four nutrient patterns (total macronutrient, fat vs. carbohydrate, and micronutrients from supplements and from foods) were identified. Education was associated positively with healthy lifestyle factors (e.g. healthy foods factor) and negatively with unhealthy factors (e.g. smoking). As expected, high body mass index (BMI) was associated with lower physical activity and higher fat vs. carbohydrate factor. Unexpectedly, BMI was positively associated with the healthy foods factor among Bethesda positive patients and men. An association between education and BMI was mediated by the healthy foods factor and by physical activity. Important poor prognostic factors, higher grade and stage, were associated with smoking and not being screened. However, unexpected associations included a positive association of physical activity with tumour grade among Bethesda positive patients and a positive association of healthy foods with stage among Bethesda negative patients. Patients with right-sided tumours were more likely to receive micronutrients from supplements, and screening and less likely to smoke, and for men, to have a high BMI, high fat diet and healthy food diet. Conclusion: Some unhealthy lifestyle factors, such as smoking and a high fat food dietary pattern, are associated with adverse CRC tumour characteristics and so may affect the prognosis. Family history may modify some associations though the findings require independent confirmation.

The work presented herein is a method optimization for biomolecule detection and identification using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI). MALDI-MSI is a unique form of mass spectrometry that is highly multiplexed; it can simultaneously retain location information of the mass of multiple ions, allowing for correlation of morphology or pathology to reconstructed ion heat maps. There were three main objectives for the research - 1) A method optimization of sample preparation techniques for bottom-up proteomic MALDI-MSI was performed. This included the optimization of tissue wash steps, trypsin digestion incubation times, and matrix deposition techniques. The results included identifying the appropriate pH for the wash steps to optimize trypsin digestion, an overnight trypsin incubation to allow for complete digestion, and the inclusion of MCAEF – Matrix Coating Assisted by an Electric Field – during matrix coating for enhanced spectra. 2) An unbiased statistical data processing workflow for simultaneous processing of multiple datasets was performed. This was done using a thyroid hormone treated tadpole dataset to gain insight into the metabolism of anuran metamorphosis. Results found included a finalized data processing workflow that detected 5000 metabolite features from five organs were detected in pre-metamorphic tadpoles. Of these detected metabolites, 136 were significantly affected upon exposure to thyroid hormone and 64 metabolites were putatively identified. 3) A sample preparation technique for metabolomic analysis of formalin-fixed paraffin embedded (FFPE) colorectal liver metastasis samples was performed. Results included the importance of using a high mass resolution mass spectrometer while emphasizing more appropriate use of fresh-frozen tissue sections for metabolomic analysis. / Graduate

Nano-DESI Mass spectrometry imaging of eicosanoids in mouse kidney tissue using selected ion monitoring. Nano-DESI mass spectrometry imaging (MSI) is a technique for label-free spatial and molecular characterization of surfaces and biological samples. Eicosanoids are lipid mediators derived from eicosapolyenoic acid- products of arachidonic acid oxidation. Eicosanoids have been of interest to the medical field for many years. Major focus on this lipid class came from the development of nonsteroidal anti-inflammatory drugs (NSAIDs), some of these including aspirin, naproxen, ibuprofen, and acetaminophen work by blocking either the formation or the effects of eicosanoids. These lipids also play important roles in various body functions (cardiovascular, renal, gastrointestinal, neuronal) and as mediators of inflammation, asthma, fever, pain, hypertension, and stroke. Typically, eicosanoids occur in subnanomolar concentrations, despite their high level of bioactivity, which makes them significantly more difficult to analyze via direct mass spectrometry. Here, selected ion monitoring (SIM) is used to increase the signal-to-noise of the identified eicosanoids compared to a broadband full scan mode.

Immune checkpoint blockade (ICB) shows remarkable clinical effects in patients with metastatic microsatellite-unstable (MSI) cancer. However, markers identifying potential non-responders are missing. We examined the prevalence of Beta-2-microglobulin (B2M) mutations, a common immune evasion mechanism, in stage IV MSI gastrointestinal cancer and its influence on metastatic pattern and patients’ survival under ICB. Twentyfive patients with metastatic, MSI gastrointestinal adenocarcinoma were included. Eighteen patients received ICB with pembrolizumab and one patient with nivolumab/ ipilimumab. Sequencing was performed to determine B2M mutation status. B2M mutations and loss of B2M expression were detected in 6 out of 25 stage IV MSI cancers. B2M mutations were strongly associated with exclusively peritoneal/peritoneal and lymph node metastases (p=0.0055). However, no significant differences in therapy response (25% vs. 46.6%, p>0.99) and survival (median PFS: 19.5 vs 33.0 months, p=0.74; median OS 39 months vs. not reached, p>0.99) were observed between B2Mmutant and B2M-wild type tumor patients. Among metastatic MSI GI cancers, B2Mmutant tumors represent a biologically distinct disease with distinct metastatic patterns. To assess ICB response in B2M-mutant MSI cancer patients, future studies need to account for the fact that baseline survival of patients with B2M-mutant MSI cancer may be longer than of patients with B2M-wild type MSI cancer.

Le soutien des militants italiens au combat « Algérie française » puis OAS entraîne, à partir de 1960, la création de réseaux franco-italiens d’extrême droite. Les solidarités internationales qui se constituent à la faveur du combat OAS, les contacts et circulations militantes, l’émergence d’une pensée d’extrême droite qui place ses objectifs au-delà du cadre strictement national, l’apparition de combats communs liés au processus de décolonisation et des échanges intellectuels inédits contribuent à mettre en place des réseaux d’extrême droite initialement fondés sur des relations personnelles. L’institutionnalisation progressive de ces contacts, la difficile reconversion des activistes OAS en exil en Europe, leurs relations troubles avec certains services de renseignements déterminés à contrer l’avancée socialiste dans le Tiers-Monde entraînent la formation de réseaux européens qui lient notamment groupes français et italiens d’extrême droite. Au-delà des renouvellements générationnels qui affectent la période, ces relations étroites permettent aux terroristes italiens d’extrême droite qui prennent part à la « stratégie de la tension », de 1969 à 1982, de bénéficier du soutien constant de leurs camerati français. Parallèlement, le Movimento sociale italiano (MSI) entretient avec la nébuleuse française d’extrême droite, entre 1960 et 1984, des relations constantes et exerce sur elle une influence certaine qui n’exclut toutefois pas certaines formes de réciprocités en matière de circulations politiques et culturelles et d’échanges militants. / The support of Italian activists in the “Algérie française” fight, and then with the OAS, gave birth to French-Italian far-right networks from 1960. International alliances that favoured the OAS struggle were formed, contacts and militants were exchanging ideas, the emergence of a far-right way of thinking whose goals went beyond strict national frameworks, a common political outlook on the issue of decolonisation, and new intellectual debates contributed to the growth of far-right networks that were initially based on personal relationships. The gradual institutionalisation of these contacts, the difficult integration of OAS activists in exile in Europe, and their ambiguous relationships with intelligence services determined to counter the socialist movement in the Third World brought about the creation of European networks that united French and Italian far-right groups. Beyond generational changes during this period, these close relationships enabled Italian far-right terrorists who were involved in the « Strategy of Tension », from 1969 to 1982, to benefit from the consistent support of their French camerati. In parallel, the Movimento sociale italiano (MSI) maintained consistent relationships with French far-right cells between 1960 and 1984. Whilst they exerted a considerable amount of influence, they maintained reciprocal alliances with regards to their political and cultural agenda, and continued to exchange militant forces.

Colorectal carcinoma (CRC) is one of the most prevalent malignancies in the Czech Republic. In general, there are two molecular pathways leading to CRC: one is characterized by chromosomal instability, the other by the deficiency in DNA mismatch repair (MMR) genes. MutL homologue 1 (MLH1) gene, a member of the MMR gene-family, represents a key component of the MMR system, responsible for recognition of nucleotide mismatches occurring during DNA replication, and for the recruitment of repair proteins to correct the replication errors. According to literature, somatic mutations in MMR genes, and MLH1 in particular, hallmark sporadic, MMR deficient, CRC cases. We aimed at analyzing somatic events in MLH1 gene and the determination of microsatellite instability (MSI) status in 99 DNA samples from 96 patients with sporadic CRC. Mutations were screened by high resolution melting (HRM) curve analysis. Positive cases in each run were subsequently verified by automated sequencing. Mainly gene variants were found in MLH1 gene: We discovered two new variants, one in exon 2 at position c. 204 C>G, p. Ile68Met (98 C/C, 1C/G) and the other in exon 11 at position c. 973 C>T, p. Arg325Trp (98 C/C, 1 C/T). Only the latter variant c. 973 C>T was identified as somatic mutation. All other variants found in MLH1 gene...

No description available.

Metabolomics is the study of metabolites and metabolic processes. Due to the diversity of structures and polarities of metabolites, no single analytical technique is able to measure the entire metabolome - instead a varied set of experimental designs and instrumental technologies are used to measure specific portions. This has led to the development of many distinct data analysis and processing methods and software. There is hope that metabolomics can be utilized for clinical applications, in toxicology and to measure the exposome. However, for these applications to be realised data must be high quality, sufficiently standardised and annotated, and FAIR (Findable, Accessible, Interoperable and Reproducible). For this purpose, it is also important that standardised, FAIR software workflows are available. There has also recently been much concern over the reproducibility of scientific research, which FAIR and open data, and workflows can help to address. To this end, this thesis aims to assess current practices and standards of sharing data within the field of metabolomics, using metascientific approaches. The types of functions of software for processing and analysing metabolomics data is also assessed. Reporting standards are designed to ensure that the minimum information required to un- derstand and interpret the results of analysis are reported. However, poor reporting standards are ignored and not complied with. Compliance to the biological context Metabolomics Standards Initiative (MSI) guidelines was examined, in order to investigate their timeliness. The state of open data within the metabolomics community was examined by investigating how much publicly available metabolomics data there is and where has it been deposited. To explore whether journal data sharing policies are driving open metabolomics data, which journals publish articles that have their underlying data made open was also examined. However, open data alone is not inherently useful: if data is incomplete, lacking in quality or missing crucial metadata, it is not valuable. Conversely, if data are reused, this can demonstrate the worth of public data archiving. Levels of reuse of public metabolomics data were therefore examined. With greater than 250 software tools specific for metabolomics, practitioners are faced with a daunting task to select the best tools for data collection and analysis. To help educate researchers about what software is available, a taxonomy of metabolomics software tools and a GitHub pages wiki, which provides extensive details about all included software, have been developed.

Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) is the most common hereditary syndrome predisposing to colorectal cancer, accounting for 1-3% of all colorectal cancer. This multi-organ cancer predisposition syndrome is caused by mutations in the mismatch repair (MMR) genes, especially MLH1 and MSH2, and to lesser extents MSH6 and PMS2, which lead to widespread genetic instability and thus microsatellite instability (MSI). Hereditary cancer often manifests in two or more tumours in a single individual; 35-40% of Lynch syndrome patients have synchronous or metachronous tumours of the two major Lynch syndrome-related cancers: colorectal and endometrial. The main purposes of the work underlying this thesis were to identify persons at risk of Lynch syndrome or other types of hereditary colorectal cancer, to estimate the cancer risks associated with these predispositions and to identify the underlying genetic causes. A population-based cohort of 78 persons with double primary colorectal or colorectal and endometrial cancer was identified. Cancer risks in their 649 first-degree relatives were estimated in relation to tumour MSI status (positive or negative) and age at diagnosis (before or after 50 years of age) in the probands. The overall standardised incidence ratio was 1.69 (95% CI; 1.39-2.03). The highest risks for Lynch syndrome-associated cancers: (colorectal, endometrial, ovarian and gastric) were found in families with young MSI-positive probands, likely representing Lynch syndrome families. Importantly, no overall risk was found in families with old probands, irrespective of MSI status. Blood samples were available from 24 MSI-positive patients for mutation screening of MLH1, MSH2 and MSH6. Sequence variants or rearrangements predicted to affect protein function were found in 16 patients. Six novel variants were found: two large rearrangements, two truncating and two missense mutations. The missense mutations were found to segregate in the families. Studies of allele frequencies, MSI and loss of immunostaning in tumours from family members further supports the hypothesis that these missense changes play a role in Lynch syndrome, as do the non-conservative nature and evolutionary conservation of the amino acid exchanges. Five families had mutations in MLH1, five in MSH2, and six in MSH6. The unexpectedly large impact of MSH6 was in genealogical studies shown to be due to a founder effect. Cumulative risk studies showed that the MSH6 families, despite their late age of onset, have a high lifetime risk for all Lynch syndrome-related cancers, significantly higher in women (89% by age 80 years) than in men (69%). The gender differences are in part due to high endometrial (70%) and ovarian cancer risk (33%) in addition to the high colorectal cancer risk (60%). These findings are of great importance for counselling and surveillance of families with MSH6 mutations. Finally, in a large family with MSI-negative hereditary colorectal cancer for which the MMR genes and APC had been excluded as possible causes, a genome-wide linkage analysis was performed, resulting in a suggested linkage to chromosome 7. Conclusions: Relatives of probands with MSI-positive, double primary colorectal and endometrial cancer diagnosed before the age of 50 years have significantly increased risks of Lynch syndrome-related cancers. MSH6 mutations, which have unusually high impact in this study population due to a founder effect, confer high cumulative risks of cancer despite the generally late age of onset.

Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry.  Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.