La galectine-3 (Gal-3), protéine de type lectine, de 29-35 kDa, étudiée comme marqueur d’aggressivité dans les gliomes, présente des caractéristiques biologiques importantes justifiant son étude dans le domaine du mélanome. En effet, la Gal-3 est une protéine qui peut se lier à la laminine, tout comme l’intégrine α6/β1 dont l’expression est réduite dans le mélanome. L’expression de cette intégrine peut d’ailleurs être modulée par la Gal-3 comme récemment montré dans des lignées cellulaires de cancer du sein (BT-549) et de glioblastome (U373). Le mélanome, véritable problème de santé publique qui est susceptible d’atteindre 1 individu sur 75 dans nos contrées, reste un tumeur mal comprise avec des évolutions parfois incertaines, et des traitements dont l’efficacité est limitée. Le diagnostic histologique du mélanome lui-même peut parfois représenter une difficulté pour le clinicien et l’expert pathologiste ou dermatopathologiste. La couleur (hyperpigmentation d’un lésion pigmentée), dont l’évaluation d’ailleurs reste subjective à défaut de standardisation, ne peut à elle seule signer la malignité d’une lésion pigmentée. Globalement l’évolution d’un patient est prédite par l’indice de Breslow qui traduit en mm l’épaisseur de la tumeur. Si cet indice dépasse 1mm, le risque métastatique augmente, justifiant la réalisation de bilans extensifs de suivi. Ceci dit, certains mélanomes épais peuvent ne pas présenter de caractéristiques d’aggressivité, alors que des mélanomes fins sont parfois mortels. L’identification de marqueurs moléculaires est donc impérative, tant pour développer des stratégies thérapeutiques ciblées, que pour affiner le diagnostic et le pronostic d’un patient. Après avoir mis en évidence par immunohistochimie une expression de Gal-3 par les mélanocytes, nous avons démontré une surexpression de cette protéine par les mélanocytes tumoraux. Nous avons démontré également sur des lésions primitives qu’à l’aggressivité mesurée selon l’indice de Breslow correspondait une diminution de cette surexpression. Cette observation a pu être confirmée par un modèle de greffe orthotopique chez la souris nude. Nous nous somme intéressés par la suite à la détection de la protéine dans le sérum, et nous avons constaté, un taux élevé de Gal-3 dans le sérum de patients en stade métastatique avancé, ce taux élevé pouvant s’expliquer tant par la charge tumorale que par la présence d’une inflammation, d’ailleurs bien connue chez le patient cancéreux en stade avancé. Le rôle antiapoptotique de la Gal-3 nous a alors amené à préciser la valeur prédictive et pronostique de cette protéine. L’hypothèse d’une potentielle action bénéfique sur la réponse immunitaire des patients atteints de mélanome qui ont été vaccinés a été rejetée. La Gal-3 sérique s’est révélée comme facteur de mauvais pronostic chez les patients métastatiques, et une analyse multivariée avec la définition d’une valeur « cut-off » de 10 ng/ml a permis de montrer une valeur pronostique indépendante, supérieure à la S100B et à la CRP.

Humans are believed to have ~20,500 protein-coding genes andmuch effort has over the last years been put into the characterizationand localization of the encoded proteins in order to understand theirfunctions. One such effort is the Human Proteome Resource (HPR)project, started in Sweden 2003 with the aim to generate specificantibodies to each human protein and to use those antibodies toanalyze the human proteome by screening human tissues and cells.The work reported in this thesis deals with structuring of data fromantibody-based proteomics assays, with focus on the importance ofaggregating and presenting data in a way that is easy to apprehend.The goals were to model and build databases for collecting, searchingand analyzing data coming out of the large-scale HPR project and tomake all collected data publicly available. A public website, theHuman Protein Atlas, was developed giving all end-users in thescientific community access to the HPR database with proteinexpression data. In 2008, the Human Protein Atlas was released in its4th version containing more than 6000 antibodies, covering more than25% of the human proteins. All the collected protein expression datais searchable on the public website. End-users can query for proteinsthat show high expression in one tissue and no expression in anotherand possibly find tissue specific biomarkers. Queries can also beconstructed to find proteins with different expression levels in normalvs. cancer tissues. The proteins found by such a query could identifypotential biomarkers for cancer that could be used as diagnosticmarkers and maybe even be involved in cancer therapy in the future.Validation of antibodies is important in order to get reliable resultsfrom different assays. It has been noted that some antibodies arereliable in certain assays but not in others and therefore anotherpublicly available database, the Antibodypedia, has been createdwhere any antibody producer can submit their binders together withthe validation data in order for end users to purchase the bestantibody for their protein target and their intended assay. / QC 20100708

Ionizing radiation induces specific proteins involved in DNA repair, cell death, inflammation and tissue injury. The radiation response of proteins within a uniform prostate cancer (PCa) radiotherapy (RT) population has been studied to a limited extent. In this thesis, the proteomic responses of patients undergoing curative RT for intermediate-risk PCa were determined for a panel of pro-inflammatory cytokines from pre-RT baseline to last treatment (39 vs. 33 fractions depending on whether the cohort received primary or post-prostatectomy RT respectively). Longitudinal proteomic research is feasible at our institution based on the study design presented herein. Interferon (IFN)-g and interleukin (IL)-6 were significantly increased during RT and these changes followed consistent patterns modeled by linear and quadratic functions respectively. Furthermore, acute gastrointestinal (GI) and genitourinary (GU) toxicities were significantly associated with IL-2 and IL-1 levels respectively during RT. Taken together this research supports cytokines as potential biomarkers of normal tissue radiation response.

Ionizing radiation induces specific proteins involved in DNA repair, cell death, inflammation and tissue injury. The radiation response of proteins within a uniform prostate cancer (PCa) radiotherapy (RT) population has been studied to a limited extent. In this thesis, the proteomic responses of patients undergoing curative RT for intermediate-risk PCa were determined for a panel of pro-inflammatory cytokines from pre-RT baseline to last treatment (39 vs. 33 fractions depending on whether the cohort received primary or post-prostatectomy RT respectively). Longitudinal proteomic research is feasible at our institution based on the study design presented herein. Interferon (IFN)-g and interleukin (IL)-6 were significantly increased during RT and these changes followed consistent patterns modeled by linear and quadratic functions respectively. Furthermore, acute gastrointestinal (GI) and genitourinary (GU) toxicities were significantly associated with IL-2 and IL-1 levels respectively during RT. Taken together this research supports cytokines as potential biomarkers of normal tissue radiation response.

This study evaluates a new serum-based biomarker for colorectal cancer (CRC) screening and diagnosis. The biomarker (GTA-446) is a member of hydroxy -polyunsaturated ultra-long chain fatty acids and was found to be reduced in CRC patients compared to CRC-free subjects. Diagnostic test performance characteristics were used to identify the effectiveness of the test. Methods: Serum levels of GTA-446 were measured in 4924 subjects who underwent colonoscopy for any reason, pathology results and clinical data were also collected. Two sets of age-matched control subjects were used; First were the lab controls (number=383) which were serum samples collected from Saskatchewan Disease Control Laboratory along with age and gender data. Second, were the endoscopy controls (number=762) which were obtained from the colonoscopy population after being determined to be cancer-free. Cut-off values were calculated using Receiver Operating Characteristic (ROC) curve. Results: Serum GTA-446 was found to be reduced in 87% of CRC patients. Compared to lab controls, the GTA-446 biomarker has a sensitivity of 87%, specificity of 75%, positive likelihood ratio of 3.6, and negative likelihood ratio of 0.16. Using endoscopy controls to calculate test performance characteristics, the biomarker has a sensitivity of 87%, specificity of 50%, positive likelihood ratio of 1.74, and negative likelihood ratio of 0.24. Also, the level of GTA-446 was found to significantly decline with age (r=-0.20, p<0.01). Conclusion: Serum GTA-446 is a potential biomarker for minimally invasive detection of colorectal cancer that compares favorably to other serum-based biomarkers.

To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analyses of blood or cerebrospinal uid. Currently, diagnosis, measurement of disease progression and response to therapeutic intervention are based on clinical observation, but the rst neuronal dysfunction precede the earliest recognition of symptom by at least 5 - 10 years. A potential diagnostic biomarker is oligomeric alpha-synuclein which in recent papers have reported a signicant quantitative dierence between PD and controls. In this master thesis, a method for measuring oligomeric levels of alpha-synuclein is presented together with a monomeric measuring commercial kit used to measure alpha-synuclein in a preclinical model of PD. A signicant dierence of monomeric levels could be detected between two weeks and four weeks post injection of a vector containing the gene for human alpha-synuclein, no signicant dierence between four and eight weeks was found.

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Zugl.: Düsseldorf, Univ., Diss., 2008

This thesis focuses on methods for quantification by liquid chromatography/mass spectrometry (LC/MS) of specific biomarkers for internal dose of chemicals which induce toxicity through their electrophilic reactivity. In vivo such compounds are short-lived, and could feasibly be measured as their reaction products (adducts) with biomacromolecules. Analysis by MS methods of stable adducts offers the specificity and accuracy required to generate data on internal dose useful in risk estimation. The primary aim was to develop a method for quantification by LC/MS of bulky adducts to serum albumin (SA) from polycyclic aromatic hydrocarbons, using the genotoxic diolepoxide (DE) of benzo[a]pyrene (BP) as a model. A method for analysis of the BPDE adducts to His146 in SA was developed which is robust, easy-to-use, has good reproducibility and which reached a high sensitivity. A method for quantification of BPDE adducts to N2-deoxyguanosine (dG) in DNA by LC/MS was also established. In mice exposed to BP, adducts to SA and DNA from stereoisomers of BPDE were identified and quantified. The adduct level was shown to be &gt;400 times higher in DNA than in SA, which from an in vitro study could be concluded to mainly depend on a large difference in the rates of adduct formation to His in SA and to dG in DNA. BPDE adduct levels to SA and DNA, and a biomarker of genotoxic effect (frequency of micronuclei), were compared in BP-exposed mice. The results were used to evaluate how these methods could be used in procedures for cancer risk estimation. An LC/MS method for analysis of valine hydantoins (VH) formed as adducts from isocyanates to N-termini in haemoglobin was established. VH, formed from urea/isocyanic acid, was investigated in mice as a potential biomarker of renal failure and for dose adjustment during treatment with a radioactive cytostatic drug. The kidney dysfunction was not severe enough to give a significant increase of VH in the experiment. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>