New affinity molecules such as nucleic acid aptamers are in demand in the science and medical fields. Current aptamer selection technologies can generate unique aptamers with desired properties to targets of interest. My thesis describes a series of investigations on the protection of an oncolytic virus, the isolation of target cells from biological fluids, and aptamer-facilitated biomarker discovery. We tested individual aptamers and constructed a tetramer aptamer structure (quadramer) to increase virus infectivity. The quadramer protects vesicular stomatitis virus (VSV) during freeze–thaw cycles, shields the virus from neutralizing antibodies and increases viral active units. In addition to aptamers, we screened carbohydrate-based ice recrystallization inhibitors for the possible elimination of the cold chain of Vaccinia virus, VSV, and Herpes virus-1. N-octyl-gluconamide provides the longest shelf life for Vaccinia virus and Herpes virus-1 as tested according to the World Health Organization’s requirements for viral vaccines efficiency during transportation and distribution. We generated switchable aptamers capable of isolating cells expressing LIFR, NRP1, DLL4, uPAR, or PTCH1. These aptamers bind to the receptor positive cells in the presence of Mg2+ and Ca2+, and release the pure cells upon addition of EDTA. The aptamers were applied for a sequential positive immunomagnetic isolation of cells from mice bone marrow. We also utilized fluorescence-activated cell sorting (FACS) in our aptamer selections to develop switchable aptamers to positive isolation of monocytes from human blood. Moreover, we have selected non-switchable aptamers as an affinity probe to the cells expressing Axl receptor for immunofluorescent analysis and cell sorting. We determined aptamers to CD107a and applied them for biomarker discovery with mass spectrometry and found that CD107a was co-expressing with PD-1. Furthermore, we identified CD91 as binding partners to our aptamers in human monocytes using FACS and orbitrap mass spectrometry.

abstract: Currently in the US, many patients with cancer do not benefit from the population-based screening, due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and get diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies is a promising class of molecules that have potential to serve as early detection biomarkers for cancers. This Ph.D thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancer, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformation on protein array. This method adopted a novel protein tag technology, called HaloTag, to covalently immobilize proteins on glass slide surface. The covalent attachment allowed these proteins to endure harsh treatment without getting dissociated from slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to significantly increase signal to noise ratio of protein array based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases / 145 controls / 70 non-basal breast cancer) by ELISA, a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. Similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed-tomography positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA level in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights on the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

abstract: Alzheimer's Disease (AD) is a progressive neurodegenerative disease accounting for 50-80% of dementia cases in the country. This disease is characterized by the deposition of extracellular plaques occurring in regions of the brain important for cognitive function. A primary component of these plaques is the amyloid-beta protein. While a natively unfolded protein, amyloid-beta can misfold and aggregate generating a variety of different species including numerous different soluble oligomeric species some of which are precursors to the neurofibrillary plaques. Various of the soluble amyloid-beta oligomeric species have been shown to be toxic to cells and their presence may correlate with progression of AD. Current treatment options target the dementia symptoms, but there is no effective cure or alternative to delay the progression of the disease once it occurs. Amyloid-beta aggregates show up many years before symptoms develop, so detection of various amyloid-beta aggregate species has great promise as an early biomarker for AD. Therefore reagents that can selectively identify key early oligomeric amyloid-beta species have value both as potential diagnostics for early detection of AD and as well as therapeutics that selectively target only the toxic amyloid-beta aggregate species. Earlier work in the lab includes development of several different single chain antibody fragments (scFvs) against different oligomeric amyloid-beta species. This includes isolation of C6 scFv against human AD brain derived oligomeric amyloid-beta (Kasturirangan et al., 2013). This thesis furthers research in this direction by improving the yields and investigating the specificity of modified C6 scFv as a diagnostic for AD. It is motivated by experiments reporting low yields of the C6 scFv. We also used the C6T scFv to characterize the variation in concentration of this particular oligomeric amyloid-beta species with age in a triple transgenic AD mouse model. We also show that C6T can be used to differentiate between post-mortem human AD, Parkinson's disease (PD) and healthy human brain samples. These results indicate that C6T has potential value as a diagnostic tool for early detection of AD. / Dissertation/Thesis / M.S. Biological Design 2013

Introduction The prognosis in oesophago-gastric cancer is poor with less than 15% patients surviving beyond 5 years after diagnosis. The addition of neoadjuvant therapy has been shown to increase survival in patients suitable for curative surgery. However, the additional gains are modest and the majority of patients do not respond sufficiently from therapy to gain any benefit. There is an urgent need to identify markers that can predict response to neoadjuvant therapy in order provide safer, more effective, individualised treatment regimes. Methods A prospective, multi-centre, collaborative study was undertaken in patients with oesophago-gastric cancer undergoing neoadjuvant therapy and potentially curative surgery. Levels of circulating biomarkers M2-Pyruvate kinase, alkaline phosphatase, CA19-9, CEA and CA 72-4 were measured in patients before and after administering the first cycle of chemotherapy. Binary logistic regression analysis was performed to assess the ability of biomarkers to predict histological response to therapy. Results 165 patients were recruited to the main study. 105 patients had complete histopathological data for analysis. There were 27 responders and 78 non-responders to neoadjuvant therapy. There were no differences in pre-therapy demographic, pathological or treatment factors between the two groups. Responders had less post-operative lymphovascular invasion (P= 0.004) and higher R0 resection rates (P=0.03). Pre-therapy M2-Pyruvate kinase levels were lower in responders compared to non-responders (P=0.037) and levels were able to predict response with each unit increase in the biomarker level being associated with a 4.1% decrease in the likelihood of response (P=0.027). M2-PK levels were not associated with any pre-operative demographic, clinical or pathological factors. Conclusions Pre-therapy dimeric M2-PK levels can predict response to neoadjuvant therapy in patients with oesophago-gastric cancer. The test could be of clinical value for 1 in every 8 patients undergoing the test.

>Magister Scientiae - MSc / Prostate Cancer is the leading cause of cancer-related death in males in the Western world. It is a common biological disease originating from the reproductive system of the male namely, the prostate gland, usually in older patients (over the age of 50) and with a family history of this disease. The disease shows clinical aggressiveness due to genetic alterations of gene expression in prostate epithelial cells. Prostate cancer is currently diagnosed by biopsy and prostate cancer screening via the Prostate-Specific Antigen (PSA) blood test. Early detection is critical and although PSA was discovered to aid in the diagnoses of this cancer at its early stages, it has a disadvantage due to its low specificity thus causing unnecessary biopsies of healthy individuals and overtreatment of patients. Although various studies and efforts have been made to identify the ideal biomarker for prostate cancer and many even being applied to clinical use, it is still challenging and has not replaced the best-known biomarker PSA. PSA test has minimal invasive characteristics, at relatively low cost together with high sensitivity but low specificity. Biomarker discovery is a challenging process and a good biomarker has to be sensitive, specific and its test highly standardized and reproducible as well as identify risk for or diagnose a disease, assess disease severity or progression, predict prognosis or guide treatment. Computational biology plays a significant role in the discovery of new biomarkers, the analyses of disease states and the validation of potential biomarkers. Bioinformatic approaches are effective for the detection of potential micro ribonucleic acid (miRNA) in cancer. Altered miRNA expression may serve as a biomarker for cancer diagnosis and treatment. Small non-protein coding RNA, miRNA are small regulatory RNA molecules that modulate the expression of their target genes. miRNAs influence numerous cancer-relevant processes such as proliferation, cell cycle control, apoptosis, differentiation, migration and metabolism. Discovery and existence of extracellular miRNAs that circulate in the blood of cancer patients has raised the possibility that miRNAs may serve as novel diagnostic markers. Since a single miRNA is said to be able to target several mRNAs, aberrant miRNA expression is capable of disrupting the expression of several mRNAs and proteins. Biomarker discovery for prostate cancer of mRNA and miRNA expression are strongly needed to enable more accurate detection of prostate cancer, improve prediction of tumour aggressiveness and facilitate diagnosis. The aim of this project was to focus on functional analyses of genes and their protein products regulated by previously identified miRNA in prostate cancer using bioinformatics as a tool. Most proteins function in collaboration with other proteins and therefore this study further aims to identify these protein-protein interactions and the biological relevance of these interactions as it relates to Prostate cancer. Various computational databases were used such as STRING, DAVID and GeneHub-GEPIS for functional analyses of these miRNA regulated genes. The main focus was on the 21 genes regulated by several miRNAs identified in a previous study. Results from this study identified six genes; ERP44, GP1BA, IFNG, SEPT2, TNFRSF13C and TNFSF4, as possible diagnostic biomarkers for prostate cancer. These results are promising, since the targeted biomarkers would be easily detectable in bodily fluids with the Gene Ontology (GO) analysis of these gene products showing enrichment for cell surface expression. The six genes identified in silico were associated to transcription factors (TFs) to confirm regulatory control of these TFs in cancer promoting processes and more specifically prostate cancer. The CREB, E2F, Nkx3-1 and p53 TFs were discovered to be linked to the genes IFNG, GP1BA, SEPT2 and TNFRSF13C respectively. The expression of these TFs show strong association with cancer and cancer related pathways specifically prostate cancer and thus demonstrates that these genes can be assessed as possible biomarkers for prostate cancer. The prognostic and predictive values of the candidate genes were evaluated to assess their relationship to prognosis of this disease by means of several in silico prognostic databases. The results revealed expression differences for the majority of the candidate genes were not significantly sufficient to be distinguished as strong prognostic biomarkers in several prostate cancer populations. Although one marker, GP1BA was supported as having prognostic value for prostate cancer based on it's statistical pvalue in one of the prostate cancer patient datasets used. Another candidate gene SEPT2 showed promise as it has some prognostic value in the early stages of the disease. Although the results yielded, based on the in silico analysis, were not the discovery of an ideal diagnostic marker based on the set criteria in this study, further analysis using a molecular approach qRT-PCR can be considered for a detailed followup study on selected candidate genes to evaluate their roles in disease initiation and progression of prostate cancer using cell lines as well as patient samples. / CSIR

Les encéphalopathies spongiformes subaigües transmissibles (ESST) sont des maladies neurodégénératives affectant l'Homme et les animaux, l'issue est toujours fatale. La détection dans le sang de l'agent pathogène responsable de l'infection (protéine prion pathologique)reste difficile à ce jour et l'identification de nouveaux biomarqueurs impliqués dans la physiopathologie des ESST constitue un projet ambitieux et risqué. Dans ce contexte, notre objectif est d'établir un profil protéique corrélé aux ESST. L'utilisation d'un modèle animalbien caractérisé : la tremblante naturelle du mouton, d'une technologie adaptée à l'analyse de profils protéiques : SELDI-TOF MS et d'un fluide biologique : le sérum, a constitué la base de nos travaux de recherche. Dans un premier temps, les protocoles expérimentaux ont été mis en place et optimisés. Puis, ils ont été évalués pour leur pertinence dans la discrimination de moutons pathologiques en phases précoce et tardive de la maladie versus des moutons contrôles par analyse des sérums fractionnés ou non. Des biomarqueurs potentiels de faibles poids moléculaires ont été sélectionnés à l'aide de la méthode statistique SAM et une signature protéique permettant un diagnostic précoce a été établie (87% de sensibilité et 90%de spécificité). Un des biomarqueurs a été identifié comme étant un fragment de la transthyrétine, puis son potentiel discriminant a été évalué en SELDI-TOF MS dans une étude cinétique de hamsters Syriens infectés par la scrapie, en western blot et par dosage ELISA.Finalement, une cohorte de validation constituée de moutons appelés « scrapie-free » a permis de valider les biomarqueurs les plus pertinents. / Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseasesoccurring in animals and humans for which no ante-mortem diagnostic test in biological fluidsis available. In such pathologies, detection of the pathological form of the prion protein (i.e.,the causative factor) in blood is difficult. Identification of new biomarkers implicated in thepathway of prion infection is relevant. In this context, our objective was to find a proteicprofile correlated to TSEs. We used a well-known TSE model: scrapie in sheep breeding, amass spectrometry technology easy-to-use for proteic profiling: SELDI-TOF MS and abiological fluid: serum. First, experimental tools have been developed and optimized. Thesetools were evaluated for their discriminating potential of control sheep and animals with earlyor late phase scrapie using a large number of serum samples (fractionated or not). Then, usingthe SAM statistical method, potential low molecular weight biomarkers were selected. Amongthese biomarkers, a protein signature pattern was identified; it can discriminate between earlyphase scrapie and control sera (sensitivity of 87% and specificity of 90%). One of theseproteins was identified as a fragment of transthyretin and evaluated as a biomarker using aSELDI-TOF MS kinetic study of sera from scrapie infected Syrian hamsters. This biomarkerwas also confirmed by western blot analysis and ELISA quantitation. Finally, a cohort of freescrapiesheep permits to validate the diagnostic potential of the candidate biomarkers.

Les hypothèses retraçant les mécanismes physiopathologiques de la maladie de Behçet (MB), une vascularite inflammatoire non liée aux anticorps anti-cytoplasme des polynucléaires neutrophiles (ANCA), sont multiples. Cette étude propose une compilation exhaustive des mécanismes immunopathologiques décrits dans la littérature contemporaine, et fournit un résumé détaillé des aspects cliniques de la maladie et de ses différents traitements. Cette étude inclut également une analyse statistique de 20 signatures de protéines proposées comme biomarqueurs potentiels de la MB. Vingt-deux patients avec une MB active (MBA) et 46 patients avec une MB inactive (MBI), répondant aux critères de l’International Criteria for Behçet Disease (2013), ainsi que 47 donneurs sains (DR) et 98 patients subissant une angiographie coronaire (AC) ont fourni des échantillons de sérums pour une étude de dosage multiplex. Les résultats indiquent que les protéines sériques ICAM-1, SAA, THBD, et VCAM-1 jouent un rôle essentiel dans la différenciation entre les patients MB et les DR. De même, Caldesmon, Clusterin, CRP, IL-8, SELP et SICMA3 permettent un tri entre les patients MB et AC. Les modèles de signatures des biomarqueurs proposés dans cette étude et qui séparent entre les patients atteints par la MB, les DR et / ou les AC, représentent une nouvelle piste pour le développement de tests sériques pour la MB, avec une sensibilité et une spécificité élevées. Ceci peut éventuellement compléter les outils de diagnostic clinique établis. Ces résultats apportent une contribution significative à l’interprétation actuelle de la pathogénie de la MB en tant que vascularite auto-immune non-ANCA. Cette enquête fournit un bilan à la fois qualitatif et quantitatif aux cliniciens et aux chercheurs dans ce domaine. / Hypotheses concerning the specific pathophysiological mechanisms of Behçet’s Disease (BD), a non-antineutrophil cytoplasmic antibodies (ANCA) inflammatory vasculitis, are numerous. This study offers an exhaustive review of the disease in an attempt to recap the immunopathological pathways described by extant literature, and provides a detailed summary of the clinical aspects of, and treatment options for the disease. In addition, this investigation completed a statistical analysis of 20 protein signatures that were proposed as potential biomarkers for BD. Twenty-two patients with active BD (BDA) and 46 patients with inactive BD (BDI) fulfilling the International Criteria for Behçet's Disease, 47 healthy donors (HD), and 98 coronary angiography patients (CA) provided serum samples for a multiplex assay study. Findings indicate that serum proteins ICAM-1, SAA, THBD, and VCAM-1 play a significant role in differentiating BD patients from HD. Likewise, Caldesmon, Clusterin, CRP, IL-8, SELP, and sICAM-3 segregate between BD and CA. The biomarker predictive models proposed in this study that segregate between BD, HD, and / or CA represent a significant avenue for the development of sera testing specific to BD with a high level of sensitivity and specificity. This may serve as a supplement to established clinical diagnostic tools. These results represent a noteworthy complement to the current interpretation of the pathogenesis of BD as an autoimmune non-ANCA vasculitis. This investigation provides expert clinicians and researchers with both qualitative and quantitative outcomes.

Le but de ce travail était de rechercher le rôle du microenvironnement dans la progression tumorale du cholangiocarcinome intrahépatique (CCIH) par une approche translationnelle, associant recherche fondamentale et clinique. Une étude transcriptomique du stroma tumoral a permis de mettre en évidence une signature spécifique de celui-ci, dont l’analyse non supervisée montrait un enrichissement dans les gènes de la matrice extracellulaire, du cycle cellulaire, de la voie TGFβ et des marqueurs de cellules souches. Ces résultats ont été validés au niveau protéique par immunohistochimie sur tissue microarrays à partir d’une cohorte indépendante. La corrélation de ces résultats avec les données cliniques a permis de démontrer que le niveau d’expression de l’Osteopontin dans le stroma était un facteur de risque indépendant de récidive et de survie. Par ailleurs, nous avons démontré que le taux sérique d’Osteopontin préopératoire des patients porteurs d’un CCIH était significativement supérieur à celui de sujets sains. Avec un seuil déterminé à 57,8 ng/ml, la sensibilité et spécificité de ce biomarqueur diagnostique était respectivement de 80 et 100%. De plus, nous avons apporté des arguments supplémentaires concernant le rôle des cellules souches cancéreuses dans la progression du CCIH, en mettant en évidence une corrélation entre le niveau d’expression de marqueurs souches tels qu’EpCAM et CD44 dans le stroma tumoral ainsi que dans le tissu fibreux du foie « sain » péri-lésionnel et le risque de récidive. Les résultats de notre étude ont confirmé le rôle central du microenvironnement dans la progression du CCIH, permis de mettre en évidence 2 nouveaux biomarqueurs pronostiques, et ouvert de nouvelles voies de recherche thérapeutiques. / The aim of this study was to specifically determine through a translational approach combining basic and clinical research, the role of the microenvironment in the tumor progression of intrahepatic cholangiocarcinoma (ICC). By gene expression profiling, we identified a signature that significantly discriminate the tumor stroma from non-tumor fibrous tissue, and the functional analysis of differentially expressed genes showed an enrichment in genes of the extracellular matrix , the cell cycle, the TGFb pathway and stem cell markers. Tissue microarray analysis using an independent cohort of ICC patients validated at a protein level the increased expression of selected candidate genes. Statistical analysis between basic and clinical data demonstrated that the stromal expression of Osteopontin was an independent prognostic marker for overall and disease-free survival. We also demonstrated that the preoperative serum level of Osteopontin was significantly higher in ICC patients than in healthy subjects. Our results identified the best diagnostic threshold to 57,8 ng/ml, associated with a sensitivity and specificity reaching to 80 and 100%, respectively. Moreover, we showed that level expression of stem cell markers such as EpCAM and CD44 in tumor stroma as well as in the fibrous non tumor liver tissue was correlated with recurrence, suggesting the pivotal role of cancer stem cells in ICC prognosis. In conclusion, our study confirmed the major involvement of the microenvironment in the progression of CCIH, allowed to identify two new prognostic tumor biomarkers, and highlighted new pathways for targeted therapeutics.

Drug induced liver injury is a leading cause of human illness and a major cause of drug withdrawals from the market. A systems biology approach has the potential to aid toxicology research since toxicological responses are a consequence of multiple non-linear and interdependent biological responses. Here such an approach is developed.The glutathione pathway is a key hepatic defence mechanism and deactivates reactive metabolites before they have the chance to damage cellular proteins. However, glutathione availability is limited and can vary between individuals. As hepatic glutathione levels cannot be measured directly, two serum-based biomarkers, i.e. 5-oxoproline and ophthalmic acid, have been proposed in literature as a means of tracking glutathione depletion. This thesis aims to test the reliability of the correlation between biomarker concentration and decreasing glutathione concentration.In this study a spiral between experiments, model predictions and falsifications, model improvement, and experimental design is described. Using this approach a kinetic model of the hepatic glutathione pathway and biomarker metabolism was constructed and subsequently expanded by adding physiologically based pharmacokinetic (PBPK) models of paracetamol and the proposed biomarkers. These models have increased the understanding of the glutathione pathway. For example, the model predicted that Glutamyl-Cysteine Synthetase induction should be a highly effective way to increase the robustness of the liver to a paracetamol challenge.In addition, it was possible to qualify with increasing precision, the correlation between biomarkers and hepatic glutathione depletion. 5-Oxoproline and ophthalmic acid provide different information about the status of the glutathione pathway. 5-Oxoproline is correlated with paracetamol-glutathione conjugate formation, but not with extreme toxicity. Ophthalmic acid is a biomarker of a more advanced stage of toxicity, where the cell is unable to protect against glutathione depletion. However, care must be taken when inferring hepatic glutathione concentration. Both models demonstrate that the sensitivity of biomarkers to exposure of paracetamol, depends on the dynamics of exposure as well as on the concentrations of intracellular metabolites, such as methionine.I discuss how the methodology of biomarker assessment could be personalised with regards to individual patients and how systems toxicology could be further developed towards reliable tools for the pharmaceutical industry.

Freshwater ecosystems have been recognized as important components of the global carbon cycle, and the flux of organic matter (OM) from freshwater to marine environments can significantly affect estuarine and coastal productivity. The focus of this study was the assessment of carbon dynamics in two aquatic environments, namely the Florida Everglades and small prairie streams in Kansas, with the aim of characterizing the biogeochemistry of OM. In the Everglades, particulate OM (POM) is mostly found as a layer of flocculent material (“floc”). While floc is believed to be the main energy source driving trophic dynamics in this oligotrophic wetland, not much is known about its biogeochemistry. The objective of this study was to determine the origin/sources of OM in floc using biomarkers and pigment-based chemotaxonomy to assess specific biomass contributions to this material, on a spatial (freshwater marshes vs. mangrove fringe) and seasonal (wet vs. dry) scales. It was found that floc OM is derived from the local vegetation (mainly algal components and macrophyte litter) and its composition is controlled by seasonal drivers of hydrology and local biomass productivity. Photo-reactivity experiments showed that light exposure on floc resulted in photo-dissolution of POC with the generation of significant amounts of both dissolved OM (DOM) and nutrients (N & P), potentially influencing nutrient dynamics in this ecosystem. The bio-reactivity experiments determined as the amount and rate of CO2 evolution during incubation were found to vary on seasonal and spatial scales and were highly influenced by phosphorus limitation. Not much is known on OM dynamics in small headwater streams. The objective of this study was to determine carbon dynamics in sediments from intermittent prairie streams, characterized by different vegetation cover for their watershed (C4 grasses) vs. riparian zone (C3 plants). In this study sedimentary OM was characterized using a biomarker and compound specific carbon stable isotope approach. It was found that the biomarker composition of these sediments is dominated by higher plant inputs from the riparian zone, although inputs from adjacent prairie grasses were also apparent. Conflicting to some extent with the River Continuum Concept, sediments of the upper reaches contained more degraded OM, while the lower reaches were enriched in fresh material deriving from higher plants and plankton sources as a result of hydrological regimes and particle sorting.