Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma (NHL). FL is clinically designated as an indolent disease with a long median survival of 8-10 years. However, the clinical and biological behavior of FL shows considerable variability, with some patients showing aggressive disease progression and very short survival. Because defects in the regulation of apoptotic cell death are fundamental in FL pathogenesis, we hypothesized that deregulated expression of components of the pRb signaling pathway may promote cell proliferation, thereby complementing antecedent anti-apoptotic mutations and producing more aggressive disease. In the present study we undertook an immunohistochemical (IHC) evaluation of expression of key cell-cycle regulatory proteins in diagnostic biopsies from 127 cases of FL using formalin-fixed, paraffin-embedded tissues (FFPE) in tissue microarray (TMA) sections immunostained for p53, pRb, p16INK4A and cyclin D3. Data analysis revealed that increased abundance of p53 or p16INK4A is associated with reduced overall survival (OS) (p=0.005 and p=0.014 respectively), and with conventional pathological markers of tumour aggressiveness including high histologic grade. Encouraged by this remarkable finding of a counterintuitive association between p16INK4A expression and clinical outcome, we analyzed CDKN2A gene deletion and methylation, as these are the most frequent mechanisms of the CDKN2A gene inactivation in NHL including FL. We determined the deletion and methylation status of CDKN2A in 105 FL cases. Laser-capture microdissection was used to enrich the samples for lymphoma cells. CDKN2A was deleted in 9 cases and methylated in 22 cases. The 29 cases (28%) with CDKN2A deletion or methylation had decreased overall survival (OS) (p=0.046) in all cases and in cases treated with rituximab (p<0.001). Our findings indicate that deleterious alterations of CDKN2A are relatively prevalent in FL at diagnosis and can predict poor clinical outcome. In summary, our data reveal novel insights into the pathogenesis of FL and suggest a relationship between increased p16INK4A expression and CDKN2A deletion or methylation and unfavorable clinical outcome in FL. We hope that the work presented herein will provide a useful prognostic tool for predicting the prognosis and choosing optimal treatment approaches to help patients suffering from FL. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2013-03-12 23:49:44.541

Environmental genotoxicants/carcinogens in the aquatic environment induce complex detrimental impacts on aquatic organisms. Integrated approach is required to comprehensively assess such impacts in terms of animal and environmental health. Biomarkers at different levels of biological organisations, including clearance rates (at individual level), histopathological alterations (at tissue level), DNA strand breaks using comet assay (at cellular level) and transcriptional alterations of key genes determined by real-time quantitative PCR (at molecular level), were designed and applied to analyse the impacts in marine bivalve: Mytilus sp., a classic bio-indicator species for environmental health monitoring, following exposure to selected environmental contaminants (i.e. Benzo(α)Pyrene (B(α)P), C60 fullerene, and tritium). The performance of biomarkers changed in response to exposures with higher sensitivity for DNA strand breaks and transcriptional alterations of selected genes. In particular, genes representing 7 gene groups (tumour suppressor gene - p53, proto-oncogene - ras, cytochrome P450 family - cyp4y1, MAPKs family - jnk, Bcl-2 family - bax, MDM2 - mdm2 and RAD family - rad51) showed tissue- and chemical-specific expression profiles under selected experimental conditions. A comparison revealed similarity of interplay among these highly conserved genes with mammalian models, suggesting Mytilus sp. could be an ideal model for signalling research in the carcinogenesis process. To my knowledge, this is the first research to detect tissue specific transcriptional alteration of p53 and ras genes in the organism for the analysis of detrimental impacts induced by different environmental genotoxicant/carcinogen exposures. Furthermore, the study is also the first one to include genes which can interplay with each other to function in the regulation of DNA repair, apoptosis and cell cycle arrest in marine organisms. The results suggested that the applied integrated biomarker approach is a comprehensive and sensitive method to monitor environmental health and, marine bivalve Mytilus sp. is an ideal model organism for research on genotoxicant induced carcinogenesis and will shed light on the mechanisms of this complex process in higher organisms, including humans.

Protein identification and quantitation using mass spectrometry has evolved as the dominant technique for studying the protein complement of a system: cell, tissue or organism. The proteomics of body fluids is a very active research area as there is great potential for protein biomarker discovery; application of such technologies would revolutionise medical practice and treatment. Saliva, through its non intrusive nature of sampling, is an ideal body fluid for disease diagnosis, screening and monitoring. Gingivitis is a gum disease with symptoms including bleeding, swollen, and receding gums. After dental decay, gingivitis is estimated to be the most common disease worldwide, and around 40% of the population in the US are reported to have gingivitis. The end point goal of this project was to identify salivary biomarkers for gingivitis. This dissertation presents an investigation of: 1) the salivary proteome; 2) developments and applications of a mass spectrometry kinase assay; and 3) salivary biomarkers for gingivitis using proteomics and kinase activities. The soluble portion of the human salivary proteome (saliva supernatant) has been studied by several research groups but very few proteomic studies have been performed on the insoluble, cellular and bacterial portion of saliva. Presented here, is the first global proteomics study performed on the saliva residue and supernatant from the same test subject. A total of 834 and 1426 proteins were identified in the saliva supernatant and residue, respectively. A global analysis of protein complexes in saliva was also performed and is the first study, to date, of such an analysis. KAYAK (‘Kinase ActivitY Assay for Kinome analysis’) was further developed for its application on a number of cell types, tissue types, and a variety of organisms. Proof of concept work for in-gel kinase activity/kinase abundance correlation profiling using blue native gels was performed, and experiments using anion exchange chromatographic kinase activity/kinase abundance correlation profiling were performed to identify kinase-substrate pairs. KAYAK applications included the analysis of kinase activities in Saccharomyces cervisiae, Drosophila, mouse, and human saliva in which significant kinase activity was detected in the saliva supernatant, a novel finding. Finally, gingivitis was induced in patients, and the saliva samples were analysed using proteomics and kinase activity profiling. Although this work is ongoing, preliminary data indicate that there are increases in various inflammatory proteins, certain bacteria and also in the activity of particular kinases as a result of the induction of gingivitis. The overall study provided insights into the salivary proteome for both the human and bacterial complement, as well as discovering the presence of significant kinase activity in saliva. In the induced gingivitis study, almost half of all the proteins identified in the residue were from bacteria (1274 bacterial proteins, 198 species identified) and there may be more potential for biomarker discovery for certain diseases in the saliva residue than in the supernatant. A very large overlap was observed between the human proteins in the saliva supernatant and residue, indicating that many of the salivary proteins originate from lysed cells. The origin of the kinase activity in the saliva supernatant is not known but is also proposed to originate predominantly from lysed cells. A range of novel KAYAK applications have been investigated, demonstrating that KAYAK has a wide variety of future uses ranging from target compound evaluation in Pharmaceutical companies to patient testing in the clinic.

Cutaneous melanoma is associated with significant morbidity and mortality representing the most significant cutaneous malignancy. As it is known that early diagnosis and treatment are the most efficient approaches to cure cutaneous melanoma, an improved understanding of the molecular pathogenesis of melanoma and exploration of more reliable molecular biomarkers are particularly essential. Two different types of molecular biomarker for melanoma have been investigated in this thesis. microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotides in length that are found in both animal and plant cells. miRNAs are involved in the RNA interference (RNAi) machinery to regulate gene expression posttranscriptionally. miRNAs have important roles in cancer: by controlling the expression level of their target genes they can affect cell signalling pathways and have been shown to have both prognostic and therapeutic potential. Importantly for melanoma research, reproducible miRNA expression profiles from formalin-fixed paraffin-embedded (FFPE) tissues can be obtained that are comparable to those from fresh-frozen samples. The aims of the miRNA project were: first, to identify a melanoma-specific miRNA expression profile; secondly, to investigate roles of some of the melanoma-specific miRNAs identified in melanomagenesis. Using miRNA microarray on FFPE samples, I obtained a melanoma-specific miRNA expression profile. 9 of these differentially expressed miRNAs between benign naevi and melanomas (7 downregulated, 2 upregulated in malignancies) were verified by qRT-PCR and the functions of four of these miRNAs were studied. Ectopic overexpression of miR- 200c and miR-205 in A375 melanoma cells inhibited colony forming ability in methylcellulose, an in vitro surrogate assay for tumourigenicity. Moreover, elevation of miR-200c resulted in increased expression levels of E-cadherin through negative regulation of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. Ectopic overexpression of miR-211 in A375 melanoma cells repressed both colony formation in methylcellulose and migratory ability in matrigel, an in vitro surrogate assay for invasiveness. These findings indicate that miR-200c, miR-205 and miR-211 act as tumour suppressors in melanomagenesis. The second biomarker investigated, mutated BRAF, has been seen in 50-70% of spontaneous cutaneous melanoma. The commonest mutation in melanoma is a glutamic acid for valine substitution at position 600 (V600E). Oncogenic BRAF controls many aspects of melanoma cell biology. The aim of this part of the work was: firstly, to study BRAF V600E mutation status in our melanoma tissue microarray (TMA) panel; secondly, to correlate this mutation to various clinicopathological features and evaluate its prognostic value through statistical analyses. BRAF V600E mutations were seen in 20% of the primary and 69% of the metastatic melanomas, respectively. More BRAF V600E mutations were seen in males relative to females. The mutation was also related to cell pigmentation, but not to age, ulceration or solar elastosis. Melanoma patients with the BRAF V600E mutation relapse earlier than patients without this mutation. However, no significant association between the BRAF V600E mutation and overall survival and melanoma specific survival was found.

La néphropathie diabétique (ND) est l'une des complications graves du diabète. Elle affecte environ 30% des patients diabétiques. La microalbuminurie est actuellement l'élément diagnostique principal de la survenue de la ND mais manque de spécificité et précocité. Plusieurs études ont été consacrées à la recherche de nouveaux biomarqueurs (BM) de la ND par des approches protéomiques. Nous avons montré dans la phase initiale de notre travail que si de nombreux candidats BM avaient été identifiés, leur nature ne faisait pas consensus et que de nombreuses études, de par leur conception, ne pouvaient identifier de BM plus précoces que l'albumine. Partant de ce constat, nous avons sélectionné une cohorte originale de diabétiques de type 1 normoalbuminuriques considérés à risque de développer la ND, sur la base de l'apparition d'une microalbuminurie consécutive à un test d'effort. Une cohorte contrôle a été aussi constituée. Dans la première partie de notre travail, nous avons comparé par électrophorèse bidimensionnelle les protéomes urinaires des patients des deux coho rtes. Les BM candidats ont été ensuite identifiés par spectrométrie de masse. L'analyse fonctionnelle de ces protéines a montré que certaines sont impliquées dans la cascade de la coagulation et les mécanismes de dysfonctionnement endothélial. Le caractère diagnostique de ces protéines a été validé dans les mêmes cohortes de patients par des expériences de Western-blot. La compréhension de la nature et de la fonction physiologique des BM candidats identifiés nous a permis de mieux appréhender les mécanismes moléculaires de la pathogénèse de la ND et d'identifier des candidats biomarqueurs plus précoces que l'albumine. Ces résultats sont présentés sous la forme d'un projet d'article .La deuxième partie de notre travail expérimental est constituée d'études préliminaires visant à la recherche de protéines spécifiques urinaires, néphrine et isoformes de l'adiponectine, afin d'évaluer leur potentiel diagnostique. 1 Ben Ameur R. et al Proteomic approaches for discovering biomarkers of diabetic nephropathy. Nephrol Dial Transplant 25, 2866-752 Ben Ameur R. et al Identification of early candidate biomarkers for diabetic nephropathy by urine proteomic analysis. To be submitted / Diabetic nephropathy (DN) is one of the most serious complications of diabetes. It affects about 30% of diabetic patients. Microalbuminuria is currently the main available marker for DN risk, but has inadequate specificity and precocity. Several published studies intended to research new biomarkers (BM) of DN by proteomic approaches. We have shown1 that, if several candidate BM were claimed, there was no consensus about their nature and that a number of studies could not identify BM earlier than albumin because of the study design. Thus, we have selected an original cohort of type 1 diabetic patients considered at risk of developing DN, on the basis of urinary albumin excretion after an exercice test. A control cohort was also enrolled. Using 2D gel electrophoresis we compared the urinary proteomes of patients from both cohorts. Then, candidate BM were identified by mass spectrometry. Functional analysis of these proteins showed that some are involved in the coagulation cascade and in mechanisms of endothelial dysfunction. The diagnostic potential of these proteins was validated by Western blotti ng. The nature and physiological function of candidate biomarkers allowed to better understand the molecular pathogenic mechanisms of DN. Results from this part of the work are shown in the form of an article2. Preliminary studies to assess the diagnostic potential research of specific urinary proteins (nephrin and different isoforms of adiponectin) are also presented.1 Ben Ameur R. et al Proteomic approaches for discovering biomarkers of diabetic nephropathy. Nephrol Dial Transplant 25, 2866-752 Ben Ameur R. et al Identification of early candidate biomarkers for diabetic nephropathy by urine proteomic analysis. To be submitted

Le devenir des pesticides en polyculture d’étang est peu étudié, en particulier la contamination de la chair des poissons proposés aux consommateurs. Cinq sites, correspondant chacun à un étang et à son bassin versant, ont été caractérisés. Ils s’échelonnent sur un gradient d’intensification agricole allant de 0% à 85% de la surface mise en culture. La somme des concentrations de pesticides quantifiés dans l’eau s’échelonne de 0,17 à 8,81 µg/L. La réponse biologique des poissons, évaluée par la recherche de huit biomarqueurs (EROD, CYP 3A, GST, CAT, SOD, GSH, GPx et AChE), a permis d’établir des différences entre les espèces et les sites étudiés. Une recherche ciblée de pesticides épandus sur grandes cultures, a été effectuée par une méthode multi-résidus, dans l’eau, les sédiments et la chair de poisson. L’isoproturon (valeur maximale 2,69µg/L) et le métazachlore (valeur maximale 0,54 µg/L) présentent les concentrations les plus élevées dans l’eau; la trifluraline (0,5 à 13 µg/kg PS) et l’isoproturon (0,7 à 56,4 µg/kg PS) dans les sédiments. Les profils de résidus dans la chair de poisson sont variables et seuls la trifluraline, l’isoproturon, le métazachlore et le carbendazime (valeurs maximales de 21 ; 0,75 ; 0,13 et 0,2 µg/kg PF, respectivement) sont quantifiés. Les facteurs de bioamplification et les demi-vies dans le muscle ont été évalués (10-6 < BMF < 10-3, 1j <DT50 < 40 j) pour la plupart des molécules. Au final, de nombreux facteurs, liés en particulier aux caractéristiques des sites (non maîtrisables) et aux pratiques de gestion (améliorables), influencent le profil des pesticides dans le muscle et la réponse biologique des poissons / Dam pond is connected to its watershed. Use of pesticides on watershed may lead to dissipation in the environment Transfer of registered pesticides remains poorly studied. Thus, the main objective of this work was to improve knowledge about the fate of pesticides in polyculture pond, especially contamination of fish flesh. Five sites, each site corresponding to a pond and its watershed, were characterized. They are placed on a gradient of crop proportion ranging from 0% to 85% of the watershed area. The sum of quantified molecules in water ranged from 0.17 to 8.81 µg / L. The biological response of fish, assessed by evaluation of eight biomarkers (EROD, CYP 3A, GST, CAT, SOD, GSH, GPx, and AChE), establish differences between species and sites studied. A study of 14 targeted pesticides was conducted by a multi-residues method in water, sediments and fish flesh. Isoproturon (maximum value 2.69 µg/L) and metazachlor (maximum value 0.54 µg/L) are the main contaminants in water; Trifluralin (0.5 to 13 µg/kg PS) and isoproturon (0.7 to 56.4 µg/kg PS) in sediments. Trifluralin, isoproturon, carbendazim and metazachlor (maximum values of 21, 0.75, 0.13 and 0.2 µg/kg PF, respectively) are quantified in muscle of fish. Low levels observed in fish can be explained by a low potential to bioaccumulate (measured ex-situ for some molecules, between 10-6 and 10-3). However, half-lives in the muscle may be several weeks depending, explaining the possibility to find molecules in fish flesh. Finally, many factors, particularly related to site characteristics (uncontrollable) and management practices (upgradable), influence profile of pesticides in muscle and biological responses of fish

Despite advances in obstetric care, hypertensive disorders continue to complicate pregnancies at a high rate. Worldwide, hypertensive disorders affect up to 10% of pregnancies. The United States has seen a 25% increase in the incidence of hypertensive disorders over the last two decades (American College of Obstetricians and Gynecologists, 2017). These complications constitute one of the greatest causes of maternal and perinatal morbidity and mortality with an estimated 50,000 to 60,000 deaths per year across the world (American College of Obstetricians and Gynecologists, 2017). Although the etiology of hypertensive disorders remains unclear, there may be an association with both maternal biological and psychological distress in the development of the disorder. Although both distress and biomarkers have been identified in association with a hypertensive disorder, little data exist examining the components of distress and the alterations in biomarkers in women developing these disorders. Due to the limited evidence, a critical need exists to examine the relationship of perceived maternal distress and biomarker measures in the development of a hypertensive disorder during pregnancy in order to better understand this phenomenon. The purposes of this dissertation were to: 1) understand the experience of having a hypertensive disorder during pregnancy; 2) to investigate the association of perceived stress and changes in immune response via biomarker measures in women who develop a hypertensive disorder during pregnancy; 3) to review, summarize, and evaluate the literature examining the relationship between perceived maternal distress (stress, anxiety, and depression) and the development of a hypertensive disorder; and 4) to investigate the association of perceived distress in the development of a hypertensive disorder during pregnancy. Data obtained from a qualitative study of women with a hypertensive disorder during pregnancy placed on bed rest reported several stressors associated with the experience. These stressors related to differing and often conflicting management plans by different providers and not feeling providers heard their concerns. The evidence supports these women experience stress during this pregnancy complication. Analysis of data obtained at each trimester of pregnancy did identify differences in biomarker levels based on perceived stress and women with a hypertensive disorder and those without a hypertensive disorder. Evidence from a systematic review of literature supporting maternal distress in the development of a hypertensive disorder was mixed. However, few studies existed and of those reviewed, most lacked rigor. Analysis of data obtained early and late in pregnancy did not indicate a relationship between psychological distress and the development of a hypertensive disorder in pregnancy. Women with a higher BMI were 12% more likely to develop a hypertensive disorder. The factors associated with the development of a hypertensive disorder are complex. Maternal perceived stress and inflammatory responses differ between women with a hypertensive disorder and those without a hypertensive disorder in pregnancy; however maternal distress did not differ between groups. Body mass index was associated with the development of hypertension in pregnancy. Clinicians need to include assessment of maternal BMI as a modifiable risk factor in the development of a hypertensive disorder during pregnancy. In addition, although psychological distress was not associated with the development of a hypertensive disorder, women still suffer with components of distress. Clinicians could identify and support women experiencing distress thereby promoting a healthier pregnancy.

In this thesis, luminescent lanthanide-doped nanocrystals, and lead-based quantum dots nanocrystals are explored as alternative bioimaging agents to fluorescent proteins and organic fluorophores for deep-tissue imaging. The first chapter gives a brief introduction on the aforementioned nanocrystals and their special optical properties. In chapter 2 the simple changes in the drying and baking temperature of the Yb3+ and Ho3+ doped LaF3 nanocrystals-silica sol-gel mixture aid in the explanation of the formation of two types of silica. The difference in the phonon energies of the two types of silica is found to control effectively the ratio of red to green emissions obtained from the upconversion process. However, the nanocrystals do not disperse in water making them unsuitable for bioimaging. Chapter 3 describe the synthesis of NaYF4 nanocrystals doped with Yb3+/Er3+ or Yb3+/Tm3+ ions followed by two surface modification strategies (intercalation and crosslinking) to disperse them in physiological buffers and biological growth media. Of the two methods, the crosslinked polymer coating of the nanocrystals alone exhibits stability in aforementioned media. In chapter 4 the applicability of lanthanide-doped NaYF4 nanocrystals are studied as bioimaging agents in two-photon upconversion laser scanning microscopy for deep-tissue imaging. Their performance as bioimaging agents was not better than fluorescent proteins and organic molecules. On the other hand with two-photon upconversion wide field microscopy (TPUWFM), brain blood vessels over a depth of 100 µm could well be separated. Furthermore, with the 800 nm emission from Tm3+ ions one can image twice as deep as the green emission with TPUWFM. In chapter 5, probing the NaYF4 nanocrystals with energy-dependent XPS shows that, the Y3+ ions on the surface of the nanocrystals are different from the ones present inside the nanocrystals. This chapter is concluded with a preliminary investigation of Yb3+ and Tm3+ doped NaYF4 with resonant XPS. Chapter 6 examines four different types of surface modification strategies to transfer hydrophobic lead-based quantum dots to physiological buffers and biological growth media. Of the four methods, the crosslinked polymer coating of quantum dots alone exhibits colloidal stability and the QDs retain their luminescence in aforementioned media over several months. The conclusions and future outlook for the work are elucidated in chapter 7. / Graduate

The tumour suppressor p53 is at the centre of the signalling pathway that controls cellular processes crucial in tumourogenesis, cancer progression and tumour clearance. Alterations in the p53 pathway that lead to cancer progression can be good candidates for molecular biomarkers that would assist in the identification of patients with different prognoses, but also serve as good predictors of appropriate targeted therapies. Patient cohorts and cancer cell panels are utilised to seek associations with the attenuation of the p53 pathway and cancer progression. Firstly, the alternatively spliced transcript of the p53 inhibitor HDMX, which is frequently found in tumours with poor prognosis, is studied. The high ratio of the alternatively spliced HDMX-S transcript over the full-length HDMX-FL transcript (HDMX-S/FL) is demonstrated to associate with p53 pathway attenuation in cancer cells and breast carcinomas, and with faster metastatic progression of osteosarcoma and breast cancer patients. Secondly, inherited polymorphism in the HDMX gene is investigated and demonstrated as a unique and highly reproducible eQTL, which identifies patients with different prognoses for metastatic disease in breast cancer and melanoma cohorts. Lastly, a screening approach to identify novel inherited polymorphisms in the p53 pathway genes that associate with metastatic progression of melanoma is developed and implemented, and subsequently in silico and in vitro functional analyses are performed to investigate a mechanism behind the FOXO3 SNP, identified as the strongest candidate, whereby the experimental evidence demonstrate that the causal SNP in the FOXO3 haplotype is controlled by the GATA3 transcription factor. Together, the work presented in this thesis provides strong support for the role of the p53 pathway in the metastatic progression of cancer, and suggests that molecular biomarkers that can detect changes in the activity of p53 pathway genes could offer a robust set of biomarkers for cancer progression applicable to different types of cancer.

We report an IgG (=immunoglobulin) electrochemical immunosensor using a newly synthesized redox-active ferrocenyl dendrimer of generation 2 (G2Fc) as a voltammetric transducer. The ferrocenyl dendrimer N(CH2CH2C(O)NHCH2CH2NHC(O)Fe(η5-C5H4)(η5-C5H5))(CH2CH2N(CH2CH2C(O)NHCH2CH2NHC(O)Fe(η5-C5H4)(η5-C5H5))2)2 (G2Fc) was used as a functional moiety to immobilize the antibody on the surface of the electrode. A sandwich immunosensor of the type IgG/Bovine serum albumin (BSA)/anti-IgG/G2Fc/glassy carbon electrode (GCE) was fabricated. The electrochemical properties of G2Fc were thoroughly studied in aqueous and non-aqueous electrolytes with varying scan rates. The incubation time was optimized for better analytical performance of the immunosensor. It is found that the developed amperometric immunosensor is sensitive to a concentration of IgG as low as 2 ng mL−1. / Dieser Beitrag ist aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.