Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and III are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.

Aptamers are single-stranded oligonucleotides, DNA or RNA, which can bind to a myriad of targets such as ions, peptides, proteins, drugs, organic and inorganic molecules with high affinity and specificity. Aptamers are derived using combinatorial libraries comprised of a variable region flanked by two primer regions used for a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The central theme of my thesis was to use this technology to develop aptamers able to bind to validated therapeutic targets, specifically the Tumour Necrosis Factor alpha (TNFα) and Carcinoembryonic Antigen (CEA), and block their biological functions. As well, I investigated the use of CEA and MUC1 binding aptamers as targeting agents to guide and detect the delivery of contrast agent-loaded liposomes in tumour-bearing mice using computed tomography (CT) imaging. Aptamer selections successfully identified a 25-base aptamer (VR11) that can bind with high affinity and specificity to TNFα. VR11 blocked TNFα signaling, prevented apoptosis, reduced nitric oxide (NO) production in cultured cells and was non-immunogenic when injected into C57BL/6 mice. As well, aptamers were derived to the IgV-like N-domain of CEA. Two DNA aptamers were isolated containing a 40-base variable region, N54 and N56, bearing anti- CEA homotypic adhesive properties. These aptamers are not cytotoxic or immunogenic and III are able to prevent CEA-mediated homotypic and heterotypic cell adhesion events. In addition, the pretreatment of murine cancer cells expressing CEA with these aptamers prior to their intraperitoneal injection into C57BL/6 mice resulted in the prevention of tumour foci formation. Finally, the in vivo targeting of nanoparticles such as pegylated liposomes to tumour cells was enhanced by introducing tumour marker-specific DNA aptamers on their surface. The CEA-specific aptamer N54 and a 40-base second generation aptamer MUC1-VR1 that recognizes the tumour-associated mucin MUC1 were incorporated into liposomes containing the CT contrast agent Omnipaque350™ and Cy5 to characterize their binding to CEA and MUC1-expressing cancer cells in vitro. Pharmacokinetic studies also revealed that the incorporation of these aptamers into pegylated liposomes significantly lenghthened their circulation half-lives to values that parrallel that of untargeted pegylated liposomes.

L’anévrysme de l’aorte abdominale (AAA) est une maladie vasculaire consistant en une dilatationde l’aorte abdominale. Son caractère asymptomatique rend importante la recherche debiomarqueurs afin de faciliter la prise en charge des patients.Le but de mes travaux de thèse a été d’étudier spécifiquement les types cellulaires impliqués dansl’AAA.Après obtention d’une banque d’échantillons d’AAA, nous avons localisé ces types cellulaires etrepéré 3 zones d’intérêt : le thrombus intraluminal contenait des neutrophiles, lymphocytes T,mastocytes et macrophages M2. Les macrophages M1 étaient localisés dans l’adventice, avec leslymphocytes B et des mastocytes. La média contenait des cellules musculaires lisses (CML) dont lephénotype variait entre les états sain, anévrysmal et apoptotique.Ces résultats suggérant une implication différente des deux types de macrophages et uneévolution des CML au cours de l’AAA, nous avons isolé ces deux types cellulaires du tissu anévrysmalpar microdissection laser afin d’y étudier l’expression de biomarqueurs potentiels. Nous avons ainsimontré que l’augmentation plasmatique de peroxiredoxine-1 provient des macrophages M1 et quel’augmentation d’ADAMTS5 provient des CML anévrysmales allongées.Nous avons ensuite comparé les protéomes des macrophages M1 et M2 afin d’élucider leur rôledans l’AAA et d’identifier de nouveaux biomarqueurs. Cinq protéines différentielles ont étéidentifiées impliquées dans la phagocytose ou la réponse au stress.L’étude des différents types cellulaires impliqués dans l’AAA pourrait nous permettre unemeilleure compréhension de ses mécanismes et l’identification de biomarqueurs potentiels de cettepathologie. / Abdominal aortic aneurysm (AAA) is a vascular disease consisting in a dilatation of the abdominalaorta. As AAA is asymptomatic, it is important to identify new biomarkers in order to improve theearly care management of patients.The aim of this work was to study specifically the cell types involved in the development of AAA.After collection of an AAA samples bank, we located these cell types among the tissue andhighlighted 3 interesting areas: the intraluminal thrombus contained neutrophils, T lymphocytes,mast cells and M2 macrophages. M1 macrophages were located in the adventitia along with Blymphocytes and other mast cells. The media contained smooth muscle cells (SMC) with a phenotypevarying from a healthy state, to an aneurysmal one and to an apoptotic one.These results suggest a different role of the two subtypes of macrophages and an evolution ofSMCs during AAA. Thus, we chose to isolate these subtypes from the aneurysmal tissue with lasermicrodissection in order to study the proteins differentially expressed between them that could bepotential biomarkers. We showed that the plasmatic increase in peroxiredoxin-1 levels in AAApatients came from pro-inflammatory M1 macrophages and the increase in ADAMTS5 levels camefrom aneurysmal elongated SMCs.We then compared the whole proteome of M1 and M2 macrophages in order to elucidate theirrole in AAA and identify new biomarkers. Five differentially expressed proteins were identified,implicated in phagocytosis and stress response.The study of the different cell types involved in AAA could enable us a better understanding of itsmechanisms and allow the identification of new potential biomarkers for this pathology

Zuzana Bílková, Biological activities of Lavandula angustifolia essential oil, Thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, thesis author: PharmDr. Jan Martin, PhD., Hradec Králové, 2013, 72 pages. The thesis called "Biological activities of Lavandula angustifolia essential oil" is interested in biological activities of Lavandula angustifolia essential oil, specifically antifungal, antioxidant, anti-inflammatory, cytotoxicity, nematicidal and repellency activities. The aim of work was to find out, if the oil can be used in these indications and become natural substitution to the chemical compound toxic to the environment. Antifungal activity was tested against the most common human patogenes, low toxicity proves that essential oil can be used on human cells without causing any harm. Antioxidant activity was tested on NO reaction, nematicidal activity was tried on proctection of crop where nematodes cause huge economic loss. Repellency activity can protect both decorative and agricultural plants. Key words: Lavandula angustifolia, biological activities, anti-inflammatory, antifungal, cytotoxicity, repellency, antioxidant, nematicidal, essential oil.

Introdução: O infliximabe é um anticorpo monoclonal quimérico que inibe o fator de necrose tumoral, sendo usado em doenças autoimunes e/ou inflamatórias, tais como a artrite reumatóide (AR), a espondilite anquilosante (EA), a psoríase e a artrite psoriásica (AP) e as doenças inflamatórias intestinais (DII). Objetivos: Avaliar se o infliximabe induz à formação de autoanticorpos e verificar a ocorrência de eventos adversos, sobretudo o lúpus induzido por este medicamento. Metodologia: Trata-se de um estudo aberto, prospectivo, de fase IV, onde dosamos os autoanticorpos antes e depois do tratamento (das doenças citadas anteriormente), o qual teve duração mínima de 6 meses (5 infusões). Resultados: No total, 286 pacientes foram avaliados para o fator anti-nuclear (FAN) por imunofluorescência indireta em células Hep2, sendo significativo o aumento de número de indivíduos (p = 0,0001), antes e depois da medicação. Além do FAN, foram dosados, em 146 pacientes, 17 outros autoanticorpos pelo método multiplex, sendo que o anti-DNA de dupla hélice (anti-dsDNA) e o anticardiolipina IgM (aCL IgM) tiveram um aumento significativo (p = 0,003 e 0,0024, respectivamente). Pacientes com AR tiveram uma variação significativa nos títulos do anticorpo anti-proteína citrulinada (ACPA) (antes e depois do tratamento) (p = 0,012). De todos os pacientes avaliados (n = 286), somente 1 (0,35%) apresentou sinais clínicos e laboratoriais de lúpus induzido pelo infliximabe. Conclusão: O estudo demonstrou que o infliximabe interferiu na formação de autoanticorpos (FAN, anti-dsDNA, aCL IgM e ACPA), sendo rara a indução de lúpus pelo medicamento. / Background: Infliximab is a chimeric monoclonal antibody that inhibits tumor necrosis factor, and is thus used in the treatment of autoimmune and/or inflammatory diseases, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis and psoriatic arthritis (PA), and inflammatory bowel diseases (IBD). Objective: To determine whether infliximab induces the formation of autoantibodies and assess the occurrence of adverse effects, mainly drug-induced lupus. Method: The study consisted of a phase IV, prospective, open-label trial, in which autoantibody levels were determined before and after treatment (of the above mentioned diseases) with a minimum duration of 6 months (5 infusions). Results: A total of 286 patients were assessed for the presence of antinuclear antibodies (ANA) by means of indirect immunofluorescence on human epithelial (HEp-2) cells, with a significant increase in the number of individuals observed after treatment (p = 0.0001). In addition to ANA, 17 other autoantibodies were assessed in 146 patients using multiplex technology, and a significant increase was observed in anti-double stranded DNA (anti-dsDNA) and anticardiolipin IgM (aCL IgM) antibody titers (p = 0.003 and 0.0024, respectively). Patients with RA showed significant variations in anti-citrullinated protein antibody (ACPA) titers (before and after treatment) (p = 0.012). Among the 286 patients assessed, only 1 (0.35%) presented clinical and laboratory signs of infliximab-induced lupus. Conclusion: The present study showed that infliximab did affect the formation of autoantibodies (ANA, anti-dsDNA, aCL IgM and ACPA), with a rare occurrence of drug-induced lupus.

This thesis was completed in order to assess and document the environmental effects that are the result of elemental phosphorus production in Pinellas County Florida. The study utilized a collection of information resources that included: personal interviews, technical references, historical documents, legal documents and field observations. By utilizing five different sources of information a broad understanding of the problem was developed. Pinellas County and Tarpon Springs officials were interested in creating a more diversified economy in the years following World War-II. The Victor Chemical Works Company responded to the interest in economic diversity by proposing to build an elemental phosphorus production facility in the area of greater Tarpon Springs, Florida. The elemental phosphorus production facility was completed and began operation in November of 1947. Three months after the facility commenced production local residents noticed damage to trees and painted surfaces on private properties. Seven months following commencement of elemental phosphorus production local residents filed suit against the Victor Chemical Works Company due to deleterious gasses and dust that appeared to be damaging to biologic health. The elemental phosphorus production facility operated from 1947 to 1981. The 34-year operational period exposed workers, residents and biologic communities to extended periods of elevated sulfur dioxide, phosphorus pentoxide gas, phosphine gas, fluorine, lead, radium-226 and asbestos. Utilizing personal interviews, technical document review, legal document review and field observations the thesis provided an amalgamation of diverse information upon which the conclusions were based. The research concludes that the production of elemental phosphorus exposed all physical and cultural environments of northwest Pinellas County to many complex adverse environmental impacts that continue to persist in 2007, approximately 26-years following the suspension of production.

Background: Psoriasis is a heterogeneous disease with several clinical manifestations; the symptoms are characterized by redness, scaliness and thickness of the skin. There are several treatment options available for psoriasis and patients with moderate to severe psoriasis generally need systemic agents. In 2004 biologics were introduced for patients with moderate to severe psoriasis in Sweden. Methods: The Swedish Health Care Registers and the Swedish registry for systemic treatment of psoriasis PsoReg, were used to; estimate the incidence of psoriasis cases in the Swedish specialist care, to examine the treatment allocation and important factors related to the initiation of especially biologic treatment. Results: On average 9000 new psoriasis patients entered specialist care in Sweden each year under study, corresponding to an incidence of 98 patients per 100,000 person-years. In the treatment allocation analysis of the incident psoriasis cases in the Swedish specialist care Patients living in a Metropolitan Area and with a University degree were more likely to initiate a biologic treatment. By analysing biologic-naïve patients enrolled in PsoReg, PASI (the physician’s assessment of the psoriasis severity) and Psoriasis Arthropathy were shown to be two important factors associated with the initiation of biologic treatment while sex was not. Furthermore, it was also shown that the decision to initiate biological treatment was more strongly associated with PASI than with DLQI (the patients’ assessment of the disease impact Quality of Life). Conclusion: These studies indicate that there are inequalities in the assignments of systemic psoriasis treatments (especially in biologic treatment). Since the allocation of treatments should not depend on sex, education or residency in a Metropolitan Area but rather the need of care, it is important that future studies continue analysing possible factors that could influence the initiation of treatment in clinical practice.

Introdução: O infliximabe é um anticorpo monoclonal quimérico que inibe o fator de necrose tumoral, sendo usado em doenças autoimunes e/ou inflamatórias, tais como a artrite reumatóide (AR), a espondilite anquilosante (EA), a psoríase e a artrite psoriásica (AP) e as doenças inflamatórias intestinais (DII). Objetivos: Avaliar se o infliximabe induz à formação de autoanticorpos e verificar a ocorrência de eventos adversos, sobretudo o lúpus induzido por este medicamento. Metodologia: Trata-se de um estudo aberto, prospectivo, de fase IV, onde dosamos os autoanticorpos antes e depois do tratamento (das doenças citadas anteriormente), o qual teve duração mínima de 6 meses (5 infusões). Resultados: No total, 286 pacientes foram avaliados para o fator anti-nuclear (FAN) por imunofluorescência indireta em células Hep2, sendo significativo o aumento de número de indivíduos (p = 0,0001), antes e depois da medicação. Além do FAN, foram dosados, em 146 pacientes, 17 outros autoanticorpos pelo método multiplex, sendo que o anti-DNA de dupla hélice (anti-dsDNA) e o anticardiolipina IgM (aCL IgM) tiveram um aumento significativo (p = 0,003 e 0,0024, respectivamente). Pacientes com AR tiveram uma variação significativa nos títulos do anticorpo anti-proteína citrulinada (ACPA) (antes e depois do tratamento) (p = 0,012). De todos os pacientes avaliados (n = 286), somente 1 (0,35%) apresentou sinais clínicos e laboratoriais de lúpus induzido pelo infliximabe. Conclusão: O estudo demonstrou que o infliximabe interferiu na formação de autoanticorpos (FAN, anti-dsDNA, aCL IgM e ACPA), sendo rara a indução de lúpus pelo medicamento. / Background: Infliximab is a chimeric monoclonal antibody that inhibits tumor necrosis factor, and is thus used in the treatment of autoimmune and/or inflammatory diseases, such as rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis and psoriatic arthritis (PA), and inflammatory bowel diseases (IBD). Objective: To determine whether infliximab induces the formation of autoantibodies and assess the occurrence of adverse effects, mainly drug-induced lupus. Method: The study consisted of a phase IV, prospective, open-label trial, in which autoantibody levels were determined before and after treatment (of the above mentioned diseases) with a minimum duration of 6 months (5 infusions). Results: A total of 286 patients were assessed for the presence of antinuclear antibodies (ANA) by means of indirect immunofluorescence on human epithelial (HEp-2) cells, with a significant increase in the number of individuals observed after treatment (p = 0.0001). In addition to ANA, 17 other autoantibodies were assessed in 146 patients using multiplex technology, and a significant increase was observed in anti-double stranded DNA (anti-dsDNA) and anticardiolipin IgM (aCL IgM) antibody titers (p = 0.003 and 0.0024, respectively). Patients with RA showed significant variations in anti-citrullinated protein antibody (ACPA) titers (before and after treatment) (p = 0.012). Among the 286 patients assessed, only 1 (0.35%) presented clinical and laboratory signs of infliximab-induced lupus. Conclusion: The present study showed that infliximab did affect the formation of autoantibodies (ANA, anti-dsDNA, aCL IgM and ACPA), with a rare occurrence of drug-induced lupus.

Background: Rheumatoid arthritis (RA) is a common complex autoimmune disorder which is influenced by both genetic and environmental factors. While multiple factors that influence susceptibility to and outcome of disease have been identified there is still a large proportion of missing heritability and limited understanding of disease pathogenesis. In recent years, biologic drug therapies have advanced treatment of RA; however good disease control is achieved in just 30% of patients, making identification of predictors of treatment response important. One area of research which is yet to be explored in relation to treatment response, and requires further evaluation in RA susceptibility, is epigenetics. Epigenetics is the study of modifications of the DNA which can influence gene expression but do not alter genetic sequence, and the most commonly studied epigenetic phenomenon, to date, is DNA methylation. Objectives: To identify DNA methylation signatures predictive of treatment response to anti-TNF biologics, to explore the role of DNA methylation in RA susceptibility using disease discordant monozygotic (MZ) twins, and to assess the effect of cryopreservation of cells on DNA methylation. Methods: Genome-wide DNA methylation levels were measured using the HumanMethylation450 BeadChip in pre-treatment whole blood DNA samples from individuals who had extremely good or extremely poor response to the anti-TNF therapies, etanercept and adalimumab, and in MZ twins discordant for RA (n=79 pairs). I also compared genome-wide methylation in cells which had been cryopreserved with fresh cells, to investigate if this technique is suitable for epigenetic investigations. Results: I identified four methylation sites which were significantly related to response to etanercept at a false discovery rate of 5%, the most significantly differentially methylated of which maps to the LRPAP1 gene (p=1.46E-8). Indeed, four other sites at the same locus also showed evidence for differential methylation indicating that this represents a differentially methylated region. No sites were significantly associated with response to adalimumab after correction for multiple testing. I identified subtle differences in DNA methylation between RA discordant twins. Although these were not statistically significant following adjustment for cell composition, one of the most differentially methylated positions mapped to the ZNF74 gene (p=4.97E-6), and replicated a methylation difference identified in the largest previous epigenome-wide association study of RA cases and unrelated healthy controls. I found that cryopreservation of cells does not significantly alter the methylome, an important observation that will impact upon design of future studies. Conclusions: In the largest studies of DNA methylation in RA treatment response and RA discordant MZ twins to date, I identified significant differential methylation in etanercept response, but not adalimumab response, and found small differences in methylation in RA discordant MZ twins. I also concluded that cryopreservation does not significantly alter the methylome.

Producers choosing to implement an early corn planting management strategy often experience several yield limiting biotic and abiotic factors. Field variability, flooding, sub-optimal soil temperatures which leads to poor nutrient uptake, delayed emergence and reduced root growth can limit grain production. Three separate experiments were conducted to address some of the negative effects associated with early corn planting. Experiment 1 evaluated flooding effects on several morpho-physiological traits including root system architecture during early crop development. Hybrids (DKC 6208, Pioneer 1197) were flooded at planting (V0) and growth stages V1, V2, V3 for 0, 6, 12, 24, 48, 96 hours. Plants flooded at V0 11% suffered the steepest decline in collar height. Plants flooded at V2 10% were more susceptible than plants flooded V1 4%. Overall, there was a linear decline in nutrient concentration if flooding occurred at planting. Tissue Na levels were the most affected by flood duration and K was the least affected. Experiment 2 evaluated biologic compounds developed to increase immobile nutrients P and K to improve fertilizer use efficiency and provide slow developing roots essential nutrients. The effectiveness of microbial products (B-300, QR, Mammoth, EM-1) with/without starter fertilizer influenced yield, emergence, plant growth, and nutrient uptake. Biologic seed treatments compared to the control, resulted in a positive yield advantage for all treatments. Yields ranged from 37 to 48% higher if biologic compounds were applied. On average, yields increased from 26 to 38% after starter fertilizer was added to the biologic compounds. Phosphorus levels at VT were significantly higher for QR and K content was higher for B300, SF-B300, QR, Mamm, and SF-Mamm compared to the control. Experiment 3 addressed soil physical/chemical properties affecting plant development and there yield plant density relationship. On average, yields significantly increased 40% as plant population increased from 49,400 to 103,740 plants ha−1. Based on the quadratic model agronomically yields would be highest at 61,360 plants ha−1. Correlation analysis among yield and soil physical and chemical properties revealed positive correlations for grain yield, sand% (r2 = 0.42), soil K (r2 = 0.17) soil Na (r2 = 0.46), and soil P (r2 = 0.49).