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Epidemiological studies have linked exposure to elevated levels of airborne particulate matter with increased incidences of several types of respiratory disease, hospital admissions and morbidity. Millions of tons of airborne particulate matter are generated and released into the atmosphere each year. However, particulate matter resulting from the combustion of fuel oil and coal are of particular concern, because they are generally composed of small particles that can easily penetrate deep into the lungs, and can contain significant concentrations of toxic transition metals, such as zinc, iron and vanadium. Pulmonary toxicity (i.e. damage caused to lung tissues) of particulate matter is currently evaluated via time-consuming in-vivo testing, or via in-vitro testing. Compared to in-vivo testing, in-vitro testing offers significant advantages in terms of time savings and sample throughput. Unfortunately, the number of in-vitro testing methods are currently very limited, and do not allow a thorough investigation of the mechanisms of particulate matter toxicity. In light of these issues, the goals of the study described here were three-fold: *To adapt several in-vitro toxicity assays currently used in other applications to use in measuring particulate matter toxicity on lung cell layers; *To use these adapted assays to quantify the toxicity of numerous types of oil and coal ashes with varying particle sizes and transition metal concentrations, and; *To use the same assays to quantify the toxicities of several transition metals found in coal and oil ashes to better understand their relative contributions to overall particulate matter toxicity. Three colorimetric in-vitro assays were chosen for adaptation, and proved effective in measuring adverse cellular response to particulate matter exposure. Particle size was shown to have a large effect on the overall cytotoxicity of particulate matter; fine (less than 2.5 μm aerodynamic diameter) particles proved substantially more toxic than coarse (larger than 2.5 μm aerodynamic diameter) particles. Dose-response experiments measuring the toxic effects of the transition metals zinc, vanadium and iron revealed that zinc was the most toxic; a concentration of 0.6 mM caused a 50% drop in cellular metabolism, compared to 3 mM and 4 mM for vanadium and iron respectively.

Studies on a series of polymethine cyanine dyes and analogs (1-24) show that certain near-infrared cyanines are capable of damaging DNA in the absence of light and external reducing agents. Experimental results imply that in this DNA thermal cleavage, the cyanine reduces Cu(II) to Cu(I) which reacts with O2 to generate the reactive oxygen species (ROS) O2∙- and ∙OH. The formation of these ROS is also thought to be responsible for the irreversible bleaching of the dyes in aqueous solutions. A correlation between structural features and DNA thermal cleavage activity as well as dye bleaching is suggested. Long polymethine chains appear to confer instability to cyanines in aqueous solutions and further contribute to undesired thermal DNA cleavage. These drawbacks can be overcome by introducing an electron-withdrawing group to the polymethine bridge of the cyanine dye.

People experience the irritating tearing and burning sensation of lachrymatory factor (LF, propanthial S-oxide) when cutting or chopping onion bulbs. LF is produced by lachrymatory factor synthase (LFS) specifically from 1-propenyl sulfenic acid, a breakdown product of trans-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) by alliinase. This thesis describes strategies to produce a tearless onion by using RNA interference (RNAi) silencing. To determine whether a gene silencing cassette can silence lfs gene transcripts from onion (Allium cepa L.), a crop recalcitrant to genetic transformation, a gene silencing assessment system was developed by using a model plant as a host for the gene of interest. Tobacco (Nicotiana tabacum) plants transgenic for LFS enzyme activity from onion were first produced by introducing a CaMV 35S-onion-lfs gene construct. These plants were then subjected to a second transformation with an RNAi construct directed against the lfs gene sequence. LFS enzyme activity assay showed that the transgenic plants, containing both the lfs gene and the RNAi construct, had significantly reduced LFS activity. This observation was supported by Western analysis for the LFS protein and further validated by quantitative RT-PCR analysis that demonstrated a significant reduction in the lfs transcript level in the dual transformants. This work demonstrated that the RNAi construct is a suitable candidate for the development of a tearless onion. This model plant RNAi system has wide reaching applications for assessment and targeting of plant secondary pathway genes, from poorly studied or recalcitrant plant species, that are important in pharmacological, food and process industries. The functional RNAi vector identified in the model system was transformed into onion. Endogenous lfs transcript levels were successfully reduced by up to 43-fold in six transgenic lines. In consequence, LFS enzyme activity was decreased by up to 1573-fold and this observation was supported by Western analysis for the LFS protein. Furthermore, the production of the deterrent LF upon tissue disruption was reduced up to 67-fold. Subjective olfactory assessment of silenced lines indicated that the pungent odour given off by the leaf and bulb material was much reduced compared with that of non-transgenic counterparts, and that this was replaced by a sweeter milder onion odour. A novel colorimetric assay demonstrated that this silencing had shifted the 1-PRENCSO breakdown pathway so that by reducing LFS protein, more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of the raised thiosulfinates levels was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer that has never previously been identified, and other volatile compounds, di-1-propenyl disulfides and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes, which had previously been reported either in small amounts or had not been detected in onions. These raised volatile sulfur compounds provide an explanation for the unique flavour notes of the LF reduced onion and are predicted to have health benefits akin to those found in garlic. These results demonstrated that silencing of LFS enzyme activity by introducing an RNAi construct directed against the lfs gene sequence simultaneously reduced levels of the deterrent LF and increased the desirable thiosulfinates in onions.

The one-humped or the dromedarian camel is a pseudo-ruminant mammal, well adapted to the hot and dry climates of the desert. Its ability to withstand torrid heat and extreme desiccation is of paramount importance to its survival. The studies presented in this thesis were designed to investigate and document the effect of dehydration in the presence or absence of angiotensin II (Ang II) AT1 receptor blocker (losartan) on blood constituents, electrolytes, hormones, neurotransmitters as well as liver and kidney enzymes in a subset of dehydrated camels and to compare them with hydrated camels. Additionally, we studied the response of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) and revealed for the first time the cardiac storage form of BNP in the camel heart. Dehydration induced significant increments in packed cell volume (PCV), white blood cells (WBC), gamma glutamyl-transferase (GGT), serum sodium, creatinine and urea levels, and a doubling in plasma cortisol and arginine vasopressin (AVP) levels. At the same time dehydration caused significant decrease in body weights, plasma insulin like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), and a 50% decrement in ANP and BNP levels. Moreover, dehydration with and without losartan resulted in significant changes in stress hormones and anti-oxidants in plasma, liver and kidney homogenates. Losartan on one hand enhanced the effect of dehydration resulting in significant increases in sodium, creatinine and urea levels. In addition losartan raised the  binding affinity of Ang II AT2 receptors in the small intestine with 8-fold and with 16-fold for liver AT1 receptors, indicating that Ang II AT1 and AT2 receptor binding sites were present in camel's small intestine while only AT1 receptor binding sites were found in the camel liver. One the other hand losartan resulted in significant decrease in body weights impaired the rise in anti-diuretic hormone and reduced aldosterone level. Finally, we showed that the proBNP is the storage form of BNP in the camel heart.

Aptamers being used as sensors is an emerging field that has capabilities of being tomorrow’s diagnostic tools. As aptameric sensors have become more popular, their visualization systems have been limited. The majority of today’s aptameric sensors require expensive machinery such as a fluorometer in order to visualize results. We propose a system that will cut the need for instrumentation and be detected via the naked eye. With the selection of an aptamer to bind the pH indicating dye bromocresol purple (BCP) this may be achieved. When rendered active, the binding towards BCP will facilitate a color change from yellow to purple or vice versa. Previous studies have shown albumin contains the ability to facilitate this role and we now intend to use a DNA aptamer to achieve this as well. The BCP aptamer has the potential to serve as a signaling domain to any already selected aptamer thus making it a universal tool for both research and diagnostic measures. We have found that an alternative structure-switching systematic evolution of ligands by exponential enrichment (SELEX) method which left the dye unaltered was not sufficient for selecting an aptamer. We believe that a classical SELEX will enable us to select an aptamer that may be used to accomplish this role as a universal visual detector.

Exogenously supplied organoselenium compounds, capable of propagating a selenium redox cycle, were shown to supplement natural cellular defenses against oxidants generated during biological activity. Phenylaminoalkyl selenides were developed in our laboratory as novel substrate analogs for the enzyme dopamine beta-monooxygenase. Recently, phenylaminoalkyl selenides were found to protect plasmid DNA and Molecular beacons from oxoperoxynitrate – mediated damage by scavenging this oxidant and forming the corresponding selenoxides as the sole selenium – containing products. Rate constants were determined for the reactions of the phenylaminoalkyl selenoxides with GSH at physiological pH and 25 degrees C. The kinetic data obtained in current and previous research was subsequently used in a MatLab simulation, which showed the feasibility of selenium redox cycling by GSH in the presence of a cellular oxidant, oxoperoxynitrate. Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests covering 11.7 million hectares. Somatic embryogenesis (SE) is an effective technique to implement production of high value genotypes of LP. SE is a multi-step process, which includes initiation of somatic embryo (SME) growth from tree tissue, maintenance and multiplication of early stage SMEs and the maturation / germination phase. In this work, we isolated a substance from stage 2 or 3 LP female gametophyte (FG) tissue that stimulates early stage SME growth, and characterized this substance as citric acid on the basis of 1H NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at p = 0.05 for 3 of the 5 genotypes tested. Phenylketonuria (PKU) is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr). A novel assay based on enzymatic - colorimetric methodology (ECA) was developed in order to detect elevated concentrations of L-Phe in undeproteinized plasma of PKU patients via continuous spectrophotometric detection. We report here that L-Phe concentrations in undeproteinized plasma measured using our ECA were comparable to those determined on an amino acid analyzer based on Pearson correlation coefficients and a Bland and Altman comparison.

Le présent travail a porté sur l’évaluation des propriétés anti-inflammatoires et anticancéreuses de Agelanthus dodoneifolius (Loranthaceae), communément appelée «gui africain». Cette plante hémiparasite est utilisée en médecine traditionnelle africaine pour le traitement de pathologies chroniques telles que l’asthme, l’hypertension, des gastroentérites et le cancer. Actuellement, les maladies chroniques représentent un problème mondial de santé publique. En effet, elles constituent la première cause de mortalité dans le monde surtout dans les pays à revenu faible ou intermédiaire. <p><p>Cette étude a été réalisée dans le but d’apporter une validation scientifique quant à certaines utilisations traditionnelles de Agelanthus dodoneifolius. <p>Pour évaluer l’effet anti-inflammatoire de Agelanthus dodoneifolius, nous avons testé les différentes fractions de la plante sur la production des espèces réactives de l’oxygène, la libération et l’activité spécifique de la myéloperoxydase (MPO), enzyme libérée par le neutrophile au cours de la phagocytose pour détruire les microorganismes. L’identification et la quantification des composés a été faite grâce à une combinaison des méthodes chromatographiques, spectrophotométriques et spectrométriques. L’activité anticancéreuse de Agelanthus dodoneifolius a consisté, d’abord, à déterminer l’effet d’inhibition de croissance de diverses fractions de la plante, de la quercétine ainsi que de ses dérivés sur des lignées cellulaires cancéreuses. Nous avons ensuite déterminé les effets de la quercétine sur l’activité de plus de 300 kinases. <p><p>Les résultats obtenus montrent qu’Agelanthus dodoneifolius est capable de moduler les activités biologiques des neutrophiles. En effet, le décocté aqueux et les fractions organiques de la plante inhibent de manière dose-dépendante la production des espèces réactives de l’oxygène, la dégranulation du neutrophile et l’activité spécifique de la myéloperoxydase. Nous avons pu identifier et quantifier dix composés polyphénoliques dont quatre acides phénoliques :l’acide gallique, l’acide coumarique, l’acide chlorogénique et l’acide ellagique et six flavonoïdes :la quercétine, le kaempférol, la catéchine, l’isoquercitrine ou quercétine 3-O-glucoside, la rutine et la miquelianine ou quercétine-3-O-glucuronide. <p>Concernant l’activité anticancéreuse, les résultats montrent que seules les fractions à l’éther diéthylique et à l’acétate d’éthyle ont une activité antiproliférative. La quercétine a des effets inhibiteurs de croissance, cytostatiques et présente un large spectre d’activité sur plusieurs kinases surexprimées dans certains cancers. <p><p>En conclusion, l’ensemble de ces résultats constitue des bases scientifiques qui pourraient justifier certaines utilisations traditionnelles de Agelanthus dodoneifolius. <p>À notre connaissance, cette étude est la première à évaluer d’une part l’effet, in vitro, des différentes fractions de Agelanthus dodoneifolius sur des neutrophiles stimulés et sur la MPO et d’autre part l’effet inhibiteur de croissance de lignées cellulaires cancéreuses par certaines fractions de la plante. En outre, cette étude a permis pour la première fois d’identifier et de quantifier des composés polyphénoliques dans Agelanthus dodoneifolius. Les nombreuses propriétés de ces composés, notamment celles anti-inflammatoires et anticancéreuses, peuvent expliquer en partie les résultats reportés dans ce travail.<p><p>This work focused on evaluating anti-inflammatory and anticancer activities of Agelanthus dodoneifolius (Loranthaceae), commonly called "African mistletoe". This plant is used in African traditional medicine for the treatment of chronic conditions such as asthma, hypertension, gastroenteritis and cancer. Currently, chronic diseases are a global public health problem. Indeed, they are the leading cause of death worldwide, especially in countries with low and middle income.<p>The study was conducted to provide scientific validation for some traditional uses of Agelanthus dodoneifolius.<p><p>To characterize the anti-inflammatory activity of Agelanthus dodoneifolius, we tested the different fractions of the plant on reactive oxygen species production, release and the specific activity of myeloperoxidase, an enzyme released by neutrophils during phagocytosis to destroy microorganisms. The identification and quantification of compounds were made through a combination of chromatographic, spectrophotometric and spectrometric techniques. The anticancer activity of Agelanthus dodoneifolius consisted, first, to determine, the antiproliferative effect of fractions of the plant, quercetin and its derivatives on cancer cell lines. Then, we determined the effects of quercetin on the activity of more than 300 kinases.<p><p>The results show that Agelanthus dodoneifolius is capable of modulating the biological activities of neutrophils. In fact, the decoction aqueous and organic fractions of the plant inhibited in a dose-dependent manner the production of reactive oxygen species, degranulation of neutrophils and specific activity of myeloperoxidase. <p>We were able to identify and quantify ten polyphenolic compounds including four phenolic acids: gallic acid, coumaric acid, chlorogenic acid and ellagic acid and six flavonoids: quercetin, kaempferol, catechin, isoquercitrin or quercetin 3-O-glucoside, rutin and miquelianin or quercetin-3-O-glucuronide.<p>Regarding the anticancer activity, the results show that only fractions with diethyl ether and ethyl acetate have antiproliferative activity. Quercetin has antiproliferative and cytostatic effects and presents a broad spectrum of activity on several kinases overexpressed in certain cancers.<p><p>In conclusion, all these findings are scientific basis that could justify some traditional uses of Agelanthus dodoneifolius. To our knowledge, this is the first study to evaluate the effect firstly, by in vitro tests, of the different fractions of Agelanthus dodoneifolius on stimulated neutrophils and the MPO and secondly the growth inhibitory effect of cancer cell lines by certain fractions. Also, this study is the first to identify and quantify the phenolic compounds in Agelanthus dodoneifolius. The many properties of these compounds, including anti-inflammatory and anticancer, may partly explain the results reported in the present work. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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