Quitooligosacáridos bifuncionales: relación entre características químico-físicas y propiedades biológicas

As the degraded products of chitosan, chitooligosaccharides (COS) have recently been produced by several methods, such as enzymatic an acidic hydrolysis. Chitosans are a family of biocompatible and biodegradable biopolymers obtained by N-deacetylation of chitin, the most abundant natural polymer after cellulose, consisting of two monomeric units, N-acetyl-2- amino-2-deoxi-D-glucose (A units) and 2-amino-2-deoxi-D-glucose (D units) linked by β (1→4) links. The degraded products COS, have a smaller molecular weight and therefore have better solubility and lower viscosity under physiological conditions because of shorter chain lengths and free amino groups in D-glucosamine units. The study of COS has been increasing not only because they come from a natural source, but also because of their biological compatibility and effectiveness. There are numerous reports on the biological activities of COS and their potential applications in food industry, pharmacy, agricultural or biomedicine. Nevertheless, in these studies it is difficult to find well defined COS in terms of physicochemical parametres, because these samples are usually poorly characterized. This makes it difficult to compare the results and to understand their mecanism of action. Degradation of the O-glycosidic linkages of chitosan by different methods, results in COS with different numbers and sequences of A and D units as well as different degrees of polymerisation (DP). Over the past few years, several technological approaches have been taken in preparing COS, including acid hydrolysis or enzymatic methods, among others. Therefore, in order to obtain COS with different physicochemical properties, different preparation methods of COS have been developed in this work. Then, the study of the relationship between physicochemical properties of these COS and their biological activities such as natural antioxidants, antibacterial agents, mucoadhesive and anti-inflammatory effects have been studied...

Novel Approaches for the Synthesis of C-5 Modified Pyrimidine Nucleosides

The antiviral or anticancer activities of C-5 modified pyrimidine nucleoside analogues validate the need for the development of their syntheses. In the first half of this dissertation, I explore the Pd-catalyzed cross-coupling reaction of allylphenylgermanes with aryl halides in the presence of SbF5/TBAF to give various biaryls by transferring multiple phenyl groups, which has also been applied to the 5-halo pyrimidine nucleosides for the synthesis of 5-aryl derivatives. To avoid the use of organometallic reagents, I developed Pd-catalyzed direct arylation of 5-halo pyrimidine nucleosides. It was discovered that 5-aryl pyrimidine nucleosides could be synthesized by Pd-catalyzed direct arylation of N3-free 5-halo uracil and uracil nucleosides with simple arenes or heteroaromatics in the presence of TBAF within 1 h. Both N3-protected and N3-free uracil and uracil nucleosides could undergo base-promoted Pd-catalyzed direct arylation, but only with electron rich heteroaromatics. In the second half of this dissertation, 5-acetylenic uracil and uracil nucleosides have been employed to investigate the hydrogermylation, hydrosulfonylation as well as hydroazidation for the synthesis of various functionalized 5-vinyl pyrimidine nucleosides. Hydrogermylation of 5-alkynyl uracil analogues with trialkylgermane or tris(trimethylsilyl)germane hydride gave the corresponding vinyl trialkylgermane, or tris(trimethylsilyl)germane uracil derivatives. During the hydrogermylation with triphenylgermane, besides the vinyl triphenylgermane uracil derivatives, 5-[2-(triphenylgermyl)acetyl]uracil was also isolated and characterized and the origin of the acetyl oxygen was clarified. Tris(trimethylsilyl)germane uracil derivatives were coupled to aryl halides but with decent yield. Iron-mediated regio- and stereoselective hydrosulfonylation of the 5-ethynyl pyrimidine analogues with sulfonyl chloride or sulfonyl hydrazine to give 5-(1-halo-2-tosyl)vinyluracil nucleoside derivatives has been developed. Nucleophilic substitution of the 5-(β-halovinyl)sulfonyl nucleosides with various nucleophiles have been performed to give highly functionalized 5-vinyl pyrimidine nucleosides via the addition-elimination mechanism. The 5-(β-keto)sulfonyluracil derivative has also been synthesized via the aerobic difunctionalization of 5-ethynyluracil analogue with sulfinic acid in the presence of catalytic amount of pyridine. Silver catalyzed hydroazidation of protected 2'-deoxy-5-ethynyluridine with TMSN3 in the presence of catalytic amount of water to give 5-(α-azidovinyl)uracil nucleoside derivatives was developed. Strain promoted Click reaction of the 5-(α-azidovinyl)uracil with cyclooctyne provide the corresponding fully conjugated triazole product.

STOPPFrail (Screening Tool of Older Persons Prescriptions in Frail Adults with Limited Life Expectancy): Consensus Validation

Background: To validate STOPPFrail, a list of explicit criteria for potentially inappropriate medications (PIMs) in frailer older adults with limited life expectancy. A Delphi consensus survey of an expert panel (n = 17) comprising specialists in geriatric medicine, clinical pharmacology, palliative care, psychiatry of old age, clinical pharmacy and general practice.
Methods: STOPPFrail criteria was initially created by the authors based on clinical
experience and appraisal of the available literature. Criteria were organised according to physiological system. Each criterion was accompanied by an explanation. Panellists ranked their agreement with each criterion on a 5-point Likert scale and invited to provide written feedback. Criteria with a median Likert response of 4/5 (agree/strongly agree) and a 25th centile of ≥4 were included in the final criteria.
Results: Three Delphi rounds were required. All panellists completed all rounds. Thirty criteria were proposed for inclusion; 26 were accepted. No new criteria were added. The first two criteria suggest deprescribing medications with no indication or where compliance is poor. The remaining 24 criteria include lipid-lowering therapies, alpha-blockers for hypertension, anti-platelets, neuroleptics, proton pump inhibitors, H-2 receptor antagonists, anti-spasmodics, theophylline, leukotriene antagonists, calcium supplements, bone anti-resorptive therapy, selective oestrogen receptor modulators, non-steroidal antiinflammatories, corticosteroids, 5-alpha reductase inhibitors, alpha-1 selective blockers, muscarinic antagonists, oral diabetic agents, ACE-inhibitors, angiotensin receptor blockers, systemic oestrogens, multivitamins, nutritional supplements and prophylactic antibiotics. Anticoagulants and anti-depressants were excluded. Despite incorporation of panellists’ suggestions, memantine and acetyl-cholinesterase inhibitors remained inconclusive.
Conclusion: STOPPFrail comprises 26 criteria, which have been judged by broad consensus, to be potentially inappropriate in frailer older patients with limited life expectancy. STOPPFrail may assist in deprescribing medications in these patients.

Modifications neurométaboliques et microstructurales à la suite d'une commotion cérébrale chez les athlètes féminines.

L’utilisation de méthodes d’investigation cérébrale avancées a permis de mettre en évidence la présence d’altérations à court et à long terme à la suite d’une commotion cérébrale. Plus spécifiquement, des altérations affectant l’intégrité de la matière blanche et le métabolisme cellulaire ont récemment été révélées par l’utilisation de l’imagerie du tenseur de diffusion (DTI) et la spectroscopie par résonance magnétique (SRM), respectivement. Ces atteintes cérébrales ont été observées chez des athlètes masculins quelques jours après la blessure à la tête et demeuraient détectables lorsque les athlètes étaient à nouveau évalués six mois post-commotion. En revanche, aucune étude n’a évalué les effets neurométaboliques et microstructuraux dans la phase aigüe et chronique d’une commotion cérébrale chez les athlètes féminines, malgré le fait qu’elles présentent une susceptibilité accrue de subir ce type de blessure, ainsi qu’un nombre plus élevé de symptômes post-commotionnels et un temps de réhabilitation plus long. Ainsi, les études composant le présent ouvrage visent globalement à établir le profil d’atteintes microstructurales et neurométaboliques chez des athlètes féminines par l’utilisation du DTI et de la SRM. La première étude visait à évaluer les changements neurométaboliques au sein du corps calleux chez des joueurs et joueuses de hockey au cours d’une saison universitaire. Les athlètes ayant subi une commotion cérébrale pendant la saison ont été évalués 72 heures, 2 semaines et 2 mois après la blessure à la tête en plus des évaluations pré et post-saison. Les résultats démontrent une absence de différences entre les athlètes ayant subi une commotion cérébrale et les athlètes qui n’en ont pas subie. De plus, aucune différence entre les données pré et post-saison a été observée chez les athlètes masculins alors qu’une diminution du taux de N-acetyl aspartate (NAA) n’a été mise en évidence chez les athlètes féminines, suggérant ainsi un impact des coups d’intensité sous-clinique à la tête. La deuxième étude, qui utilisait le DTI et la SRM, a révélé des atteintes chez des athlètes féminines commotionnées asymptomatiques en moyenne 18 mois post-commotion. Plus spécifiquement, la SRM a révélé une diminution du taux de myo-inositol (mI) au sein de l’hippocampe et du cortex moteur primaire (M1) alors que le DTI a mis en évidence une augmentation de la diffusivité moyenne (DM) dans plusieurs faisceaux de matière blanche. De iii plus, une approche par région d’intérêt a mis en évidence une diminution de la fraction d’anisotropie (FA) dans la partie du corps calleux projetant vers l’aire motrice primaire. Le troisième article évaluait des athlètes ayant subi une commotion cérébrale dans les jours suivant la blessure à la tête (7-10 jours) ainsi que six mois post-commotion avec la SRM. Dans la phase aigüe, des altérations neuropsychologiques combinées à un nombre significativement plus élevé de symptômes post-commotionnels et dépressifs ont été trouvés chez les athlètes féminines commotionnées, qui se résorbaient en phase chronique. En revanche, aucune différence sur le plan neurométabolique n’a été mise en évidence entre les deux groupes dans la phase aigüe. Dans la phase chronique, les athlètes commotionnées démontraient des altérations neurométaboliques au sein du cortex préfrontal dorsolatéral (CPDL) et M1, marquées par une augmentation du taux de glutamate/glutamine (Glx). De plus, une diminution du taux de NAA entre les deux temps de mesure était présente chez les athlètes contrôles. Finalement, le quatrième article documentait les atteintes microstructurales au sein de la voie corticospinale et du corps calleux six mois suivant une commotion cérébrale. Les analyses n’ont démontré aucune différence au sein de la voie corticospinale alors que des différences ont été relevées par segmentation du corps calleux selon les projections des fibres calleuses. En effet, les athlètes commotionnées présentaient une diminution de la DM et de la diffusivité radiale (DR) au sein de la région projetant vers le cortex préfrontal, un volume moindre des fibres de matière blanche dans la région projetant vers l’aire prémotrice et l’aire motrice supplémentaire, ainsi qu’une diminution de la diffusivité axiale (DA) dans la région projetant vers l’aire pariétale et temporale. En somme, les études incluses dans le présent ouvrage ont permis d’approfondir les connaissances sur les effets métaboliques et microstructuraux des commotions cérébrales et démontrent des effets délétères persistants chez des athlètes féminines. Ces données vont de pair avec la littérature scientifique qui suggère que les commotions cérébrales n’entraînent pas seulement des symptômes temporaires.

Investigating the role of glutathione and glutathione biosynthetic genes in the adaptation of Anopheles gambiae to insecticides

Malaria remains a serious public health challenge in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in Africa, and mostly in pregnant women and children under the age of five. Anopheles gambiae (An. gambiae) is the principal malaria vector in Africa, where vector control measures involve the use of insecticides in the forms of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The development of insecticides resistance mitigates these approaches. Glutathione (GSH) is widely distributed among all living organisms, and is associated with detoxification pathways, especially the Glutathione S-transferases (GSTs). Its direct involvement and relevance in insecticide resistance in An. gambiae has not been determined. Thus, this work examines the contribution of GSH, its biosynthetic genes (GCLM, GCLC) and their possible transcriptional regulator Nrf2 in insecticide resistance in An. gambiae sampled from agricultural setting (areas of intensive agriculture) and residential setting (domestic area). Bioinformatics analysis, W.H.O. adult susceptibility bioassays and molecular techniques were employed to investigate. Total RNA was first isolated from the adults An. gambiae mosquitoes raised from agricultural and residential field-caught larvae which had been either challenged or unchallenged with insecticides. Semi-quantitative RT-PCR using gel image densitometry was used to determine the expression levels of GCLM, GCLC genes and Nrf2. Bioinformatics’ results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) transcription factor binding sites in An. gambiae GCLC and GCLM promoters in silico. An. gambiae s.l. studied here were highly resistant to DDT and permethrin but less resistant to bendiocarb. Both knockdown resistance (kdr) mutation variants L1014S and L1014F that confers resistance to pyrethroid insecticides were identified in both An. coluzzii and An. arabiensis sampled from northern Nigeria. The L1014F was much associated with An. coluzzii. A significant positive correlation (P=0.04) between the frequency of the L1014F point mutation and resistance to DDT and permethrin was observed. However, a weak or non-significant correlation (P=0.772) between the frequency of the L1014S point mutation and resistance was also found. L1014S and L1014F mutations co-occurred in both agricultural and residential settings with high frequencies. However, the frequencies of the two mutations were greater in the agricultural settings than in the residential settings. The levels of total, reduced and oxidized GSH were significantly higher in mosquitoes from agricultural sites than those from residential sites. Increased oxidized GSH levels appears to correlate with higher DDT resistance. The expression levels of GCLM, GCLC and Nrf2 were also significantly up-regulated in adults An. gambiae raised from agricultural and residential field-caught larvae when challenged with insecticide. However, there was higher constitutive expression of GCLM, GCLC and Nrf2 in mosquitoes from agricultural setting. The increased expression levels of these genes and also GSH levels in this population suggest their roles in the response and adaptation of An. gambiae to insecticide challenges. There exists the feasibility of using GSH status in An. gambiae to monitor adaptation and resistance to insecticides.

Acetilação do exopolissacarídeo (1→6)-β-D-glucana (lasiodiplodana): derivatização química e caracterização

Lasiodiplodan is an exocellular β-glucan with biological functionalities such as antioxidant, antiproliferative, hypocholesterolemic, protective activity against DNA damage induced by doxorubicin and hypoglycemic activity. Chemical derivatization of polysaccharide macromolecules has been considered as a potentiating mechanism for bioactivity. In this context, this work proposes the derivatization of lasiodiplodan by acetylation. Acetic anhydride was used as derivatizing agent and pyridine as catalyst and reaction medium. The derivatives obtained were evaluated by its water solubility, degree of substitution (DS), antioxidant potential, and characterized by infrared spectroscopy (FT-IR), thermal analysis, differential scanning calorimetry, X-ray diffraction and scanning electron microscopy. Acetylated derivatives with different degrees of substitution (1.26; 1.03; 0.66 and 0.48) were obtained, and there was correlation between the concentration of derivatizing agent and DS. FT-IR spectroscopy analysis confirmed the insertion of acetyl groups into derivatized macromolecules (LAS-AC) through of specific bands concerning to carbonyl group (C = O) and increase in C-O vibration. SEM analysis indicated that native lasiodiplodan presents morphological structure in the form of thin films with translucent appearance and folds along its length. Derivatization led to morphological changes in the polymer, including aspects thickness, translucency and agglomeration. Thermal analysis indicated the native sample and derivative with DS 0.48 presented three weight loss stages. The first stage occurred until 125 ° C (loss of water) and there were two consecutive events of weight loss (200 ° C - 400 ° C) attributed to molecule degradation. Samples with DS 1.26; 1.03 and 0.66 demonstrated four weight loss stages. The first stage occurred until 130 ° C (loss of water), following by two consecutive events of weight loss (200 ° C - 392 ° C) attributed to degradation of the biopolymer. The fourth stage was between 381 ° C and 532 ° C (final decomposition) with exothermic peaks between 472 ° C and 491 ° C. X-ray diffraction patterns showed that native and acetylated lasiodiplodan have amorphous structure with semicrystalline regions. Derivatization did not contribute to increased solubility of the macromolecule, but potentiated its antioxidant capacity. Acetylation of lasiodiplodan allowed to obtaining a new macromolecule with higher antioxidant potential than the native molecule and with technological properties applicable in various industrial sectors.

In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

The structural and mechanical integrity of historic wood

Little is known about historic wood as it ages naturally. Instead, most studies focus on biological decay, as it is often assumed that wood remains otherwise stable with age. This PhD project was organised by Historic Scotland and the University of Glasgow to investigate the natural chemical and physical aging of wood. The natural aging of wood was a concern for Historic Scotland as traditional timber replacement is the standard form of repair used in wooden cultural heritage; replacing rotten timber with new timber of the same species. The project was set up to look at what differences could exist both chemically and physically between old and new wood, which could put unforeseen stress on the joint between them. Through Historic Scotland it was possible to work with genuine historic wood from two species, Oak and Scots pine, both from the 1500’s, rather than relying on artificial aging. Artificial aging of wood is still a debated topic, with consideration given to whether it is truly mimicking the aging process or just damaging the wood cells. The chemical stability of wood was investigated using Fourier-transform infrared (FTIR) microscopy, as well as wet chemistry methods including a test for soluble sugars from the possible breakdown of the wood polymers. The physical properties assessed included using a tensile testing machine to uncover possible differences in mechanical properties. An environmental chamber was used to test the reaction to moisture of wood of different ages, as moisture is the most damaging aspect of the environment to wooden cultural objects. The project uncovered several differences, both physical and chemical, between the modern and historic wood which could affect the success of traditional ‘like for like’ repairs. Both oak and pine lost acetyl groups, over historic time, from their hemicellulose polymers. This chemical reaction releases acetic acid, which had no effect on the historic oak but was associated with reduced stiffness in historic pine, probably due to degradation of the hemicellulose polymers by acid hydrolysis. The stiffness of historic oak and pine was also reduced by decay. Visible pest decay led to loss of wood density but there was evidence that fungal decay, extending beyond what was visible, degraded the S2 layer of the pine cell walls, reducing the stiffness of the wood by depleting the cellulose microfibrils most aligned with the grain. Fungal decay of polysaccharides in pine wood left behind sugars that attracted increased levels of moisture. The degradation of essential polymers in the wood structure due to age had different impacts on the two species of wood, and raised questions concerning both the mechanism of aging of wood and the ways in which traditional repairs are implemented, especially in Scots pine. These repairs need to be done with more care and precision, especially in choosing new timber to match the old. Within this project a quantitative method of measuring the microfibril angle (MFA) of wood using polarised Fourier transform infrared (FTIR) microscopy has been developed, allowing the MFA of both new and historic pine to be measured. This provides some of the information needed for a more specific match when selecting replacement timbers for historic buildings.

3-Dichloroacetyl oxazolidine protect maize from imazethapyr herbicide injury

Safeners are an important tool used to ensure the safe using of herbicide. The objective of this paper was to investigate the protective effect of four 3-dichloroacetyl oxazolidine safeners (3-dichloroacetyl-2,2-dimethyl-1,3-oxazolidine [R-28725], racemate of 3-dichloroacetyl-2,2-dimethyl-4- ethyl-1,3-oxazolidine, and its two chiral stereoisomers) in reducing the injury caused by imazethapyr. Physiological and biochemical tests were conducted under laboratory condition, by using seed treatment with safeners and soil treatment with imazethapyr, respectively. The interaction of two safeners (R-28725 and R-stereoisomer) and imazethapyr reduced the injury of maize significantly, and also increased glutathione content, activity of glutathione S-transferases, and activity of acetolactate synthase in maize. When induced by R-stereoisomer, the GSH content in root and in shoot increased 100.7% and 73.6%, respectively. When induced by R-28725, the GST activity in vivo increased threefold and the GST activity in vitro more than doubled. The kinetic parameter Vmax of GST in the maize treated with R-28725 and R-stereoisomer increased by 102.2% and 81.9%, respectively, compared with the control. The results also showed that R-28725 and R-stereoisomer induced glutathione S-transferases affinity for the substrate of conjugation reaction significantly.

Herbicides cross resistance of a multiple resistant short-awn foxtail ( Alopecurus aequalis Sobol.) population in wheat field

Alopecurus aequalis Sobol. is a common grass weed, which has become increasingly troublesome to control in China wheat fields. One A. aequalis population, collected from Anhui Province China, was suspected to be resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl. This study aimed to establish the cross-resistance pattern using the purified subpopulation and explore the potential targetsite and non-target-site based resistance mechanisms. Sequencing results showed that a single nucleotide change of ATT to AAT was present in acetyl-CoA carboxylase (ACCase) gene of the resistant (R) plants, resulting in an Ile2041Asn amino acid substitution. Besides, another single nucleotide change of CCC to CGC was present in acetolactate synthase (ALS) gene of the R plants, resulting in a Pro197Arg amino acid substitution. The homozygous resistant plants were isolated and the seeds were used in whole-plant herbicide bioassays. Compared with the susceptible (S) population, R population displayed high level resistance to fenoxaprop-P-ethyl and mesosulfuronmethyl. Cross resistance patterns showed that the R population was highly resistant to clodinafop-propargyl, moderately resistant to pyroxsulam and flucarbazoncsodium, lowly resistant to pinoxaden, and susceptible to tralkoxydim, sethoxydim, and isoproturon. The pretreatment of piperonyl butoxide reduced the 50% growth reduction (GR50) value of fenoxaprop-P-ethyl, suggesting that target-site resistance and non-target-site resistance mechanisms were both present in fenoxaprop- P-ethyl-resistance of A. aequalis. This is the first report of ACCase Ile2041Asn and ALS Pro197Arg mutation in A. aequalis.

Wood-derived biomaterials for biomedical applications

Driven by the global trend in the sustainable economy development and environmental concerns, the exploring of plant-derived biomaterials or biocomposites for potential biomedical and/or pharmaceutical applications has received tremendous attention. Therefore, the work of this thesis is dedicated to high-value and high-efficiency utilization of plant-derived materials, with the focus on cellulose and hemicelluloses in the field of biomedical applications in a novel biorefinery concept. The residual cellulose of wood processing waste, sawdust, was converted into cellulose nanofibrils (CNFs) with tunable surface charge density and geometric size through 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-mediated oxidation and mechanical defibrillation. The sawdust-based CNFs and its resultant free-standing films showed comparable or even better mechanical properties than those from a commercial bleached kraft pulp at the same condition, demonstrating the feasibility of producing CNFs and films thereof with outstanding mechanical properties from birch sawdust by a process incorporated into a novel biorefinery platform recovering also polymeric hemicelluloses for other applications. Thus, it is providing an efficient route to upgrade sawdust waste to valuable products. The surface charge density and geometric size of the CNFs were found to play key roles in the stability of the CNF suspension, as well as the gelling properties, swelling behavior, mechanical stiffness, morphology and microscopic structural properties, and biocompatibility of CNF-based materials (i.e. films, hydrogels, and aerogels). The CNFs with tunable surface chemistry and geometric size was found promising applications as transparent and tough barrier materials or as reinforcing additive for production of biocomposites. The CNFs was also applied as structural matrices for the preparation of biocomposites possessing electrical conductivity and antimicrobial activity by in situ polymerization and coating of polypyrrole, and incorporation of silver nanoparticles, which make the material possible for potential wound healing application. The CNF-based matrices (films, hydrogels, and aerogels) with tunable structural and mechanical properties and biocompatibility were further prepared towards an application as 3D scaffolds in tissue engineering. The structural and mechanical strength of the CNF matrices could be tuned by controlling the charge density of the nanocellulose, as well as the pH and temperature values of the hydrogel formation conditions. Biological tests revealed that the CNF scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells, suggesting the usefulness of the CNF-based 3D matrices in supporting crucial cellular processes during cell growth and proliferation. The CNFs was applied as host materials to incorporate biomolecules for further biomedical application. For example, to investigate how the biocompatibility of a scaffold is influenced by its mechanical and structural properties, these properties of CNF-based composite matrices were controlled by incorporation of different hemicelluloses (O-acetyl galactoglucomanan (GGM), xyloglucan (XG), and xylan) into CNF hydrogel networks in different ratios and using two different approaches. The charge density of the CNFs, the incorporated hemicellulose type and amount, and the swelling time of the hydrogels were found to affect the pore structure, the mechanical strength, and thus the cells growth in the composite hydrogel scaffolds. The mechanical properties of the composite hydrogels were found to have an influence on the cell viability during the wound healing relevant 3T3 fibroblast cell culture. The thusprepared CNF composite hydrogels may work as promising scaffolds in wound healing application to provide supporting networks and to promote cells adhesion, growth, and proliferation.

Isolamento de uma quitinase extraída do cefalotórax do camarão marinho Litopenaeus schmitti (BURKENROAD, 1936) : avaliação de suas atividades antimicrobiana e larvicida

Chitinases are enzymes involved in degradation of chitin and are present in a range of organisms, including those that do not contain chitin, such as bacteria, viruses, plants and animals, and play important physiological and ecological roles. Chitin is hydrolyzed by a chitinolytic system classified as: endo-chitinases, exo-chitinases and N-acetyl-b-D-glucosaminidases. In this study a Litochitinase1 extracted from the cephalotorax of the shrimp Litopenaeus Schmitt was purified 987.32 times using ionexchange chromatography DEAE-Biogel and molecular exclusion Sephacryl S-200. These enzyme presented a molecular mass of about 28.5 kDa. The results, after kinetic assay with the Litochitinase1 using as substrate p-nitrophenyl-N-acetyl-b-Dglucosaminideo, showed apparent Km of 0.51 mM, optimal activity at pH ranging from 5.0 to 6.0, optimum temperature at 55°C and stability when pre-incubated at temperatures of 25, 37, 45, 50 and 55°C. The enzyme showed a range of stability at pH 4.0 to 5.5. HgCl2 inhibited Litochitinase1 while MgCl2 enhances its activity. Antimicrobial tests showed that Litochitinase1 present activity against gram-negative bacterium Escherichia coli in the 800 μg/mL concentration. The larvicidal activity against Aedes aegypti was investigated using crude extracts, F-III (50-80%) and Litochitinase1 at 24 and 48 hours. The results showed larvicidal activity in all these samples with EC50 values of 6.59 mg/mL for crude extract, 5.36 mg/mL for F-III and 0.71 mg/mL for Litochitinase1 at 24 hours and 3.22 and 0.49 mg/mL for the F-III and Litochitinase1 at 48 hours, respectively. Other experiments confirmed the presence of chitin in the midgut of Aedes aegypti larvae, which may be suffering the action of Litochitinase1 killing the larvae, but also the absence of contaminating proteins as serine proteinase inhibitors and lectins in the crude extract, F-III and Litochitinase1, indicating that the death of the larvae is by action of the Litochitinase1. We also observed that the enzymes extracted from intestinal homogenate of the larvae no have activity on Litochitinase1. These results indicate that the enzyme can be used as an alternative to control of infections caused by Escherichia coli and reducing the infestation of the mosquito vector of dengue.

Effects of gamma and electron-beam irradiation on the individual phenolics of Viola tricolor L. edible flowers.

Edible flowers are being used in culinary preparations to improve the sensorial and nutritional qualities of food, besides improving human health due to the profusion in bioactive compounds [1]. Nevertheless, edible flowers are highly perishable and must be free of insects, which is difficult because they are usually cultivated without using pesticides [2]. Food irradiation is an economically viable technology to extend shelf life of foods, improving their hygiene and quality, while disinfesting insects [3]. The efficiency and safety of radiation processing (using Co-60 or electronaccelerators) have been approved by legal authorities (FDA, USDA, WHO, FAO), as also by the scientific community, based on extensive research [4]. Viola tricolor L. (heartseases), from Violaceae family, is one of the most popular edible flowers. Apart from being used as food, it has also been applied for its medicinal properties, mainly due to their biological activity and phenolic composition [5]. Herein, the phenolic compounds were analyzed by HPLC-DAD-ESI/MS and linear discriminant analysis (LDA) was performed to compare the results from flowers submitted to different irradiation doses and technologies (Co-60 and electron-beam). Quercetin-3-O-(6-O-rhamnosylglucoside)-7-O-rhamnoside (Figure 1) was the most abundant compound, followed by quercetin-3-O-rutinoside and acetyl-quercetin-3-O (6-O-rhamnosylglucoside)-7-O-rhamnoside. In general, irradiated samples (mostly with 1 kGy) showed the highest phenolic compounds content. The LDA outcomes indicated that differences among phenolic compounds effectively discriminate the assayed doses and technologies, defining which variables contributed mostly to that separation. This information might be useful to define which dose and/or technology optimizes the content in a specific phenolic compound. Overall, irradiation did not negatively affect the levels of phenolic compounds, providing the possibility of its application to expand the shelf life of V. tricolor and highlighting new commercial solutions for this functional food.

Purificação e caracterização parcial de duas N-acetil-ß-hexosaminidases do Equinoderma marinho Echinometra lucunter

Two b-N-acetylhexosaminidases (F11 e F15) were purified from Echinometra lucunter gonads extracts. The purified enzymes were obtained using ammonium sulfate fractionation, followed by gel filtration chromatographies (Sephacryl S-200, Sephadex G-75 and Sephacryl S-200). The F11 fraction was purified 192.47 -fold with a 28.5% yield, and F15 fraction 85.41 -fold with a 32.3% yield. The molecular weights of the fractions were 116 kDa for F11 and 42 kDa for F15 using SDS-PAGE. In Sephacryl S-200, F15 was 84 kDa, indicating that it is a dimeric protein. When p-nitrophenyl-β-D-glycosaminide was used as substrate, we determined an apparent Km of 0.257 mM and Vmax of 0.704 for F11 and for F15 the Km was 0.235 mM and Vmax of 0.9 mM of product liberated by hour. Both enzymes have optimum pH and temperature respectively at 5.0 and 45 °C. The enzymes showed inhibition by silver nitrate, while the glucuronic acid was a potent activator. The high inhibition of F15 by N-etylmaleimide indicates that sulphydril groups are involved in the catalysis of synthetic substrate

Modifications neurométaboliques et microstructurales à la suite d'une commotion cérébrale chez les athlètes féminines

L’utilisation de méthodes d’investigation cérébrale avancées a permis de mettre en évidence la présence d’altérations à court et à long terme à la suite d’une commotion cérébrale. Plus spécifiquement, des altérations affectant l’intégrité de la matière blanche et le métabolisme cellulaire ont récemment été révélées par l’utilisation de l’imagerie du tenseur de diffusion (DTI) et la spectroscopie par résonance magnétique (SRM), respectivement. Ces atteintes cérébrales ont été observées chez des athlètes masculins quelques jours après la blessure à la tête et demeuraient détectables lorsque les athlètes étaient à nouveau évalués six mois post-commotion. En revanche, aucune étude n’a évalué les effets neurométaboliques et microstructuraux dans la phase aigüe et chronique d’une commotion cérébrale chez les athlètes féminines, malgré le fait qu’elles présentent une susceptibilité accrue de subir ce type de blessure, ainsi qu’un nombre plus élevé de symptômes post-commotionnels et un temps de réhabilitation plus long. Ainsi, les études composant le présent ouvrage visent globalement à établir le profil d’atteintes microstructurales et neurométaboliques chez des athlètes féminines par l’utilisation du DTI et de la SRM. La première étude visait à évaluer les changements neurométaboliques au sein du corps calleux chez des joueurs et joueuses de hockey au cours d’une saison universitaire. Les athlètes ayant subi une commotion cérébrale pendant la saison ont été évalués 72 heures, 2 semaines et 2 mois après la blessure à la tête en plus des évaluations pré et post-saison. Les résultats démontrent une absence de différences entre les athlètes ayant subi une commotion cérébrale et les athlètes qui n’en ont pas subie. De plus, aucune différence entre les données pré et post-saison a été observée chez les athlètes masculins alors qu’une diminution du taux de N-acetyl aspartate (NAA) n’a été mise en évidence chez les athlètes féminines, suggérant ainsi un impact des coups d’intensité sous-clinique à la tête. La deuxième étude, qui utilisait le DTI et la SRM, a révélé des atteintes chez des athlètes féminines commotionnées asymptomatiques en moyenne 18 mois post-commotion. Plus spécifiquement, la SRM a révélé une diminution du taux de myo-inositol (mI) au sein de l’hippocampe et du cortex moteur primaire (M1) alors que le DTI a mis en évidence une augmentation de la diffusivité moyenne (DM) dans plusieurs faisceaux de matière blanche. De iii plus, une approche par région d’intérêt a mis en évidence une diminution de la fraction d’anisotropie (FA) dans la partie du corps calleux projetant vers l’aire motrice primaire. Le troisième article évaluait des athlètes ayant subi une commotion cérébrale dans les jours suivant la blessure à la tête (7-10 jours) ainsi que six mois post-commotion avec la SRM. Dans la phase aigüe, des altérations neuropsychologiques combinées à un nombre significativement plus élevé de symptômes post-commotionnels et dépressifs ont été trouvés chez les athlètes féminines commotionnées, qui se résorbaient en phase chronique. En revanche, aucune différence sur le plan neurométabolique n’a été mise en évidence entre les deux groupes dans la phase aigüe. Dans la phase chronique, les athlètes commotionnées démontraient des altérations neurométaboliques au sein du cortex préfrontal dorsolatéral (CPDL) et M1, marquées par une augmentation du taux de glutamate/glutamine (Glx). De plus, une diminution du taux de NAA entre les deux temps de mesure était présente chez les athlètes contrôles. Finalement, le quatrième article documentait les atteintes microstructurales au sein de la voie corticospinale et du corps calleux six mois suivant une commotion cérébrale. Les analyses n’ont démontré aucune différence au sein de la voie corticospinale alors que des différences ont été relevées par segmentation du corps calleux selon les projections des fibres calleuses. En effet, les athlètes commotionnées présentaient une diminution de la DM et de la diffusivité radiale (DR) au sein de la région projetant vers le cortex préfrontal, un volume moindre des fibres de matière blanche dans la région projetant vers l’aire prémotrice et l’aire motrice supplémentaire, ainsi qu’une diminution de la diffusivité axiale (DA) dans la région projetant vers l’aire pariétale et temporale. En somme, les études incluses dans le présent ouvrage ont permis d’approfondir les connaissances sur les effets métaboliques et microstructuraux des commotions cérébrales et démontrent des effets délétères persistants chez des athlètes féminines. Ces données vont de pair avec la littérature scientifique qui suggère que les commotions cérébrales n’entraînent pas seulement des symptômes temporaires.