Heritage apple cultivars for cider-making are often distinguished by a high concentration of tannins (phenolic compounds), and/or acid. The phenolic content of some cider apples far exceeds that of white wine, however most cider fermentation practices are directly taken from white winemaking, not accounting for effects of high concentrations of phenolic compounds on yeast fermentation. The objective of this study was to determine the impact of ferulic acid, p-coumaric acid, and chlorogenic acid—at concentrations reported in apples—and their interactions with yeast assimilable nitrogen (YAN) on fermentation kinetics and cider aroma. Our hypothesis was that the phenolic compounds present in high-tannin cider apples would negatively impact fermentation kinetics, but not alter the aroma, and that added YAN would reduce these effects. Ferulic acid negatively affected fermentation performance (p < 0.05), but p-coumaric acid and chlorogenic acid did not. p-Coumaric acid led to the greatest changes in cider aroma. Differences were also detected for different concentrations of ferulic acid. Chlorogenic acid did not affect aroma. Yeast strain influenced fermentation performance and cider aroma. Finally, addition of exogenous YAN improved fermentation performance for the low concentration ferulic acid condition, but not for the high concentration. Adding YAN also changed cider aroma in the presence of p-coumaric acid. / Master of Science in Life Sciences / Hard cider is increasingly popular in the United States. Heritage apple cultivars are traditional cider apples, often distinguished by a high concentration of tannins (phenolic compounds), and/or acid, unlike dessert apples that are typically higher in sugar than tannins. While the phenolic content of some cider apples far exceeds that of white wine, most cider fermentation practices are directly taken from white winemaking, and do not account for the effects of high concentrations of phenolic compounds on fermentation performance. The objective of this study was to determine whether three phenolic compounds—ferulic acid, p-coumaric acid, and chlorogenic acid—at concentrations reported in apples, would inhibit fermentation or alter the aroma of the cider, and if adding yeast assimilable nitrogen (YAN), a yeast nutrient, would reduce these effects. Our hypotheses were that the phenolic compounds present in high-tannin cider apples would negatively impact fermentation performance, but not alter the aroma, and that added YAN would reduce these effects. Ferulic acid negatively affected fermentation performance (p < 0.05), but p-coumaric acid and chlorogenic acid did not. Addition of nitrogen improved fermentation performance for the low concentration ferulic acid condition, but not for the high concentration. p-Coumaric acid led to the greatest changes in cider aroma, with differences in aroma also detected for ferulic acid ciders. Chlorogenic acid did not affect aroma or fermentation performance. Yeast strain and YAN addition also influenced fermentation performance and aroma.

4-Ethylphenol (4-EP) is a malodorant of swine waste and is derived from a component of lignin called p-coumaric acid (p-CA). The production of 4-EP from lignin in swine waste is untested. Additionally, the effect of Fe (III) on 4-EP levels is unknown. Four experiments were performed to determine if Lactobacillus sp. pep8 cultures, as well as enriched swine lagoon slurries, could liberate p-CA from lignin and convert p-CA to 4-EP. Furthermore, it was tested if the addition of Fe (III) influences the conversion of p- CA to 4-EP. Experiment 1 tested Lactobacillus sp. pep8 cultures to determine if the addition of 10 mM Fe (III) and 0.2% sulphite lignin to Lactobacillussp. pep8 cultures would stimulate production of 4-EP. Experiment 2 tested the effect of 0.2% sulphite lignin and 10 mM Fe (III) on 4-EP production in the presence of enriched swine lagoon slurries. On day 0 there was no detectable 4‐EP, for either 0.2% sulphite lignin addition or the 10 mmol l‐1 Fe (III) additions. Experiment 3 tested alternative forms of lignin, including 0.2% sulphite, indulin, or sigma lignin as potential source compounds for 4-EP production in enriched swine lagoon slurries. 4-EP produced in all three conditions are likely endogenous to the lagoon slurry additions. Experiment 4 was designed to measure the degradation of exogenous 4-EP with varying final concentrations of 4-EP in enriched swine lagoon slurries. Data in Figure 7 indicate immediate degradation of 4-EP by day 5, however, by day 7 synthesis of 4-EP occurred until day 14 where 4-EP levels remained in a steady state. Our results suggest that when both Lactobacillus sp. pep8 cultures and enriched swine lagoons are supplemented with p-CA, 4-EP is produced indicating that p-CA serves as a source of 4-EP. However, when supplemented with Fe (III) and/or sulphite, indulin, or sigma lignin, 4-EP production was not stimulated. This data indicates that, 4- EP production is not enhanced by the presence of Fe (III) in either Lactobacillus sp. pep8 cultures or in enriched swine lagoon slurries. Furthermore, lignin did not serve as a source of 4-EP.

Orientador: Marcos Antonio Correa / Resumo: O ácido p-cumárico (APC) é um composto fenólico não flavonóide que apresenta segurança e eficácia bem descritas na ciência dos alimentos devido suas propriedades antioxidante, antitumoral, anti-inflamatória, antimicrobiana e por apresentar baixa toxicidade oral. Essas propriedades são pouco exploradas em produtos para cuidados com a pele. O objetivo do presente estudo foi avaliar de forma in vitro a segurança e a eficácia do APC e incorporá-lo em emulsão cosmética óleo em água (O/A). Nossos estudos in vitro e alternativos ao uso de animais foram conduzidos para verificar a citotoxicidade do APC em células de queratinócitos (HaCat) e fibroblastos humanos (HDFa), o potencial de irritação ocular (HET-CAM) e a atividade antimicrobiana em diferentes cepas. Emulsões contendo APC foram submetidas ao estudo de estabilidade e de liberação, retenção e permeação cutâneas. Resultados: o IC50 do APC em células HaCat e HDFa foi 693,57 e 2.814,75 μg/mL, respectivamente. Além disso 100 μg de APC foram levemente tóxicos em membrana corioalantóica de ovo embrionado. As concentrações bactericidas mínimas em cepas de E. coli, S. aureus, P. aeruginosa e S. epidermidis, foram 3.000 μg/mL, 1.875 μg/mL, 3.750 μg/mL e 3.750 μg/mL, respectivamente. Emulsões contendo diferentes concentrações de APC foram estáveis durante o período de estabilidade acelerada. Após 12 horas 91,08 ± 0,03% do APC foi liberado da formulação e após 24 horas, 0,52 ± 0,18% e 0,94 ± 0,37% ficaram retidos no estrato córneo e na e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: p-Coumaric acid (PCA) is a non flavonoid phenolic compound whose safety and efficacy widely described in food science papers due to antioxidant, antitumor, anti inflammatory properties, and low oral toxicity. These characteristics are poorly or no described when PCA is added to skin care products. The aim of the present study was to evaluate the safety and efficacy of PCA through in vitro assays, and then add it in oil in water cosmetic emulsion (O/W). Our in vitro trials, without animal use were conducted to predict PCA´s cytotoxicity in HaCat and HDFa cells, ophthalmic irritation potential, and antibacterial activity against differents strains. Emulsions with PCA has been tested to predict shelf life, dermal absorption, and dermal retention. Results: PCA´s IC50 in HaCat and HDFa cells was 693.57 and 2,814.75 μg.mL-1, respectively. In addition 100 μg of APC were slightly toxic in chorioallantoic membrane assay. The minimum bactericidal concentrations against E. coli, S. aureus, P. aeruginosa and S. epidermidis strains were 3,000 μg.mL-1, 1,875 μg.mL-1, 3,750 μg.mL-1, and 3,750 μg.mL-1 , respectively. Emulsions containing 0.04%, 0.06%, 0.08% and 0.10% of PCA were stable during self life testing for ninety days. After 12 hours 91.08 ± 0.03% of PCA was released of the emultion, and after 24 hours 0.52 ± 0.18% and 0.94 ± 0.37%, were present in stratum corneum and epidermis/dermis, respectively. Our findings indicate that PCA concentrations would be effective and safe to use in c... (Complete abstract click electronic access below) / Mestre

Most of the biological systems in nature are sustained by molecular self-assemblies which are the finest examples of supramolecular architectures. Non-covalent interactions are key concepts which govern these molecular assemblies. Inspired by these examples crystal engineering emerged as an important tool in supramolecular chemistry which aids in the invention of new molecular structures with desired properties. Understanding of how the molecules interact at the molecular levels enables one to rationally design novel solid forms with modulated physicochemical properties. This feature of crystal engineering has heightened its position in materials chemistry and is currently one of the most well studied fields for generating novel compounds with pre-defined composition and supramolecular architectures. One such class of compounds that has immensely attracted the scientific community and is under continuous study for wider applications is cocrystals. The applications include various interdisciplinary fields such as pharmaceutics, catalysis, organic conductors, explosives etc. Distinctly on the other side, cocrystals also provide a means to discover new supramolecular synthons which is the ultimate key to molecular assembly. Many robust supramolecular synthons have been discovered and hierarchies are also being developed which can serves as a design tool for cocrystal synthesis. The Cambridge Structural Database (CSD) is an important accessory in determining the robustness of a supramolecular synthon but, this does not preclude us from discovering new synthons. The work presented here explores new persistent supramolecular synthons in polyphenols utilizing the basic concepts of crystal engineering and the CSD statistical analysis. This contribution also includes the implementation of cocrystallization for various categories of compounds which includes nutraceuticals, pharmaceuticals and ionic salts for the design and synthesis of molecular and ionic cocrystals. Chapter 1 highlights how supramolecular synthon approach can be used to design and synthesize multi-component crystals, namely, cocrystals. The role of the CSD and its importance in crystal engineering has also been discussed. Chapters 2 and 3 focus on new persistent supramolecular synthons in the context of nutraceuticals. The cocrystals isolated in the study are also compared with the existing cocrystals in the CSD supramolecularly in terms of synthon formation. These persistent supramolecular synthons are helpful in developing hierarchies which could be utilized and applied to similar and analogous compounds. The main feature of Chapter 4 is expanding the field of cocrystallization by studying the properties of cocrystals. Some of the properties which have been examined here include effects of cocrystallization on solubility and correlations between the solubility of cocrystal with cocrystal former (CCF) and melting point of the cocrystal. The extension of cocrystals to the active pharmaceutical ingredients (APIs) has been explored in the context of pharmaceutical cocrystals by selecting a BCS class IV drug, hydrochlorothiazide in Chapter 5. Chapter 6 highlights the hybridization of organic and inorganic components for the synthesis of ionic cocrystals and is exemplified by considering lithium salts with achiral and homochiral amino acids for the generation of 1:1 and 1:2 cocrystals.

Tyrimo tikslas: sumodeliuoti puskietes sistemas su bičių produktais ir įvertinti jų kokybę biofarmaciniais tyrimais. Uždaviniai: 1. Atrinkti tinkamą propolio, kaip veikliosios medžiagos, formą įvedimui į modeliuojamas puskietes sistemas; 2. Pagaminti stabilias puskietes formas su bičių produktais (bičių vašku, propoliu, medumi) ir įvertinti jų kokybę, tiriant šiuos parametrus: vienalytiškumas, pH reikšmė, antioksidacinis aktyvumas; 3. Atlikti puskiečių sistemų su bičių produktais biofarmacinį vertinimą ir identifikuoti veiksnius, darančius įtaką bendro fenolinių junginių kiekio atpalaidavimui; 4. Įvertinti puskiečių formų su bičių produktais kokybę, remiantis atliktais veikliųjų junginių (fenolinių rūgščių, vanilino) atpalaidavimo in vitro tyrimo rezultatais. Bičių produktų puskiečių sistemų analizei pasirinktas fenolinių junginių nustatymas spektrofotometriniu ir ESC metodais bei spektrofotometrinis metodas laisvųjų DPPH• radikalų surišimo aktyvumui pagal troloksą nustatyti. Vaisto formų kokybė vertinta nustatant pH reikšmę ir atliekant veikliųjų junginių atpalaidavimo tyrimą in vitro. Į puskietes sistemas veiklioji medžiaga (propolis) įterpiama tirštojo arba skystojo (gelyje) ekstrakto forma. Stabilios I grupės sistemos pagaminamos tada, kai bičių vaško ir saulėgrąžų aliejaus santykis ne didesnis kaip 1:5. Bičių vaškas ir medus daro įtaką atpalaiduojamam veikliųjų junginių kiekiui – didėjant vaško kiekiui, veikliųjų junginių iš sistemų atsipalaiduoja mažesnis kiekis, o iš... [toliau žr. visą tekstą] / The aim of work: Formulate and analyze semisolid preparations with bees products and evaluate their quality by biopharmaceutical researches. Main tasks: 1. Select the proper form of propolis as an active material in formulated semisolid systems; 2. Formulate stable semisolid systems with bees products (beeswax, propolis, honey) and evaluate their quality by investigation of these parameters: homogenity, pH value, antioxidant activity; 3. Perform biopharmaceutical evaluation of semisolid systems with bees products and identify the factors that influence release the total amount of phenolic compounds; 4. Evaluate quality of semisolid systems with bees products based on active compounds (phenolic acids, vanillin) in vitro release test results. Semisolid systems with bees products are analyzed by using spectrophotometric and capillary HPLC methods for analysis of total phenolic compounds measuring, DPPH• free radical scavenging activity measuring according to trolox. Quality of the systems was evaluated by measuring pH values and by testing release of active compounds in vitro. The active substance propolis is inserted into semisolid systems by thick or liquid (in gels) extract form. Stable system of the Group I semisolid systems is formulated when beeswax and sunflower oil ratio is less than 1:5. Beeswax and honey in systems affect the release of active compounds - increasing the wax content, the active compounds release of the systems is reduced, adequate quantity of honey... [to full text]

Orientadoresr: Daniele Ribeiro de Araujo, Eneida de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T08:56:23Z (GMT). No. of bitstreams: 1 Guilherme_VivianeAparecida_M.pdf: 3020599 bytes, checksum: ce06e2f6da5344128ba90d18a9b569f2 (MD5) Previous issue date: 2012 / Resumo: Compostos anti-inflamatórios não esteroídais (AINEs) são amplamente utilizados para o combate da inflamação, mas frequentemente acarretam efeitos adversos que impedem a continuação do tratamento. Atualmente, a terapia a base de plantas tem sido bastante empregada a fim de desenvolver novos fármacos que apresentem eficácia analgésica e anti-inflamatória. Nesse contexto, a utilização de sistemas carreadores, como as nanopartículas de hidróxidos duplos lamelares (HDL), visa melhorar propriedades biofarmacêuticas e farmacológicas dos compostos nelas intercalados. Dessa forma, este estudo teve como objetivo caracterizar a interação entre o ácido coumárico (AC), um composto fenólico extraído de plantas, e hidróxido duplo lamelares (HDL), nanopartículas inorgânicas, considerando parâmetros como a cinética de liberação in vitro, a citotoxicidade e as atividades analgésica-antiinflamatória em relação ao fármaco não intercalado. Os ensaios de liberação in vitro foram realizados utilizando uma célula de difusão vertical (com membrana de acetato de celulose, MWCO 1000 Da) e a viabilidade celular avaliada em células 3T3 pelo teste de incorporação do vermelho neutro (VN). Os testes farmacológicos realizados em camundongos swiss foram: a determinação do número de contorções abdominais, teste tail-flick, ensaios de peritonite e teste de formalina para o composto livre e intercalado nas concentrações de 10, 20 ou 30 mg/kg. Os ensaios de liberação in vitro mostraram que a intercalação reduziu significativamente a constante de liberação (Klib) do AC, em relação ao fármaco livre, sendo os valores de Klib de 41,6 ± 1,5 %.h-1 e 32,4 ± 1,5 %.h-1 , para o AC e AC-HDL, respectivamente. A viabilidade celular foi reduzida apenas em concentrações mais elevadas de AC e AC-HDL (10 e 12,5 mM). Porém, mesmo nestas concentrações foi observada porcentagem de viabilidade celular maior que 50%. Por fim, a avaliação farmacológica apontou o AC-HDL como um sistema de liberação com atividades analgésica e anti-inflamatória mais pronunciadas do que as observadas para anti-inflamatórios não esteroidais como a indometacina (p< 0,001). Esses efeitos foram obtidos para teste de tail-flick, quando o AC foi intercalado em HDL aumentou a duração da analgesia (~ 1,7 vezes) quando comparado com o grupo de controle indometacina. Assim, os resultados indicam que o AC intercalado em nanopartículas inorgânicas de HDL apresentou uma taxa de liberação lenta e também induziu uma atividade analgésica - anti-inflamatória, possivelmente, por um mecanismo semelhante ao observado para um anti-inflamatório não esteroidais como a indometacina / Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to avoid inflammation, but these compounds can evoke some side effects, considering these as an important limitation to the treatment. Currently, the plant-based therapy has been widely employed to develop new drugs which have analgesic and anti-inflammatory pharmacological activities. In this context, the use of inorganic nanoparticles is important to improve biopharmaceutical and pharmacological properties of the intercalated molecules. Thus, this study aimed to characterize the interaction between coumaric acid (CA), a phenolic compound extracted from plants, and layered double hydroxide (LDH), inorganic nanoparticles, considering parameters such as the in vitro release kinetics cytotoxicity and analgesic-antiinflammatory activities compared to the non-intercalated-CA. In vitro release tests were performed using a vertical diffusion cell (with cellulose acetate membrane, MWCO 1000 Da) and cell viability assessed in 3T3 cells by the neutral red (NR) uptake test. Pharmacological assays were carried in Swiss mice out in order to determine the number of writhings, tail-flick test, peritonitis test and formalin test for the free compound at three different concentrations (10, 20 or 30 mg/kg). In vitro release tests showed that the release constant (Krel) value was significantly reduced when compared to the non-intercalated CA (Krel values of 41.6 ± 1.5 %.h-1 and 32.4 ± 1.5 %.h-1 for the free CA and CA-HDL, respectively). Cell viability was reduced only at higher concentrations (10 and 12.5 mM) of CA and CA-HDL. However, even at these concentrations the percentage of cell viability was more than 50%. Finally, the pharmacological evaluation reveal the CA-HDL as a drug-release system with more pronounced analgesic-antiinflammatory effects than those observed for classic NSAIDs, such as indomethacin (p <0.001). Those effects were obtained, specially, for tail-flick test, when the treatment with CA-LDH increased the duration of analgesia (~ 1.7 times), when compared with the control group (indomethacin). Thus, the results pointed out that the system CA-LDH showed a slow release rate and also induced in vivo analgesic-anti-inflammatory activities, possibly using similar mechanisms to that observed for classic non-steroidal anti-inflammatory drugs, such as indomethacin / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Marlus Chorilli / Resumo: A candidíase vulvovaginal (CVV) é o tipo mais comum de vaginite aguda entre as mulheres, causada por fungos do gênero Candida spp. A Candida albicans é a espécie responsável por 80-90% dos casos, contudo nos últimos anos, houve um aumento no número de casos envolvendo outras espécies, sendo a Candida glabrata, a segunda mais reportada e mais resistente aos tratamentos convencionais. As terapias existentes para CVV apresentam desvantagens devido aos efeitos colaterais dos agentes antifúngicos utilizados e resistência fúngica prevalente. Como alternativa, compostos naturais como o ácido p-cumárico (p-AC) vêm sendo estudados devido à sua potencial atividade antifúngica. Todavia, seu caráter lipofílico dificulta sua solubilidade em água, diminuindo sua eficácia. A incorporação do p-AC em um sistema de liberação de fármacos (SLF), como os cristais líquidos (CLs), pode auxiliar na sua administração tópica, e assim aumentar sua solubilidade, eficácia, reduzir efeitos colaterais sistêmicos e potencializar sua ação na mucosa vaginal. Dessa forma, este trabalho teve como objetivo desenvolver sistemas liquido-cristalinos com propriedades mucoadesivas para liberação controlada do ácido p-cumárico no tratamento de candidíase vulvovaginal, via administração tópica. A atividade antifúngica do p-AC foi avaliada a partir dos ensaios de concentração inibitória mínima (CIM), concentração fungicida mínima (CFM) , formação de biofilme e ensaio in vivo contra cepas de C. albicans (SC5314), C. glab... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Vulvovaginal candidiasis (VVC) is the most common type of acute vaginitis among women, caused by fungi of the genus Candida spp. Candida albicans is the species responsible for 80-90% of cases, however in recent years, there has been an increase in the number of cases involving other species, with Candida glabrata being the second most reported and most resistant to conventional treatments. Existing therapies for VVC have disadvantages due to the side effects of the antifungal agents used and the prevailing fungal resistance. As an alternative, natural compounds such as p-cumaric acid (p- CA) have been studied due to their potential antifungal activity. However, its lipophilic character hinders its solubility in water, reducing its effectiveness. The incorporation of p-CA in a drug delivery system (DDS), such as liquid crystals (LCs), can assist in its topical administration, and thus increase its solubility, effectiveness, reduce systemic side effects and enhance its activity on the vaginal mucosa. Thus, this work aimed to develop liquid-crystalline systems with mucoadhesive properties for controlled release of p-cumáric acid in the treatment of vulvovaginal candidiasis, via topical administration. The antifungal activity of p-CA was analysed by assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), biofilm formation and in vivo assay against strains of C. albicans (SC5314), C. glabrata (ATCC 2001) and C. krusei (ATCC 6528). For the develop... (Complete abstract click electronic access below) / Mestre

Les altérations sensorielles des vins dues à la présence des levures du genre Brettanomyces se caractérisent par une augmentation de la teneur en phénols volatils tel que le 4-éthylphénol. Le premier objectif de ce travail était de faire un état des lieux sur le risque « éthylphénol » au Liban en s'intéressant à la présence d'un précurseur (acide p-coumarique), du microorganisme responsable (Brettanomyces) et du produit final (4-éthylphénol) dans les vins élaborés dans ce pays. Une forte hétérogénéité de concentrations en acide p-coumarique a été observée avec des valeurs variant de 0 à 31,4 mg.L-1. Des niveaux importants de 4-éthylphénols de l'ordre de 1,367 mg.L-1 ont été détectés sur certains vins. Un dépistage du contaminant microbien a permis de confirmer pour la première fois la présence de Brettanomyces au Liban, les proportions restant toutefois assez faibles (3 % des échantillons testés). Une étude génétique a caractérisé les souches retenues qui se sont montrées diverses au sein de l'espèce. Le travail a porté ensuite sur l'analyse cinétique des étapes réactionnelles constituant le processus enzymatique de la bioconversion des substrats acide p-coumarique et 4-vinlphénol en 4-éthylphénol pour 5 souches de Brettanomyces bruxellensis d'origines libanaises et françaises. La variabilité entre les souches s'est exprimée aux niveaux génétique et cinétique. Des profils hétérogènes de bioréaction ont été mis en évidence en fonction de la nature des souches. L'analyse du bilan-matière a révélé l'existence probable de phénomènes d'adsorption sur les parois des Brettanomyces qui sont souche-dépendants. La dernière partie a été consacrée à l'évaluation du lien entre quantité de biomasse et production de 4-éthylphénol ainsi qu'à l'influence de quelques paramètres environnementaux (pH, source d'ammonium et milieu de culture) sur la cinétique réactionnelle. / Wine sensory alterations due to the presence of Brettanomyces yeasts are characterized by an increased content of volatile phenols such as 4-ethylphenol. The first aim of this work was to make an inventory of the "ethylphenol" risks in Lebanon by focusing on the presence of one precursor (p-coumaric acid), the microorganism provoking these risks (Brettanomyces) and the final product (4-ethylphenol) in wines produced in this country. High heterogeneity of p-coumaric acid concentration was observed with values ranging from 0 to 31,4 mg.L-1. Significant levels of 4-ethylphenols of about 1,367 mg L-1 have been detected in some wines. Screening of microbial contaminants confirmed the presence of Brettanomyces for the first time in Lebanon, with proportions remaining relatively low (3 % of samples tested). A genetic study has characterized the selected strains which are shown to be various within the species. The second objective of this study was the kinetic analysis of the reaction steps constituting the bioconversion enzymatic process of both substrates p-coumaric acid and 4-vinlphenol into 4-ethylphenol for 5 strains of Brettanomyces bruxellensis of different origins (Lebanon and France). Variability between strains was expressed at both levels, genetic and kinetic. Heterogeneous bioreaction profiles were identified according to strain's nature. The mass balance analysis revealed the possible existence of adsorption phenomena on the cell walls of Brettanomyces which are strain-dependent. The last part was devoted to the evaluation of the relationship between biomass concentration and production of 4-ethylphenol as well as the influence of some environmental parameters (pH, ammonium source and culture medium) on the reaction's kinetic.

Philosophiae Doctor - PhD / The role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants.

Biologiska processer hos olika organismer påverkas av ultraviolett-B-strålning (UV-B). Kunskapen om förändringar i UV-B-flödet som nått planetens yta under det geologiska tidsspann sedan flercelligt liv uppstod är dock begränsad. Följaktligen har olika biologiska proxies använts som ett sätt att försöka rekonstruera UV-B-flödet som når jordens yta. Växter reagerar på fluktuationen av UV-B genom att variera produktionen av fenolföreningar såsom parakumarsyra (pCA) och ferulinsyra, och därför kan dessa beståndsdelar användas som UV-B-proxys. UV-B-absorberande föreningar (UAC) finns i växternas yttersta skikt, den så kallade kutikulan men även i pollen och sporer, vilka oftaär välbevarade i det geologiska arkivet. Ändå är användningen av kutikula som UV-B-proxy inte lika undersökt. Därför syftar denna magisteruppsats till att analysera kemiska förändringar i fossila växters från trias – jura-gränsen (c. 200 miljoner år gamla)  och deras potential som UV-B-proxys. Eftersom de fossila löven behövde rengöras från sediment och från förkolnat mesofyll, gjordes också en analys av de kemiska förändringarna som orsakades av de olika rengöringsstegen. Icke-destruktiva tekniker, som mikro-FTIR och konfokalt Raman-mikroskop, användes för analysen. Försöken visade att analysen endast kunde utföras med mikro-FTIR på grund av den höga auto-fluorescensen hos de fossila bladen  vilketförhindrade analys med Raman. Signifikanta förändringar orsakades då Schulzes reagens användes för borttagning av mesofyllet, vilket ledde till nitrering av kutikulan. Ett mindre aggressivt och mer hållbart alternativ till denna process har visat sig vara väteperoxid, vilket orsakade mindre förändringar av den fossila kutikulans kemiska sammansättning. Dessutom upptäcktes indikation på förekomst av UAC endast i Ginkgoites regnellii, vilket indikerar att eventuell nedbrytning av dessa föreningar inträffade under diagenesen av de andra analyserade fossilerna. / Biological processes of different organisms are impacted by ultraviolet-B radiation (UV-B). However, knowledge about alterations in the UV-B flux reaching the planet’s surface during the geological past is limited. Consequently, different biological proxies have been used as an approach to attempt reconstructing the UV-B flux reaching Earth’s surface. Plants respond to the fluctuation of UV-B by varying the production of phenolic compounds such as p-coumaric acid (pCA) and ferulic acid, and therefore these constituents can be used as UV-B proxies. UV-B-absorbing compounds (UACs) can be found in the cuticle, pollen, and spores, which are all well-preserved in the geological record. Nevertheless, the use of cuticles as UV-B proxy is not as explored. Hence, this Master’s thesis aimed to analyse chemical changes in the plant cuticles from the Triassic–Jurassic boundary and their potential as UV-B proxies. Moreover, as the cuticles needed to be cleaned from rocks and isolated from coalified mesophyll, an analysis of the chemical changes caused by the cleaning steps was also done. Non-destructive techniques, as micro-FTIR and confocal Raman microscope, were used for the analysis.  Yet, the analysis could only be conducted with micro-FTIR due to auto-fluorescence of the leaves preventing success with Raman. Main changes during the cleaning steps were caused using Schulze’s reagent, which led to nitration of the cuticles. A less aggressive and more sustainable alternative to this process is shown to be hydrogen peroxide, which caused minor alteration of the fossil cuticle’s chemical composition.  Moreover, indication of the presence of UACs was detected only in Ginkgoites regnellii, indicating that possible degradation of these compounds occurred during diagenesis of the other fossils analysed.