Bio-plastics have emerged as a promising alternative to conventional petrochemical derived plastics particularly over the past few decades. Numerous production methods for bio-plastics have been researched; however, work remains towards developing a commercially viable and economical process. The purpose of this research was to develop a sustainable fermentation strategy for production and scale-up of medium-chain length polyhydroxyalkanoates (mcl-PHAs), or bio-plastics, using a novel strain of the gram negative bacterium Pseudomonas putida, LS46, with biodiesel derived waste glycerol (WG) as feedstock. Experiments were conducted to gain a basic understanding of the general growth patterns exhibited by LS46. Thereafter, flask-batch experiments were conducted to study effects of variation in media conditions upon cell biomass production and mcl-PHA accumulation. Subsequently, optimal medium conditions observed within flasks were scaled-up and employed in the operation of a pilot-scale fermenter to increase production capacity for mcl-PHAs. It was concluded that mcl-PHA production at commercial levels could be viable with advanced process optimization. / October 2015

El rápido y marcado progreso que se ha venido dando en los últimos años en la ciencia y tecnología de los polímeros ha creado nuevos materiales plásticos con excelentes propie-dades físicas y durabilidad. Sin embargo, los productos plásticos tienen usualmente aplicaciones para un solo uso, especialmente en el envasado de alimentos y en aplicaciones médicas. Dado que estos materiales no son biodegradables pueden permanecer en la superficie de la tierra por cientos de años sin cambios considerables en su estructura. Debido a esto, se han incrementado, en los últimos años, los intereses sociales y gubernamentales de modo tal de contrarrestar los efectos nocivos para el medio ambiente; enfocándose los esfuerzos hacia la producción de nuevos materiales biodegra-dables a partir de estructuras económica y ambientalmente sostenibles. Los poli(hidroxialcanoato)s, PHAs, una gran familia de biopoliésteres naturales, son una de las alternativas más viable para la sustitución de plásticos de origen petroquímico. En esta tesis se hace una contribución tanto a nivel experi-mental como teórica con el objetivo de mejorar las técnicas actuales asociadas a la producción de PHAs. Fueron emplea-das diferentes cepas productoras de PHAs bajo diversas condiciones nutricionales, en medios sintéticos como comple-jos. Además fueron obtenidos nuevos esquemas de biorrefi-nerías para la producción de PHAs mediante el uso de mate-rias primas renovables como topinambur, residuos de semillas de canola y glicerol. Por otro lado, se emplearon técnicas de optimización dinámica enfocadas a la maximización de la productividad de los PHAs en fermentaciones semicontuinuas. Finalmente, los PHAs obtenidos a partir del trabajo experimen-tal fueron identificados y caraterizados mediante el uso de diversas técnicas analíticas de polímeros. En general, los aportes experimentales y teóricos de esta tesis constituyen una potencial aplicación a esquemas actuales de producción de biopoliésteres naturales de origen microbiano. Además, los resultados experimentales obtenidos representan un importan-te punto de partida en el estudio de la biosíntesis de dife-rentes copolimeros de PHAs a partir de sistemas biotecno-lógicos económicos y complejos, como los desarrollados a partir de los esquemas de bioproceso que emplean topina-mbur, residuos de semillas de canola y glicerol. / The rapid and marked progress that has been occurring in polymer science and technology during the last years has created new plastic materials with excellent physical properties and durability. However, plastics are usually single-use, especially in food packaging and medical applications. Since these materials are not biodegradable, they can remain on the earth surface for hundreds of years without significant changes in their structure. Because of this, nowadays, social and governmental interests have conside-rably increased aiming to offsetting the adverse effects of synthetic polymers on the environment. These efforts are being focused on the production of new biodegradable materials from economically and environmentally sustainable structures. Poly(hydroxyalkanoate)s, PHAs, a large family of natural biopolyesters, are one of the more viable alternative in order to replace petrochemical-based plastics. This thesis makes a contribution both at experimental and theoretical level to improve current techniques associated to the whole PHAs production process. Different PHA-producer strains were used by implementing different nutritional culture conditions on both synthetic and complex media. In addition, new biore-finery structures for PHAs production were obtained through renewable feedstock material such as Jerusalem artichoke, rapeseed cake, and glycerol. On the other hand, dynamic optimization techniques were used in order to maximize the productivity of PHAs. Finally, the PHAs obtained from the experimental work were identified and characterized by several analytical techniques. Overall, the experimental and theore-tical contributions developed in this thesis represent a potential application to current developments in production of natural microbial polyesters. In addition, the experimental results represent an important start point in the study of the biosynthesis of different hydroxyalkanoates-based copolymers from inexpensive and complex biotechnological systems, such as those developed from bioprocess schemes based on Jerusalem artichoke, rapeseed cake, and waste glycerol

Los biopolímeros se presentan como una opción promisoria a los polímeros sintéticos convencionales, ya que son biodegradables y se obtienen de fuentes renovables. Entre ellos, los poli(hidroxialcanoato)s (PHAs) son biopoliésteres especialmente atractivos por su similitud en propiedades finales con algunos de los polímeros sintéticos, pero con la ventaja adicional de ser biodegradables. Sin embargo, el costo de producción de los PHAs es alto debido, principalmente, al costo de la fuente de carbono. Por lo que buscar materias primas más económicas es crucial para reducir los costos asociados a la producción de estos biopoliésteres. El objetivo de esta tesis fue estudiar la producción de PHAs utilizando vinaza (residuo de la industria del bioetanol) como sustrato y Bacillus megaterium como microorganismo productor. En este sentido, se optimizó la composición del medio de cultivo y las condiciones de fermentación mediante un diseño experimental y la metodología de superficie de respuesta, lográndose una productividad de 0,0105 g.L-1.h-1 Mediante optimización matemática se demostró que producir PHAs a partir de vinaza es económicamente viable, especialmente si el microorganismo productor posee una alta capacidad de acumulación del biopolímero, si la vinaza es pretratada o mezclada con otras fuentes de carbono más accesibles y si se agregan sales minerales al medio. Por otra parte, se exploró la obtención de vinaza seca mediante secado por aspersión, empleando maltodextrina como aditivo coadyuvante y se demostró que su uso como sustrato resultó en una mayor productividad de PHAs (0,0227 g.L-1 h-1) en comparación con la vinaza líquida. Este incremento podría atribuirse a la presencia de la maltodextrina que resultaría en una fuente de carbono más accesible en la fermentación empleado como cepa Bacillus megaterium, en concordancia con los resultados del estudio de factibilidad técnico-económica. Los PHAs biosintetizados se sometieron a una caracterización molecular y térmica mediante Espectroscopía Infrarroja con Transformada Fourier (FTIR), Calorimetría Diferencial de Barrido (DSC) y Análisis Termogravimétrico (TGA). Los valores de la temperatura de fusión de los PHAs obtenidos sugieren que el biopolímero podría estar formado por 3HB con comonómeros de cadena mas larga como el 3HV, entre otros. En función de los resultados reportados en la literatura los PHAs obtenidos resentan una temperatura de fusión similar al P(3HB-co-3HV) con una concentración aproximada del 9 mol% HV. Los avances en la optimización de la producción de PHAs utilizando vinaza como sustrato, la evaluación económica, la obtención de vinaza seca y su uso para la síntesis de PHAs y la caracterización de los PHAs permiten avanzar hacia una producción más sostenible de materiales poliméricos biodegradables y renovables. Además, los resultados experimentales obtenidos representan una importante contribución al estudio de la biosíntesis de PHAs empleando procesos biotecnológicos más económicos. / Biopolymers are presented as a promising option to conventional synthetic polymers, since they are biodegradable and they can be obtained from renewable resources. Among them, poly(hydroxyalkanoates)s (PHAs) are especially attractive biopolyesters due to their similarity in final properties to some synthetic polymers, but with the additional advantage of being biodegradable. However, the production cost of PHAs is high, mainly due to the cost of the carbon source. Therefore, looking for more economical raw materials is crucial to reduce the costs associated with the production of these biopolyesters. This thesis focused on studying the production of PHAs using vinasse (residue from the bioethanol industry) as substrate and Bacillus megaterium as the producing microorganism. In this sense, the composition of the culture medium and fermentation conditions were optimized through an experimental design and the response surface methodology, achieving a productivity of 0.0105 g.L-1.h-1. Through mathematical optimization, it was shown that producing PHAs from vinasse is economically viable, especially if the producing microorganism has a high capacity for biopolymer accumulation, if the vinasse is pretreated or mixed with more accessible carbon sources, and if mineral salts are added to the medium. On the other hand, spray drying of vinasse by was explored, using maltodextrin as a coadjuvant additive, in order to obtain dry vinasse. The use of dry vinasse as substrate resulted in a higher productivity of PHAs (0.0227 g.L-1 h-1) compared to the liquid vinasse. This increase could be attributed to the presence of maltodextrin, which would result in a more accessible carbon source for fermentation by Bacillus, in accordance with the results of the technical-economic feasibility study. The biosynthesized PHAs were subjected to molecular and thermal characterization using Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), and Thermogravimetric Analysis (TGA). The values of melting temperature of PHAs suggest that the biopolymer obtained could be identified as a copolymer of 3HB with longer monomers such as 3-HV, among others. Comparing the melting temperature with values reported in the literature the obtained PHAs could be a P(3HB-co-3HV) with an approximate concentration of 9 mo % HV. The advances in the optimization of the production of PHAs using vinasse as a substrate, the economic evaluation, the obtaining of dry vinasse and its use for the PHAs synthesis, and the biopolymer characterization allow progress towards more sustainable production of biodegradable polymers using renewable resources. In addition, experimental results obtained represent an important contribution to the biosynthesis of PHAs by cost- effective biotechnological processes.

Actualment, es pot considerar que vivim a l'era dels plàstics. En els últims 50 anys els plàstics d'origen petroquímic han esdevingut un dels materials més emprats. La seva versatilitat, propietats tècniques i un cost relativament baix (0,70$US per Kg) són alguns dels factors que han influït en la proliferació del seu ús. Fins al moment han estat descrites moltes i molt diverses aplicacions per aquest material (Zinn et al., 2001).El principal desavantatge dels plàstics convencionals és la seva baixa biodegradabilitat. Els residus plàstics no són biodegradables i els costos dels processos de reciclatge són molt elevats (Yu et al., 1998). D'altra banda, la disponibilitat, l'accessibilitat i l'explotació de les fonts de carboni d'origen fòssil són cada vegada més limitades. El fet que sigui una font d'energia no renovable, conjuntament amb l'augment del seu cost ha fet necessari buscar altres fonts d'energia alternatives, per la qual cosa l'aplicació de processos biotecnològics ha despertat gran interès pel seu potencial.Per aquest motiu, al començament dels anys 80s, es començaren a realitzar diferents aproximacions en el desenvolupament i en la producció de plàstics biodegradables com per exemple: la cel·lulosa, el midó, la quitina, la poliamida, l'àcid polilàctic (PLA) i els polihidroxialcanoats (PHAs), ja que són considerats com una alternativa als "petroplàstics".Els Polihidroxialcanoats (PHAs) són polièsters biològics sintetitzats per nombrosos microorganismes i acumulats intracel·lularment en forma de grànuls com a reservori d'energia i de font de carboni. Aquest fenomen, sovint, està associat a un excés de la font de carboni i a la limitació d'algun altre nutrient (p.e: el nitrogen, el fòsfor, etc.). Els PHAs són materials biodegradables, biocompatibles i totalment estereoespecífics (tots els estereocentres tenen configuració [R]). Aquests biopolímers presenten propietats mecàniques semblants a alguns termoplàstics sintètics (p.e. el polipropilè), per altra banda el fet de ser materials biocompatibles fa que siguin bons canditats en el camp de la biomedicina. D'altra banda els PHAs tenen una gran versatilitat en termes de composició, de tal manera que es poden obtenir mcl-PHAs funcionalitzats susceptibles de ser modificats químicament a posteriori per tal de modular les propietats del polímer original.Els objectius d'aquesta tesi són:1. L'obtenció de nous polihidroxialcanoats (PHAs) funcionalitzats a partir de cultius de Pseudomonas aeruginosa 42A2 emprant com a substrats fonts de carboni definides i olis vegetals.2. La determinació estructural, caracterització físico-química i l'estudi microscòpic dels polièsters obtinguts.3. La derivatització dels PHAs funcionalitzats.En el present treball s'ha demostrat que Pseudomonas aeruginosa 42A2 es capaç de créixer i acumular PHA a partir dels següents substrats: àcid nonanoic, àcid undecanoic, àcid undecenoic, extracte monodi, àcid linoleic i oli de llinosa. Tots els PHAs obtinguts han estat caracteritzats mitjançant RMN, CG-EM i FTIR, de la mateixa manera també s'han analitzat les propietats tèrmiques (DSC i TGA) i s'han determinat els pesos moleculars (GPC). Quan s'utilitza l'oli de llinosa com substrat s'obté un PHA altament funcionalitzat que conté un 16% d'unitats monomèriques poliinsaturades (8,3% de C14:3 i 7,6% de (C14:2+C12:2). Aquest polímer funcionalitzat s'ha derivatitzat mitjançant tractament fotoquímic amb radiació UV per tal d'induir l'entrecreuament de les cadenes laterals poliinsaturades i generant així un nou polímer.Els canvis estructurals deguts a la reacció d'entrecreuament han estat seguits mitjançant diverses tècniques analítiques (RMN, FTIR, CG-EM, DSC i TGA). En aquest treball, també es presenta un estudi tant del bacteri com del PHA com a biomaterial emprant diverses tècniques microscòpiques (TEM i AFM), essent la primera vegada que es recullen imatges d'un polihidroxialcanoat reticulat.Paral·lelament, es va realitzar l'escalat a bioreactor dels cultius amb àcid undecanoic i àcid undecenoic, com a co-substrats. En els diferents experiments realitzats en bioreactor s'han obtingut polímers amb diferent contingut de monòmers insaturats (15-70%), indicant així que, el règim d'alimentació continu en els cultius en bioreactor permet modular la composició del polímer i augmentar el percentatge d'acumulació de PHA fins a un 50% en front del 20% que s'obtenia en els cultius amb matrassos. / Nowadays one may consider that we are living in the age of the plastics. The global dependence on petroleum-derived plastics has increased over the last 50 years. However the rapid depletion of crude oil, the increasing problems with the biodegradation of these plastics and the environmental effects has prompted much interest in biologically derived polymers, particularly of the biodegradable class, produced by biotechnological processes. Among the different types of biodegradable polymers, a class that is drawing considerable attention is the polyhydroxyalkanoates (PHAs). Polyhydroxyalkanoates are optically active biopolyesters synthesized by a large number of microorganisms using a wide range of substrates such as fatty acids, sugars, oily wastes, etc. These polymers exhibit thermoplastic and elastomeric properties, depending on their composition. Moreover their high biocompatibility allows these polymers to be good candidates for medical applications.This doctoral thesis deals in a first part with the biosynthetic production and characterization of functionalized PHA, using defined substrates (nonanoic, undecanoic and undecenoic acid) and blends of fatty acids (linoleic acid (60%), linseed oil and an extract of hydroxylated fatty acids) as well as the undecanoic and undenoic acid has been used as a cosubstrates to obtained a block copolymer. The scale up to a 10L bioreactor of this process allows to improve the yield of PHA-accumulation reaching up to 50% versus the 20% obtained in the flasks experiments. Furthermore, the continuous feeding strategy let us to improve and to modulated the final composition of the biopolymers. Several experiments have been carried out rendering in polymers with different content of unsaturated monomers (15-70%). In the second part of this work, the functionalized PHA obtained from linseed oil, which contain up to 16% of polyunsaturated monomers (8,3% C14:3 i 7,6% (C14:2+C12:2), were modified by UV-irradiation to enhance the crosslinking reaction among the polyunsaturated side chains, in order to obtain a novel polymer with new and improved properties. The structural modifications as well as the thermal properties have been followed during the reaction by various analytical techniques (NMR, FTIR, GC, GC-MS, HPLC-MS, GPC, DSC and TGA). Besides the analytical techniques used for the characterization of these biopolymers, in this work is also showed a study of the bacterial cells as well as the PHA as a biomaterial using microscopical techniques (TEM and AFM), being the first time that a crosslinked PHA micrograph is acquired.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-14T14:31:03Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Luiz Eduardo Alves Bezerra do Nascimento.pdf: 898152 bytes, checksum: 6a9c77c38d08afcb99b849e2f5b50493 (MD5) / Made available in DSpace on 2016-07-14T14:31:03Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Luiz Eduardo Alves Bezerra do Nascimento.pdf: 898152 bytes, checksum: 6a9c77c38d08afcb99b849e2f5b50493 (MD5) Previous issue date: 2015-11-30 / CAPEs / Poecilia vivipara apresenta distribuição ampla na costa brasileira, e sua reprodução por viviparidade viabiliza a utilização de jovens recém-nascidos com 24 horas de vida em estudos ecotoxicológicos para avaliar o potencial de toxicidade de contaminantes ambientais. Este trabalho teve por objetivos avaliar os efeitos letais e subletais nestes estágios iniciais de vida do peixe estuarino Poecilia vivipara após exposição a diferentes concentrações dos HPAs fenantreno e pireno dissolvidos na água. Os jovens recém-nascidos foram obtidos de plantel de reprodutores mantidos em laboratório, expostos ao fenantreno nas concentrações 0, 10, 50, 200 e 500 μg L-1 durante 7 dias, e ao pireno nas concentrações 0, 1, 10, 50 e 100 μg L-1 durante 14 dias. Após a exposição foi avaliada a taxa de mortalidade, e nos sobreviventes foram quantificados os parâmetros subletais atividade da Glutationa S-Transferase (GST), velocidade de natação espontânea, captura de presas e crescimento. Jovens expostos a 500 μg fenantreno L-1 apresentaram 40% de mortalidade, e não foi detectada mortalidade nas demais concentrações. Jovens expostos ao pireno nas concentrações 50 e 100 μg L-1 apresentaram 44% e 33% de mortalidade, respectivamente, e não foi detectada mortalidade nas demais concentrações. Com relação aos parâmetros subletais, a concentração de efeito observado (CEO) para atividade de GST, velocidade média de natação, distância linear nadada e incremento de peso foi igual a 500 μg fenantreno L-1. Já para a exposição ao pireno a (CEO) para atividade de GST foi igual a 10 μg pireno L-1, 50 μg pireno L-1 para velocidade média de natação e distância linear nadada, e 1 μg pireno L-1 para incremento de peso. O aumento da atividade da GST após exposição a 500 μg fenantreno L-1 sugere um aumento dos gastos energéticos com os processos de biotransformação de fase 2, e esta alteração esteve associada a decréscimos na atividade locomotora, na habilidade de capturar presas, e também no crescimento. A exposição ao pireno também apresentou um padrão semelhante, com alterações detectadas em concentrações menores, indicando uma maior potência do pireno comparado ao fenantreno para induzir a GST, gerar hipoatividade locomotora e inibir o crescimento, parâmetro de maior relevância para o recrutamento da espécie. Estes resultados sugerem que o uso de jovens recém-nascidos de Poecilia vivipara como modelos ecotoxicológicos para a compreensão dos efeitos de contaminantes em parâmetros de diferentes níveis de organização biológica relacionados ao recrutamento de peixes é promissor. / Poecilia vivipara has a wide distribution pattern along the Brazilian coast, and its reproduction by viviparity enables the use of newborn in ecotoxicological studies to evaluate the potential toxicity of environmental contaminants. This study aimed to assess the lethal and sublethal effects on these early stages of life of the estuarine fish Poecilia vivipara after exposure to different concentrations of the PAHs phenanthrene and pyrene dissolved in water. The newborn were obtained from a breeding stock kept under controlled conditions, and exposed to phenanthrene concentrations of 10, 50, 200 and 500 μg L-1 and control for 7 days, and pyrene concentrations of 1, 10, 50 and 100 μg L-1 and control for 14 days. After exposure, the mortality rate was calculated, and the survivors were evaluated for the sublethal parameters glutathione S-transferase activity (GST), spontaneous swimming speed, prey capture rate and growth. Young individuals exposed to 500 μg phenanthrene L-1 exhibited 40% mortality, only detected in this concentration. Juveniles exposed to pyrene concentrations of 50 and 100 μg pyrene L-1 exhibited 44% and 33% mortality, respectively, and no mortality was detected in other tested concentrations. Regarding sublethal parameters, the lowest observed effect concentration (LOEC) for GST activity, average swimming speed, linear swimming distance and weight increase was equal to 500 μg phenanthrene L-1. Already for an exposition to pyrene the LOEC for GST activity was equal to 10 μg pyrene L-1, 50 μg pyrene L-1 for average swimming speed, and 1 μg pyrene L-1 for weight increase. The increase in GST activity after exposure to 500 μg phenanthrene L-1 suggests an increase in energy expenditure with the biotransformation processes of phase 2, and this change was associated with decreases in locomotor activity, the ability to catch prey, and also in growth. Exposure to pyrene also exhibited a similar pattern, with changes detected in lower concentrations, indicating a greater pyrene potency compared to phenanthrene to induce GST and to generate locomotor hypoactivity and inhibit growth, the most relevant parameter for the recruitment of the species. These results suggest the potential use of newborn young Poecilia vivipara as ecotoxicological models for understanding the effects of contaminants on parameters of distintic biological organization levels related to the recruitment of fish.

The unveiling of small RNA (sRNA)-mediated gene regulatory pathways has profoundly shaped our understanding of the complexity of gene regulation. In eukaryotes, sRNAs have been found to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. To gain insight into the roles of small RNAs in apple and peach, we conducted sRNA-seq, computational analysis and molecular experiments to genome-widely characterize their microRNAs (miRNAs) and trans-acting siRNA (tasiRNA) pathways. We identified totally 75 miRNAs or families, including 23 conserved, 10 less-conserved and 42 apple-specific ones, and 118 miRNA target genes in apple. Two classical trans-acting siRNA (tasiRNA) pathways, miR390-TAS3 and miR828-TAS4, were characterized with similar but unique tasiRNA biogenesis profiles and target specificities. Importantly, miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. In contrast to the location of the miR159 target site in a sequence-divergent region, the target sites of miR828 and miR858 are located in the region encoding the conserved R3 repeat domain of MYB proteins. 10 out of the 19 miR828-targeted MYBs undergo the biogenesis of various phased siRNA (phasiRNA), which potentially regulate diverse genes outside the MYB family. In peach, totally 94 miRNAs or families and 80 target genes were identified. Similar pathways of tasiRNA (miR828-TAS4 and miR390-TAS3) or phasiRNA (miR828-MYB-siRNA) processing were also characterized in peach. Taking advantage of reverse computation and public available deep-sequencing data, we demonstrated that the miRNA-TAS-PPR-siRNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to instigate phasiRNA production from PPR �genes, which are triggered by different 22-nt miRNAs, including miR7122, miR1509, and fve-PPRtri1/2 and through distinct mechanistic strategies, like miRNA direct-targeting or indirect-targeting through TAS-like genes, one-hit or two-hit, or even two layers of tasiRNA-TAS interactions. We found that the MIRNA genes of these miRNA triggers show great identity with the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Combined results from phylogenetic analyses and conservation extent profiling revealed that the super-miR7122 was potentially evolved from another miRNA superfamily (super-miR4376), which probably originated from the miR390. Additionally, the miR482/2118-NB-LRR-siRNA pathway was found to be conserved, but evolved with distinct features, in apple and peach. Taken together, widespread and complex miRNA and tasiRNA regulatory networks have been adapted in apple and peach. They add another crucial layer of regulation on gene activity and stability, and must exert essential functions in all aspects of plant life. / Ph. D.

Made available in DSpace on 2014-12-17T15:41:52Z (GMT). No. of bitstreams: 1 PatriciaMFS_DISSERT.pdf: 2351084 bytes, checksum: 3084e21b3350befdebe1abcad298bc5a (MD5) Previous issue date: 2010-02-23 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Polycyclic aromatic hydrocarbons (PAHs) constitute a family of compounds characterized by having two or more condensed aromatic rings and for being a class of substances that are widely distributed in the environment as a complex mixture, being very persistent in the environment due to its low solubility in water. The application of chemometric methods to analytical chemistry has provided excellent results in studying the solubility of PAHs in aqueous media in order to understand the mechanisms involved in environmental contamination. The method consists in analyzing the solubilization of PAHs from diesel oil in water varying parameters such as stirring time, volume of oil added and pH, using a full factorial design of two levels and three factors. PAHs were extracted with n-hexane and analyzed by fluorescence spectroscopy because they have molecular characteristics fluorescent due to the large number of condensed rings and links, and gas chromatography coupled to a mass spectrometer (GC-MS). The results of fluorescence analysis showed that only the stirring time and pH influenced the solubility of PAHs in diesel fuel. How is a non-selective technique for the study of fluorescence was performed on form and semi-quantitative. And for the chromatographic analysis the results showed that the solubility of the different PAHs is influenced differently so that you can classify them into groups by the results of the effects / Os hidrocarbonetos polic?clicos arom?ticos (HPAs) constituem uma fam?lia de compostos caracterizada por possu?rem dois ou mais an?is arom?ticos condensados e por ser uma classe de subst?ncias que est?o amplamente distribu?das no ambiente como uma mistura complexa, sendo bastante persistentes no ambiente devido a sua baixa solubilidade em ?gua. A aplica??o de m?todos quimiom?tricos ? qu?mica anal?tica vem proporcionando excelentes resultados no estudo da solubilidade de HPAs em meio aquoso, no sentido de entender os mecanismos envolvidos na contamina??o do ambiente. O m?todo consiste na an?lise da solubiliza??o dos HPAs de ?leo diesel em ?gua variando par?metros como tempo de agita??o, volume de ?leo adicionado e pH, atrav?s de um planejamento fatorial completo de dois n?veis e tr?s fatores. Os HPAs foram extra?dos com n-hexano e analisados atrav?s da espectroscopia de fluoresc?ncia molecular por possu?rem caracter?sticas fluorescentes devido ao grande n?mero de an?is condensados e liga??es conjugadas e cromatografia gasosa acoplada a um espectr?metro de massa (CG-EM). Os resultados da an?lise por fluoresc?ncia mostraram que, apenas o tempo de agita??o e o pH influenciaram no processo de solubiliza??o dos HPAs presentes no ?leo diesel. Como ? uma t?cnica n?o seletiva o estudo por fluoresc?ncia foi realizado de forma relativa e semi-quantitativa. E para a an?lise cromatogr?fica os resultados mostraram que a solubilidade dos diferentes HPAs ? influenciada de forma diferenciada de modo que ? poss?vel classific?-los em grupos atrav?s dos resultados dos efeitos

Polyhydroxyalkanoates (PHAs) are polyesters, produced by many bacteria and some archaea. The most commonly characterised is polyhydroxybutyrate (PHB). Produced when nutrients are growth limiting and carbon available in excess, PHA serves as a carbon-energy storage material and forms generally spherical insoluble inclusions between 50-500nm in diameter in the cytoplasm. The key enzyme for PHA synthesis is the PHA synthase and this enzyme catalyses the polymerisation of (R)-3-hydroxy fatty acids into PHA. PHA synthase remains covalently attached to the growing polyester chain and is displayed on the surface of the PHA granule. Other proteins associated with PHA granules include depolymerases for mobilisation or degradation of granules, regulatory proteins and phasins, proteins that aid in PHA granule stability. PHA bio-beads displaying an IgG binding protein were produced and used to purify IgG from serum demonstrating that the PHA synthase can be engineered to display functional synthase fusion proteins at the PHA granule surface. Correctly folded eukaryotic proteins were also produced and displayed at the PHA granule surface as phasin fusion proteins. Multiple-functionality was also achievable by co-expression of various hybrid genes suggesting that this biotechnological bead production strategy might represent a versatile platform technology. The production of functional eukaryotic proteins at the PHA bead surface represents a novel in vivo matrix-assisted protein folding system. Protein engineering of PHA granule surface proteins provides a novel molecular tool for the display of antigens for FACS based analysis and offers promising possibilities in the development of future biotechnological production processes. Overall, the results obtained in this study strongly enhance the applied potential of these polyester beads in biotechnology and medicine.

This thesis focuses on the structural and behavioural factors that placed South Asian immigrant women living with HIV/AIDS in the Greater Toronto Area at risk. Informed by Connell's social theory of gender (1987), this study examined the role of hegemonic masculinity in legitimizing male power and contributing to the HIV risk of these women. By conducting one-on-one interviews with 12 HIV-positive immigrant women, meaningful constructions of the women's narratives and accounts of their experiences relative to HIV were created. This study examined the intersection of power ideologies such as gender, race and class in specific contexts as they generated particular experiences that affected women's risk for HIV. Following a community-based research approach, a collaborative relationship was established with the Alliance for South Asian AIDS Prevention where qualitative methods of analysis and an inductive approach with an iterative process were followed. Factors such as isolation, economic dependence on their husbands, discrimination, racism, investment in psychologically and emotionally abusive relationships, combined with the absence of support from their family of origin exacerbated the women's risk of HIV infection. The strong ties exhibited by most of the women to their religious/ethnic communities helped sustain a gender-based social hierarchy. To facilitate dialogue and social change for South Asian women, gender and culture need to be situated in social and historical contexts. As such, programs should be understood within a larger critical understanding of the social power relations and history of Canadian immigration patterns. Using anti-racist frameworks, initiatives should address violence against women, while tackling interrelated issues (i.e., housing, poverty, etc.). This work draws attention to oppressions through the experiences of a community of women who are rarely given a voice within the context of research on HIV/AIDS. It will be also helpful for Ontario’s HIV prevention strategy and the field of women's sexual health.

This thesis focuses on the structural and behavioural factors that placed South Asian immigrant women living with HIV/AIDS in the Greater Toronto Area at risk. Informed by Connell's social theory of gender (1987), this study examined the role of hegemonic masculinity in legitimizing male power and contributing to the HIV risk of these women. By conducting one-on-one interviews with 12 HIV-positive immigrant women, meaningful constructions of the women's narratives and accounts of their experiences relative to HIV were created. This study examined the intersection of power ideologies such as gender, race and class in specific contexts as they generated particular experiences that affected women's risk for HIV. Following a community-based research approach, a collaborative relationship was established with the Alliance for South Asian AIDS Prevention where qualitative methods of analysis and an inductive approach with an iterative process were followed. Factors such as isolation, economic dependence on their husbands, discrimination, racism, investment in psychologically and emotionally abusive relationships, combined with the absence of support from their family of origin exacerbated the women's risk of HIV infection. The strong ties exhibited by most of the women to their religious/ethnic communities helped sustain a gender-based social hierarchy. To facilitate dialogue and social change for South Asian women, gender and culture need to be situated in social and historical contexts. As such, programs should be understood within a larger critical understanding of the social power relations and history of Canadian immigration patterns. Using anti-racist frameworks, initiatives should address violence against women, while tackling interrelated issues (i.e., housing, poverty, etc.). This work draws attention to oppressions through the experiences of a community of women who are rarely given a voice within the context of research on HIV/AIDS. It will be also helpful for Ontario’s HIV prevention strategy and the field of women's sexual health.