No / This review summarises the recent developments in the synthesis and applications of polymers derived from malic acid. There has been an increased interest in the design of sustainable and biodegradable polymers as a result of the drive to use renewable feedstocks as an alternative to petrochemicals in addition to their significant potential in biomedical applications. Synthetic strategies to access polymers from malic acid based on both condensation and ring-opening polymerization, across a broad range of conditions, are reviewed along with their advantages and limits. The role that such materials are studied for in biomedical applications is discussed, and their environmental impact based on the biodegradability of the malic polymer backbone is outlined. / The Royal Society, EPSRC, BBSRC

Magnesium and its alloys have been considered for load-bearing implant materials due to their similar mechanical properties to the natural bone, excellent biocompatibility, good bioactivity, and biodegradation. Nevertheless, the uncontrollable corrosion rate in biological environment restrains their application. Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is a widely used bio-ceramic which has bone-like mineral structure for bone fixation. Poor fracture toughness of HA makes it not suitable for load-bearing application as a bulk. Thus, HA is introduced into metallic surface in various forms for improving biocompatibility. Recently friction stir processing (FSP) has emerged as a surface modification tool for surface/substrate grain refinement and homogenization of microstructure in biomaterial. In the pressent efforts, Mg-nHA composite surface on with 5-20 wt% HA on Mg substrate were fabricated by FSP for biodegradation and bioactivity study. The results of electrochemical measurement indicated that lower amount (~5% wt%) of Ca in Mg matrix can enhance surface localized corrosion resistance. The effects of microstructure,the presence of HA particle and Mg-Ca intermetallic phase precipitates on in vitro behavior of Mg alloy were investigated by TEM, SEM, EDX,XRD ,and XPS. The detailed observations will be discussed during presentation.

<p>U okviru disertacije, kori&scaron;ćenjem ve&scaron;tačkih neuronskih mreža razvijeni su modeli za predviđanje količina ambalažnog i biorazgradivog komunalnog otpada u Republici Srbiji do kraja 2030. godine. Razvoj modela baziran je na zavisnosti između ukupne potro&scaron;nje domaćinstva i generisane količine dva posmatrana toka otpada. Pored toga, na bazi zavisnosti sa bruto domaćim proizvodom (BDP), definisan je i model za projekciju zastupljenosti osnovnih opcija tretmana komunalnog otpada u Republici Srbiji za isti period. Na osnovu dobijenih rezultata, stvorene su polazne osnove za procenu potencijala za reciklažu ambalažnog otpada, kao i za procenu u kojoj meri se može očekivati da određene količine biorazgradivog otpada u narednom periodu ne budu odložene na deponije, &scaron;to je u skladu sa savremenim principima upravljanja otpadom i postojećim zahtevima EU u ovoj oblasti.</p> / <p>By using artificial neural networks, models for prediction of the quantity of<br />packaging and biodegradable municipal waste in the Republic of Serbia by<br />the end of 2030, were developed. Models were based on dependence<br />between total household consumption and generated quantities of two<br />observed waste streams. In addition, based on dependence with the Gross<br />Domestic Product (GDP), a model for the projection of share of different<br />municipal solid waste treatment options in the Republic of Serbia for the same<br />period, was created. Obtained results represent a starting point for assessing<br />the potential for recycling of packaging waste, and determination of<br />biodegradable municipal waste quantities which expected that in the future<br />period will not be disposed at landfills, in accordance with modern principles<br />of waste management and existing EU requirements in this area.</p>

<p>Предмет докторске дисертације је развој јестивих, биоразградивих, активних филмова на бази скроба са додатком есенцијалних уља зачинског биља и њихова примена за паковање одабраног прехрамбеног производа. Глобални допринос овог рада иде у прилог развоју области биоразградивих, јестивих, активних амбалажних материјала.<br />Основно истраживање се базира на испитивању могућности добијања скробних филмова влажним поступком (&quot;casting&quot; метода), модификацији врста и количина додатих компонената које улазе у састав филмогене смеше и карактеризацији добијених филмова ради побољшања особина формираног филма. Својства значајна за примену добијених материјала су физичко-хемијске особине (садржај влаге, способност бубрења), механичке особине (дебљина, затезна јачина и издужење при кидању), баријерне особине (пропустљивост водене паре), структурне (АТР-ФТИР спектрометрија и дифракција X зрака), термалне, оптичке и биолошке особине (антимикробна и антиоксидативна активност).<br />Обзиром на велики број параметара који утичу на формирање филма, као и на особине формираних филмова, испитано је међусобно деловање више фактора на могућност продукције и особине добијеног филма. Избор и оптимизација процесних параметара и моделовање продукције филмова изведено је имплементирањем компјутерске и аналитичке методологије.<br />На основу приказаних резултата, може се закључити да је синтетисан активан јестиви скробни филм оптимизованих физичко-хемијских, механичких, баријерних, термалних и биолошких карактеристика. Синтетисаном скробном филму смањена је способност бубрења, унапредјене су механичке особине (вредности затезних јачина опадају, уз истовремено повећање вредности издужења при кидању). Значајан је утицај додатка есенцијалних уља на вредности пропустљивости водене паре, а њиховим додатком постигнута је и<br />изражена биолошка активност.<br />На основу детаљне карактеризације, одабрани су филмови оптимизованих својстава за примену у заштити осмотски дехидрираног свињског меса током периода складиштења. Осмотски дехидрирано месо је упаковано у комерцијалан високо-баријеран амбалажни материјал у атмосферским условима и у условима модификоване атмосфере, док је други део узорака претходно заштићен скробним премазом са оптимизованим особинама, а затим упакован на исти начин. Утицај оптимизоване скробне превлаке на осмотски дехидрирано месо испитан је праћењем промена параметара квалитета током<br />складиштења.<br />Резултати су показали да јестиви, активни скробни премаз значајно доприноси стабилности упакованог осмотски дехидрираног меса и то у погледу оксидације липида, микробиолошке стабилности и позитивног утицаја на сензорне карактеристике осмотски дехидрираног меса током периода складиштења.</p> / <p>Predmet doktorske disertacije je razvoj jestivih, biorazgradivih, aktivnih filmova na bazi skroba sa dodatkom esencijalnih ulja začinskog bilja i njihova primena za pakovanje odabranog prehrambenog proizvoda. Globalni doprinos ovog rada ide u prilog razvoju oblasti biorazgradivih, jestivih, aktivnih ambalažnih materijala.<br />Osnovno istraživanje se bazira na ispitivanju mogućnosti dobijanja skrobnih filmova vlažnim postupkom (&quot;casting&quot; metoda), modifikaciji vrsta i količina dodatih komponenata koje ulaze u sastav filmogene smeše i karakterizaciji dobijenih filmova radi poboljšanja osobina formiranog filma. Svojstva značajna za primenu dobijenih materijala su fizičko-hemijske osobine (sadržaj vlage, sposobnost bubrenja), mehaničke osobine (debljina, zatezna jačina i izduženje pri kidanju), barijerne osobine (propustljivost vodene pare), strukturne (ATR-FTIR spektrometrija i difrakcija X zraka), termalne, optičke i biološke osobine (antimikrobna i antioksidativna aktivnost).<br />Obzirom na veliki broj parametara koji utiču na formiranje filma, kao i na osobine formiranih filmova, ispitano je međusobno delovanje više faktora na mogućnost produkcije i osobine dobijenog filma. Izbor i optimizacija procesnih parametara i modelovanje produkcije filmova izvedeno je implementiranjem kompjuterske i analitičke metodologije.<br />Na osnovu prikazanih rezultata, može se zaključiti da je sintetisan aktivan jestivi skrobni film optimizovanih fizičko-hemijskih, mehaničkih, barijernih, termalnih i bioloških karakteristika. Sintetisanom skrobnom filmu smanjena je sposobnost bubrenja, unapredjene su mehaničke osobine (vrednosti zateznih jačina opadaju, uz istovremeno povećanje vrednosti izduženja pri kidanju). Značajan je uticaj dodatka esencijalnih ulja na vrednosti propustljivosti vodene pare, a njihovim dodatkom postignuta je i<br />izražena biološka aktivnost.<br />Na osnovu detaljne karakterizacije, odabrani su filmovi optimizovanih svojstava za primenu u zaštiti osmotski dehidriranog svinjskog mesa tokom perioda skladištenja. Osmotski dehidrirano meso je upakovano u komercijalan visoko-barijeran ambalažni materijal u atmosferskim uslovima i u uslovima modifikovane atmosfere, dok je drugi deo uzoraka prethodno zaštićen skrobnim premazom sa optimizovanim osobinama, a zatim upakovan na isti način. Uticaj optimizovane skrobne prevlake na osmotski dehidrirano meso ispitan je praćenjem promena parametara kvaliteta tokom<br />skladištenja.<br />Rezultati su pokazali da jestivi, aktivni skrobni premaz značajno doprinosi stabilnosti upakovanog osmotski dehidriranog mesa i to u pogledu oksidacije lipida, mikrobiološke stabilnosti i pozitivnog uticaja na senzorne karakteristike osmotski dehidriranog mesa tokom perioda skladištenja.</p> / <p>The subject of the doctoral thesis is the development of<br />edible biodegradable active films based on starch with<br />the addition of herbal essential oils and their<br />application in packaging of selected food products.<br />Basic research is examination of the possibility of<br />obtaining starch films by wet method (&quot;casting&quot;<br />method), modification of the types and amounts of<br />added components that constitute the film forming<br />mixture and characterization of films in order to<br />improve the characteristics. Characteristics important<br />for the implementation of the obtained materials<br />include the physico-chemical properties (moisture<br />content, solubility), mechanical properties (thickness,<br />tensile strength and elongation at break), barrier<br />properties (water vapor permeability), structural (ATRFTIR<br />spectroscopy and X ray diffraction), thermal,<br />optical and biological properties (antimicrobial and<br />antioxidant activity).<br />Considering the large number of parameters that affect<br />the film formation, as well as the properties of the<br />films, interactions of multiple factors on the possibility<br />of production and properties of the produced film were<br />examined. Selection and optimization of process<br />parameters and modeling of film production were<br />performed by implementing computer and analytical<br />methodology.<br />Based on these results, it can be concluded that it were<br />synthesized active edible starch films with optimized<br />physico-chemical, mechanical, barrier, thermal and<br />biological characteristics. Synthesized starch film had<br />reduced solubility, improved mechanical properties (tensile strength values declined, while elongation at</p><p>break increased). There is a significant influence of the<br />addition of essential oils on the water vapor<br />permeability value with expressed biological activity.<br />The global contribution of this investigation favors the<br />development of the biodegradable edible active<br />packaging materials scientific field.<br />Based on the detailed characterization, film with<br />optimized characteristics was selected for protection of<br />osmotically dehydrated pork during storage.<br />Osmotically dehydrated meat is wrapped in commercial<br />high-barrier packaging material in atmospheric<br />conditions and in conditions of modified atmosphere,<br />while the second part of the sample is packaged in the<br />same way, but previously protected with optimized<br />starch edible coating. Influence of optimized starch<br />coating on the osmotic dehydrated meat was examined<br />by monitoring the quality change during storage in<br />order to preserve the quality and shelf life of packed<br />meat.<br />The results showed that an edible starch based active<br />coating contributes significantly to the stability of the<br />packaged osmotically dehydrated meat during the<br />storage period in terms of lipid oxidation,<br />microbiological stability and positive influence on the<br />sensory characteristics of the osmotically dehydrated<br />meat during the storage period.</p>

Les essais préliminaires pour préparer des alcoolates de fer à partir du bichlorure ou bibromure de fer (II), en les combinant avec des ligands de type diimino pyridine, ont engendré la formation de complexes homoleptiques et hétéroleptiques, dépendant des substituants sur les branches imines du ligand. Ces complexes homoleptiques octaédriques et paramagnétiques ont été étudiés par rapport à leurs propriétés spectroscopiques et cristallographiques. De plus, la synthèse des complexes de fer hétéroleptique a engendré de bons précurseurs penta-coordonnés pour les réactions de substitution de ligands avec des alcoolates de métaux alcalins, de manière à produire les dialcoolates de fer (II) désirés. Des techniques d’analyse telles que la spectroscopie UV-vis, l’analyse élémentaire, la spectrométrie de masse à haute résolution et la cristallographie aux rayons X ont été utilisées pour caractériser ces complexes de fer. L’activité catalytique de ces complexes de fer (II) a aussi été étudiée par rapport à la polymérisation du lactide; les dialcoolates convoités ont été générés in-situ en raison de la difficulté à produire et à isoler les dérivés alcoolates des complexes diimino pyridine de fer. Une étude approfondie a aussi été faite sur les réactions de polymérisation, surtout par rapport aux valeurs de conversion à l’échelle du temps, ainsi qu’à la tacticité des chaines de polymères obtenues. Ces analyses ont été effectuées par l’entremise de la spectroscopie de résonance magnétique nucléaire, de la chromatographie d’exclusion stérique, et de la spectrométrie de masse MALDI (désorption-ionisation laser assistée par matrice). / Initial attempts to prepare iron alkoxide complexes, from iron (II) dichloride or dibromide, in combination with various bis(imino) pyridine ligands, led to the formation of homoleptic and heteroleptic complexes depending on the N-substituents on the imino moieties. A study was made of the resulting paramagnetic octahedral homoleptic complexes, with respect to their spectroscopic properties, as well as their crystallographic parameters. Alternatively, the synthesis of penta-coordinate heteroleptic iron (II) complexes provided good precursors for the ligand substitution reactions with alkaline alkoxides, to produce the desired iron bis(alkoxide) derivatives. Methods such as UV-vis spectroscopy, elemental analysis, high resolution mass spectrometry and X-ray crystallography were used for the characterization of these iron complexes. The catalytic activity of these iron (II) complexes was investigated with respect to lactide polymerization; the desired bis(alkoxide) species were generated in-situ due to the difficulties in isolating pure alkoxide derivatives of the bis(imino) pyridine iron (II) complexes. An informative study was made of both time-scale conversion values, as well as tacticity properties of the resulting polylactic acid chains, through proton nuclear magnetic resonance spectroscopy, gel permeation chromatography, as well as MALDI (matrix-assisted laser desorption/ionization) mass spectrometry measurements.

La biomasse représente l’une des principales alternatives à l’utilisation du pétrole dans la plasturgie. Grâce à leurs propriétés fonctionnelles, les caséinates sont une matière première prometteuse pour la fabrication de films plastiques pour des applications dans l’emballage biodégradable et hydrosoluble. La transformation du caséinate de sodium par les techniques habituellement utilisées dans la plasturgie a été démontrée. Des extrudats de caséinate plastifié au glycérol ont été obtenus au moyen d’une extrudeuse bi-vis corotative. La caractérisation physico-chimique du matériau obtenu a confirmé la thermostabilité de cette protéine et a montré la dépendance du comportement du matériau vis-à-vis de l’humidité ambiante. Pour une variation du taux d’Humidité Relative, de 40 à 90% et une augmentation de la concentration du glycérol, plastifiant hygroscopique, le matériau passe d’un état vitreux (rigide) à un état caoutchouteux (mou). Des films fins ont été ensuite réalisés, à partir des extrudats thermoplastiques, par extrusion gonflage. La perméabilité à la vapeur d’eau des films de caséinate de sodium a été étudiée et a montré que ces matériaux sont de mauvaises barrières à l’humidité. La deuxième partie a été consacré à l’étude de mélanges caséinate de sodium/caséinate de calcium d’un côté et caséinate de sodium/PBAT de l’autre. Le mélange des deux caséinates a permis d’augmenter la tenue mécanique du mélange, à partir de 50% de caséinate de calcium, et de retarder le transfert hydrique à travers le film. Dans le même sens, l’ajout du PBAT, a augmenté jusqu’à deux fois plus, le module d’Young des mélanges mais a baissé la résistance au transfert d’humidité du film à cause de l’incompatibilité des deux polymères / Biomass is one of the main alternatives to the use of oil in plastics field. Due to their various functional properties, caseinates are considered as an interesting raw material for making biodegradable and water-soluble packaging. The transformation of sodium caseinate by the processes used for synthetic plastics industry has been demonstrated. A corotating twin-screw extruder was used to get glycerol plasticized caseinate pellets. The physicochemical properties of the obtained material have confirmed the thermal stability of this protein and demonstrated the influence of surrounding moisture on material behavior. With the Relative Humidity varying from 40 to 90% and increasing the glycerol content, an hydrous plasticizer, the mechanical properties of sodium caseinate based materials changed from those of glassy (rigid) to rubbery (soft) plastics. The pellets were then taken over to make film using a blown-film extruder. The water vapour permeability of blown film was studied and showed that sodium caseinate based films are a poor moisture barrier. The second part dealt with the sodium caseinate blend. Sodium caseinate/calcium caseinate and sodium caseinate/PBAT blends was performed. Adding calcium caseinate, started from 50%, improved the mechanical properties and delayed hydrous transfer through the film. Concerning the sodium caseinate/PBAT blends, adding PBAT increased until twice more the material’s Young modulus but decreased the moisture transfer resistance because of the incompatibility between the two polymers

L’absence de croissance en monocouche des cellules endothéliales sur la paroi des prothèses vasculaires est une des causes d’échec de leur implantation chez l’humain. Des études précédentes ont montré que le recouvrement de ces prothèses par un échafaudage de nanofibres d’acide polylactique (PLA), fabriqué par un système de filage par jet d’air innovant, peut être utilisé pour promouvoir la croissance des cellules endothéliales de façon adéquate. Ainsi, le caractère dégradable d’un matériau comme le PLA permettrait son remplacement graduel par la matrice extra-cellulaire produite par les cellules. D’autre part, la réussite d’une transition entre les nanofibres dégradables et la matrice extra-cellulaire nécessite un remplacement contrôlé et approprié. Or, la dégradation des nanofibres de PLA, dépendant de ses séquences stéréochimiques, est généralement trop longue et peut induire une cytotoxicité relative pendant sa dégradation. Dans ce contexte, les études de cette thèse ont pour objectifs de mieux comprendre la formation des fibres lors du filage, d’optimiser la fabrication des échafaudages permettant ainsi la création de nanofibres d’autres polymères, puis, de concevoir des nanofibres provenant d’un polymère mieux adapté à nos besoins, d’évaluer leur mécanisme de dégradation et sa cytotoxicité durant sa dégradation. Les travaux d’optimisation du système de filage ont démontré que la concentration avec un effet prépondérant. Ainsi, la mesure de la viscosité permet de trouver les paramètres adéquats pour le filage de polymère. Ensuite, un poly(L-lactide) semi-cristallin (PLLA) et un terpolymère de poly(lactide-co-ε-caprolactone) (PLCL) dédié pour des applications vasculaires ont été synthétisés et filés par jet d’air. Ces échantillons ont été dégradés en solution aqueuse et caractérisés par des méthodes physico-chimiques afin de mieux comprendre leurs mécanismes de dégradation et mis en présence de cellules endothéliales pour évaluer leur cytotoxicité. La comparaison entre les échafaudages des deux polymères a montré des comportements singuliers en dégradation, dépendants des caractéristiques thermiques des polymères. De plus, ces mécanismes de dégradation des nanofibres ont une influence directe sur la sensibilité des cellules endothéliales face aux produits de dégradation. En conclusion, ces travaux de doctorat présentent une solution prometteuse pour améliorer les prothèses vasculaires et qui pourrait être appliquée pour résoudre plusieurs problématiques en médecine régénératrice. / The absence of neo-endothelium on the intimal surface of vascular substitutes is known to be one cause of failure upon implantation of these prostheses in humans. Previous studies have shown that the coating of these substitutes with a nanofiber scaffold, made with an innovative air spinning device, can be used to promote a suitable endothelial cells growth. On one hand, the degradable feature of material as PLA enable the progressive replacement of the scaffold by the extracellular matrix of cells. On the other hand, the success of this replacement between degradable nanofibers and the extracellular matrix requires to be appropriate and controlled. Yet, the PLA nanofiber degradation process, which depends on its stereosequences, is generally too long for this application and could involve cell sensitivity during the degradation. In this context, studies from this thesis aim to understand the fibers formation during spinning, optimizing the scaffold fabrication as well as to promote the making of novel polymer scaffolds, then, design solution to polymeric nanofiber scaffolds for vascular application, evaluate its degradation mechanism and cytotoxicity during degradation process. The work on spinning device optimisation has demonstrated that the concentration had a dominant effect. Thus, viscosity measurements enable to find suitable parameters for polymer spinning. Then, a semi-cristalline poly(L-lactide) (PLLA) and a poly(lactide-co-ε-caprolactone) (PLCL) terpolymer specifically made for vascular application have been synthesized and air-spun. These samples were degraded in aqueous solution and characterized by physical and chemical methods to better understand their degradation mechanisms and seeded with endothelial cells to evaluate their cytotoxicity. The comparison between the two polymers scaffolds have shown surprising degradation behaviors depending on thermal properties of polymers. Moreover, these nanofiber degradation mechanisms have a direct influence on endothelial cells sensitivity with degradation by-products. To conclude, these works of doctorate display a promising solution to improve vascular prostheses and which could be applied to solve several issues in regenerative medicine field.

El presente trabajo de investigación se realizó para validar la necesidad de la población de Lima de contar con un coach de la salud que le brinde asesoramiento para cuidar su salud a través de la alimentación. Necesidad que surge a causa de problemática actual sobre obesidad y sobrepeso que la población peruana viene enfrentando por los malos hábitos de alimentación y la falta de ejercicios en su rutina de vida por un factor tiempo. Luego de haber recabado información necesaria se elabora el presente trabajo de investigación con la finalidad de poner en marcha un proyecto empresarial para la creación de una empresa que brinda el servicio de asesoramiento nutricional que está estrechamente relacionado con el bienestar del individuo, el equilibrio, la atención plena y la eficiencia de sus metas personales. Para el desarrollo de este proyecto se realizó entrevistas de profundidad con la finalidad de delimitar nuestro segmento objetivo. La metodología que se ha implementado, han sido las entrevistas cualitativas abiertas, a personas que ya cuentan con estilo de vida sano, segmento al que atenderá Life Coaching. Los resultados de las entrevistas efectuadas nos muestra que existe un mercado potencia interesado en nuestra idea de negocio. Para la viabilidad de este proyecto se requiere de una inversión inicial de S/ 57,595,00 soles que será financiado por un Angel Investor. Del análisis financiero efectuado nos arroja que la Tasa Interna de Retorno es del 145% con un Valor Actual Neto de S/ 143,749.68 soles. Estos resultados nos muestran que el proyecto analizado devuelve el capital invertido más una ganancia de S/ de 143,749.68 soles. Por lo antes señalado este trabajo de investigación nos indica que el proyecto de negocio es viable y rentable. / The present research work was conducted to validate the need of the population of Lima to have a health coach to provide advice to take care of their health through food. Need that arises because of current problems about obsessiveness and overweight that the Peruvian population has been facing due to poor eating habits and lack of exercises in their routine for a time. After having gathered the necessary information, the present research work is chosen in order to start up a business project for the creation of a company that provides the nutritional advice service that is closely related to the wellbeing of the individual, the balance, the full attention and the efficiency of your personal goals. For the development of this project, in-depth interviews were conducted in order to define our target segment. The methodology that has been implemented has been qualitative open interviews, to people who already have a healthy lifestyle, a segment that Life Coaching will attend. The results of the interviews carried out show us that there is a potential market interested in our business idea. For the viability of this project, an initial investment of S / 57,595.00 soles will be required and will be financed by Angel Investor. From the financial analysis carried out, we find that the Internal Rate of Return is 145% with a Net Present Value of S / 143,749.68 soles. These results show us that the project analyzed returns the capital invested plus a profit of S/. 143,749.68 soles. As indicated earlier this research work indicates that the business project is viable and profitable. / Trabajo de investigación

A capacidade de B. sacchari acumular poli-3-hidroxibutirato-co-3-hidroxihexanoato (P3HB-co-3HHx) foi confirmada, com até 2 mol% de 3HHx no PHA total (<10% do 3HHx máximo teórico a partir do ácido), indicando flexibilidade da PHA sintase por substratos, porém alta eficiência nas vias catabólicas do hexanoato. Análise da estabilidade térmica do PHA indicou uma temperatura de degradação reduzida, compatível com a presença de unidades 3HHx. Mutantes incapazes de crescer em ácido hexanóico foram obtidos com UV e transposon mini-Tn5, que ainda acumulavam 3HHx a partir de hexanoato mas com redução na capacidade do acúmulo de 3HB e 3HHx. Foram construídos recombinantes abrigando o gene phaB (codificador de 3-cetoacil-CoA redutase) de Ralstonia eutropha ou phaJ1 e phaJ2 (codificadores de enoil-CoA hidratases R-específicas) de Pseudomonas aeruginosa. A expressão de phaB ou phaJ1 aumentou a canalização de 3HB para a PHA sintase, apesar de não aumentar as frações de 3HHx. Monômeros de 3HHx e 3HO foram detectados a partir de ácidos butírico e octanóico, respectivamente. / The ability of B. sacchari to accumulate poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P3HB-co-3HHx) from glucose and hexanoic acid was confirmed. 3HHx content was up to 2 mol% of PHA (<10% of the maximum theoretical 3HHx from the acid), indicating a substrate flexibility of B. sacchari PHA synthase, but high efficiency of hexanoate catabolic pathways. Thermal stability analysis of the copolymer indicated a reduced degradation temperature compatible with 3HHx units. Mutants unable to grow on hexanoic acid were obtained with UV and mini-Tn5 transposon. They still accumulated 3HHx from hexanoate, but the ability to accumulate 3HB and 3HHx was reduced. Recombinants harboring the Ralstonia eutropha phaB (encoding 3-ketoacyl-CoA reductase) and Pseudomonas aeruginosa phaJ1 and phaJ4 genes (encoding R-specific enoyl-CoA hydratases) were constructed. Expression of both phaB and phaJ1 increased the channeling of 3HB to the PHA synthase, despite no increase on 3HHx fraction was observed. 3HHx and 3HO monomers were detected from butyric and octanoic acids, respectively.

O processo de micrencapsulação de proteínas em microesferas (MS) de PLGA [poli (ácido lactico-co-glicolico)] é fácil de fazer e é uma ferramenta útil para melhorar tanto uma formulação quanto para aumentar a atividade imunológica de vacinas de novas gerações. A MS-PLGA têm caráter adjuvante porque é um sistema particulado e, além disto, controla a liberação do antígeno. O escopo desta tese foi o de dar uma nova roupagem para um antígeno vacinal tradicional e muito bem estudado- o toxoide diftérico (Dtxd). Estudaram-se a produção de MS de tamanho desejado; os mecanismos que controlam danos nas proteínas durante o processo de micrencapsulação; a produção de microesferas com características de liberações em tempos distintos e ensaios biológicos. O tamanho de MS é um determinante fundamental para controlar a velocidade de liberação de um soluto. Para se produzir MS com tamanhos controlados usou-se um desenho fatorial experimental com três fatores distintos e três pontos centrais, para se determinar a influência das variáveis (concentração de poli álcool vinílico; velocidade de agitação e relação fase dispersa/fase contínua) na determinação do tamanho das MS. Foram obtidas MS esféricas e lisas de 4- 15 &#181;m de diâmetro. Estes resultados abrem a possibilidade de se formular PLGA-MS com tamanhos planejados através de um mínimo de experimentos. O mecanismo de danos conformacionais nas proteínas nas várias fases do processo de produção de PLGA-MS é ainda uma questão em aberto. Usaram-se várias técnicas biofísicas (HPLC, espectroscopias no uv, fluorescência e CD) além de ELISA para se testar a interferência dos sais da série de Hofmeister sobre a solubilidade e estabilidade da proteína durante a emulsificação e do contacto com a interface água/cloreto de metileno (primeira etapa do processo de preparação de MS). Estudaram-se também a influência de oligômeros de PLGA e SDS sobre a estrutura da proteína no meio de liberação (etapa de liberação do soluto). A emulsificação de Dtxd na presença de Mg2+ induziu agregação protéica, com exposição de resíduos hidrofóbicos para o meio; variações no ângulo diédrico do S-S proteico e perda de identidade imunológica. Esta agregação foi quase abolida pelo caotrópico SCN- (toxicidade = 30 g/ homem adulto de 70 kg). A conformação \"nativa\" do Dtxd e sua atividade biológica foram protegidas pelo KSCN. Os oligômeros de PLGA e o SDS induziram uma conformação de Dtxd nova. A adição de KSCN na fase aquosa aumentou a eficiência de encapsulação de Dtxd pela PLGA-MS em 20 %. Esta foi a solução mais simples quando comparada com aquelas descritas na literatura. Produziram-se seis formulações diferentes (diferentes massas molares e carboximetilações do PLGA) com pelo menos três cinéticas de liberações distintas. Imunizaram-se camundongos com 5 &#181;g de Dtxd encapsulado em MS-PLGA usando-se dois polímeros de 12 kDa (-COOH livre ou metilado) e um outro de 63 kDa (metilado). O padrão de resposta e a maturidade imunológicas foram medidos por titulações de IgG1 e IgG2a. Mantiveram-se os mesmos padrões de resposta humoral (desejável). Menores quantidades de antígenos foram necessárias para se obter os mesmos benefícios gerados pela vacina tradicional de Dtxd. Aumentaram-se a produção e a seletividade de anticorpos através de duas manipulações simples: a formulação e o tempo da aplicação da dose de reforço. Estes resultados colocam estas formulações na área de vacinas de sucesso uma vez que também foram obtidas memórias imunológicas. / The protein microencapsulation within microspheres (MS) of PLGA (Poly-lactide-co-glycolide) is easy to do and, it is a useful tool to enhance formulation and immunologic performances for new generation vaccines. MS-PLGA has adjuvant character because it is a particulate system and can control the antigen release. The question addressed in this thesis was to give this new dress for the traditional and well studied vaccine antigen - the diphtheria toxoid (Dtxd). The steps of MS control size production; mechanism to control protein damages; MS production with different polymers and biological assay were addressed here. MS size is a primary determinant of solute release velocity. A full factorial experimental design 23 with triplicate at the central point was used to determine the influence of variables (polyvinyl alcohol concentration, stirring velocity and the relationship between dispersed /continuous phase) on MS size. Uniformly spherical and smooth microspheres (4 - 15 &#181;m of diameter) were obtained. These results open the possibility of formulating PLGA microspheres with custom sizes performing a minimum of experiments as required for specific applications. It stills an open question to detail the conformational mechanism of protein damages during the various steps of the PLGA microencapsulation process. Various techniques (HPLC gel filtration, ELISA, Fluorescence, UV and Circular dichroism spectroscopies) were tested on the interference of the Hofmeister ion series over protein solubility and stability during the emulsification and contact with the interface water/CH2Cl2 interface (First step on MS preparation). The interference of SDS and PLGA olygomers over protein structure in the liberation media was also studied (solute liberation step). The Dtxd emulsification in the presence of Mg2+ was followed by protein aggregation, with exposition of hydrophobic residues and changes on the dihedral S-S protein angle and loses on immunological identity. This aggregation is 95% avoided by the chaotropic and little toxic salt KSCN (30g/ adult human of 70 kg). All the \"native\" Dtxd conformation and biological properties were maintained by KSCN. MS with different liberation kinetics profile and different erosion characteristics were obtained by using six different polymers. The SDS and PLGA olygomers exerted a generation of new Dtxd molecular organization. The KSCN increased Dtxd encapsulation within PLGA-MS in more than 20 %. This was the simplest solution used to solve protein aggregation compared with others solutions used in the literature. The six different formulations produced (differing in molar mass and carboxymethylation) produced, at least, three different Dtxd liberation profiles. Mice were primed with 5 µg of Dtxd microencapsulated within MS prepared with 12 kDa (ended carboxymethylated or free PLGA) and with 63 kDa (methylated) PLGA. The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The humoral pattern was maintained, but fewer antigens were needed to obtain the same traditional Dtxd vaccine benefits. The simple change on Dtxd-PLGA formulation and timing of the booster enhanced both, antibody production and selectivity. An immunological memory was also obtained, putting so, these formulations in the field of successful vaccine.