<p>U radu su predstavljeni rezultati&nbsp; ispitivanja biolo&scaron;ke&nbsp;<br />stabilnosti vode za piće primenom rotirajućih prstenastih reaktora (eng.&nbsp;<br />rotating annular reactor, RAR). Pomoću RAR praćen je uticaj odabranih&nbsp;<br />faktora na suspendovani i pričvr&scaron;ćeni mikrobni rast pri kontrolisanim&nbsp;<br />laboratorijskim uslovima koji su simulirali distributivne sisteme. Pored&nbsp;<br />toga, u cilju potpunog razumevanja biolo&scaron;ke stabilnosti vode za piće&nbsp;<br />izvr&scaron;eno je ispitivanje metoda za određivanje biodegradabilne frakcije<br />organske materije, BDOC i AOC test.</p><p>Poređenjem BDOC testova sa nativnom populacijom bakterija&nbsp;<br />pričvr&scaron;ćenom za pesak iantracit utvrđena je efikasnija primena biolo&scaron;kog&nbsp;<br />aktivnog peska. Primenom peska za inokulaciju uzorka, vrednost BDOC&nbsp;<br />u podzemnoj vodi sa teritorije srednjeg Banata iznosila je 1,27 mgC/l. U<br />ispitivanju AOC testa sa suspendovanim standardnim kulturama&nbsp;<br />Pseudomonas fluorescens P17 (ATCC 49642) i Spirillum sp. NOX&nbsp;<br />(ATCC 49643), AOC iskori&scaron;ćen od strane NOX je imao veću vrednost u&nbsp;<br />odnosu na P17, &scaron;to pokazuje da su karboksilne kiseline dominantan&nbsp;<br />supstrat za rast. Vrednost AOC u tretiranoj podzemnoj vodi iznosila 32&nbsp;<br />&mu;g acetat-C/l. Veće vrednosti BDOC ukazuju na prisustvo jedinjenja&nbsp;<br />veće molekulske mase u ovoj frakciji u odnosu na AOC frakciju&nbsp;<br />biodegradabilne organske materije.</p><p>U ispitivanju biostabilnosti podzemne vode sa teritorije srednjeg&nbsp;<br />Banatatokom tretmana, najveća količina biofilma je razvijena u RAR sa&nbsp;<br />ozoniranom vodom (13,30 CFU/cm²), &scaron;to potvrđuje efekte ozonizacije na&nbsp;<br />biodegradabilnost organske materije i ponovni rast mikroorganizama.&nbsp;<br />Tokom tretmana zabeleženo je variranje razvoja biofilma, pri čemu je&nbsp;<br />najmanji stepen rasta biofilma uočen u RAR sa vodom nakon GAC&nbsp;<br />filtracije (1,10 CFU/cm²).</p><p>Tokom mikrobne kolonizacije test-pločica RAR pri laminarnom i&nbsp;<br />turbulentnom protoku uočen je sličan trend rasta bakterija u biofilmu u sintetičkoj sme&scaron;i organskih i neorganskih nutrijenata. Međutim, tokom dominacije suspendovanog rasta u sistemu voda-biofilm pri neprotocnim uslovima, pona&scaron;anje biofilmova razvijenih pri protočnim (laminarnim i turbulentnim) uslovima je bilo različto. Uočen je manji udeo bakterija prisutnih u vodenoj fazi kod biofilma nastalim pri turbulentnom protoku u odnosu na veće oslobađanje ćelija iz biofilma nastalim pri laminarnim uslovima, nakon 48h 78% i 89% suspendovanih bakterija, redom. Ovi rezultati pokazuju da su biofilmovi razvijeni pri turbulentnom protoku snažniji, stabilniji &nbsp; i jače pričvr&scaron;ćeni nego pri laminarnom protoku.</p><p>Tokom praćenja sekundarne kolonizacije odvojenih ćelija biofilma&nbsp;<br />uočeno je da se primarni biofilm pona&scaron;a kao stalni rezervoar ćelija koje&nbsp;<br />su sposobne da zauzmu nove povr&scaron;ine vrlo brzo pri različitim&nbsp;<br />hidrodinamičkim uslovima. Brojnost bakterija u biofilmu razvijenom na&nbsp;<br />sekundarnim test-pločicama nakon 24 h iznosila je 32000&plusmn;1200&nbsp;CFU/cm^2&nbsp;<br />u odnosu na 16&plusmn;1 CFU/cm^2&nbsp;primarne test-pločice. Ustanovljen&nbsp;<br />je izraženiji potencijal kolonizacije novih povr&scaron;ina odvojenim želijama iz&nbsp;<br />primarnog biofilma koji je formiran pri turbulentnim uslovima u odnosu&nbsp;<br />na laminarne uslove.</p><p>Proces stvaranja biofilma na test-pločicama od nerđajućeg čelika&nbsp;<br />(SS), polivinilhlorida(PVC) i polietilena(PE) je bio vrlo sličan. Samo&nbsp;<br />debljina biofilma je bila veća na PE (4,0-5,5 kg/m³) nego na SS i PVC&nbsp;<br />test-pločicama (2,7-3,6 kg/m^3&nbsp;i 2,8-3,9 kg/m³, redom). Ustanovljen je&nbsp;<br />uticaj materijala cevi na rast biofilma u RAR snabdevanim vodom iz&nbsp;<br />distributivnog sistema grada Novog Sada, naročito za vreme početne faze&nbsp;<br />procesa. Biofilmovi formirani na različitim materijalima u RAR su uticali&nbsp;<br />na mikrobiolo&scaron;ki kvalitet vode, zavisno od njihove bakterijske gustine.&nbsp;</p><p>SEM analiza potvrdila je prisustvo biofilma na test-pločicama u&nbsp;<br />eksperimentima, ukazujući na uspe!nu primenu RAR u ispitivanju&nbsp;<br />biofilma i biostabilnosti vode za piće.</p><p>Rezultati dobijeni tokom ispitivanja ukazuju da se primenom&nbsp;<br />odabranih i optimizovanih, zavisno od cilja ispitivanja i vrste uzorka,&nbsp;<br />BDOC i AOC testova može efikasno definisati biolo!ka stabilnost vode&nbsp;<br />za piće. Pored toga, sistematski pristup odabiru odgovarajućih materijala&nbsp;<br />cevi, koji je zasnovan na pouzdanim testovima i definisanim&nbsp;<br />kriterijumima, može se primeniti u cilju osiguravanja kvaliteta vode sa&nbsp;<br />mikrobiolo&scaron;kog aspekta.</p><p>Ispitivanja su pokazala uspe&scaron;nu primenuRAR u simulaciji&nbsp;<br />mikrobnog rasta u cevima pri kontrolisanim laboratorijskim uslovima i&nbsp;<br />značaj istovremenog ispitivanja imobilisanog i suspendovanog rasta u&nbsp;<br />definisanom hidrodinamičkom sistemu.&nbsp; Primena RAR kao model biofilma je ukazala na njegov značaj u ispitivanju potencijala ponovnog naseljavanja povr&scaron;ina otkinutih delova i/ili pojedinačnih ćelija biofilma za uspostavljanje efikasne kontrole mikrobnih problema u distributivnim sitemima vode za piće.</p> / <p>This thesis presents theresults of investigation of drinking water biostability using the&nbsp; rotating annular reactor (RAR). The effects of selected factors on the suspended and attached microbial growth under controlled laboratory conditions that simulated the&nbsp;distribution systems were monitored using RAR. In addition, in order to completeunderstanding of the drinking water biological stability,&nbsp;methods for determining biodegradable organic matter fractions, BDOC&nbsp;and AOC methods were tested.</p><p>Comparing the BDOC tests with the native population of bacteria&nbsp;attached to sand and anthracite, a better application of biologically active&nbsp;sand was established. Using BDOC test with sand, the BDOC value in&nbsp;groundwater from the territory of the Central Banat (Republic of Serbia)&nbsp;was 1.27 mgC/L. In the investigation of AOC tests with standard&nbsp;<br />suspended culture of Pseudomonas fluorescens P17(ATCC 49642) and&nbsp;Sprillumsp. NOX (ATCC 49643), AOC utilized by NOX had a higher&nbsp;value compared to the P17, which shows that the carboxylic acid are&nbsp;dominant substrate for growth. The AOC value in the treated ground&nbsp;water was 32 &mu;g acetate-C/L. The higher BDOC values indicate the&nbsp;presence of higher molecular weight compounds in this fraction&nbsp;compared to the AOC biodegradable fraction of organic matter.&nbsp;</p><p>In the investigation of biostability during treatment of&nbsp;groundwater from the territory of the Central Banat (Republic of Serbia),&nbsp;the largest amount of biofilm was developed in the RAR with ozonated&nbsp;water (13.30 CFU/cm²), confirming the effects of ozonation on organic&nbsp;matter biodegradability and microbial regrowth. During treatment the&nbsp;<br />variation of biofilm development was detected and in the RAR with water after GAC filtration the lowest level of biofilm growth (1.10 CFU/cm²) was observed.&nbsp;</p><p><span style="font-size: 12px;">During the microbial colonization of the RAR test-coupons under&nbsp;</span><span style="font-size: 12px;">laminar and turbulent flow, the similar trend of growth of bacteria in the&nbsp;</span><span style="font-size: 12px;">biofilm in synthetic mixture of organic &nbsp;and inorganic nutrients was&nbsp;</span><span style="font-size: 12px;">observed. However, the higher numbers of immobilized bacteria under&nbsp;</span><span style="font-size: 12px;">the turbulent conditions in the same growth period (16 days) were&nbsp;</span><span style="font-size: 12px;">recorded. During the domination of suspended growth in water-biofilm system under &nbsp;non-flow conditions, the behavior of biofilms developed under flow (laminar and turbulent) conditions were different. There was a smaller proportion of bacteria present inthe aqueous phase of the biofilm formed under the turbulent flow over a larger release of cells &nbsp;from biofilms formed under laminar conditions, after 48h 78% and 89% of the suspended &nbsp;bacteria, respectively. These results indicate that biofilms developed under turbulent flow &nbsp;were stronger, more stable and more strongly attached than the ones under laminar flow.</span></p><p>During monitoring of the secondary colonization of the dettached&nbsp;biofilm cells, it was noted that the primary biofilm was a constant&nbsp;reservoir of cells that are able to occupy the new areas very quickly&nbsp;under different hydrodynamic conditions. The abundance of bacteria in&nbsp;the biofilm developed on the secondary test-coupons after 24 h was 32&nbsp;<br />000&plusmn;1200 CFU/cm² compared to 16&plusmn;1 CFU/cm²on the primary test-coupons. A stronger potential for colonization of new areas by dettached&nbsp;cells from the primary biofilm formed under the turbulent conditions in&nbsp;comparison to laminar conditions was found.&nbsp;</p><p>The biofilm formation processes on SS, PVC and PE test-coupons&nbsp;werevery similar. Only biofilm thickness was greater on the PE (4.0 to&nbsp;5.5 kg/m³) than the SS and PVC &nbsp;test-coupons (2.7 to 3.6 kg/m³ and 2.8 to&nbsp;3.9 kg/m³, respectively). There was a slight influence of pipe material on&nbsp;the biofilm growth, especially during the initial stages of the process.&nbsp;Biofilms formed on different materials in the RAR with water from the&nbsp;distribution system of the city of Novi Sad (Vojvodina, Republic of&nbsp;Serbia) have showed the influence on the microbiological quality of&nbsp;water, depending on their bacterial density.</p><p>SEM analysis confirmed the biofilm presence on test-coupons in&nbsp;the experiments, indicating the successful implementation of RAR in the&nbsp;examination of biofilm and drinking water biostability.&nbsp;</p><p><span style="font-size: 12px;">The results obtained during the investigations indicate that the&nbsp;</span><span style="font-size: 12px;">application of selected and optimized, depending on the test objective&nbsp;</span><span style="font-size: 12px;">and sample types, BDOC and AOC tests can effectively define the&nbsp; biological stability. In addition, a systematic approach for &nbsp;electing appropriate pipe materials, which is based on reliable tests and defined criteria, specific for the investigated drinking water distribution system can be applied to ensure &nbsp;he water quality from a microbiological point of view.</span></p><p>The investigations have shown the successful application of RAR&nbsp;in the simulation of microbial growth in the pipes under controlled&nbsp;laboratory conditions as well as the importance of simultaneous&nbsp;examination of immobilized and suspended growth under defined&nbsp;hydrodynamic system. Implementation of RAR as amodel biofilm&nbsp;indicated its importance in examining the potential resettlement area by&nbsp;dettached parts and/or individual biofilm cells to establish effective&nbsp;control of microbial problems in the drinking water distribution system.&nbsp;</p>

L’auto-assemblage de copolymères à blocs amphiphiles est un outil puissant de la chimie supramoléculaire pour la conception de nano-objets complexes et fonctionnels. Dans ces travaux de thèse, l’étude approfondie d’un copolymère à blocs « hybride » synthétique-b-peptidique poly(triméthylène carbonate)-b-poly(acide glutamique) pour des applications de vectorisation a été menée. Des morphologies vésiculaires, obtenues par auto-assemblage en voie « co-solvant » et présentant une grande stabilité ainsi qu’un caractère stimulable ont été mises en évidence. Une transition inédite en température, par des phénomènes de fusion et de fission, a pu être observée. L’encapsulation dans ces vésicules polymères d’un principe actif anti-tumoral et de nanoparticules magnétiques, à des taux très élevés, permet d’améliorer le contraste en IRM ainsi que de moduler la libération de la molécule par une variation des paramètres environnementaux (pH, T) ou par un effet d’hyperthermie magnétique. / Block copolymer self-assembly is a powerful tool within supramolecular chemistry to design smart and functional nano-objects. In this thesis work, comprehensive study of hybrid poly(trimethylene carbonate)-b-poly(glutamic acid) block copolymers for drug delivery applications has been conducted. Highly stable vesicular morphologies presenting stimuli-responsive behaviour were prepared using a solvent-injection method. In particular, original temperature responsiveness mediated by fusion and fission events has been evidenced. Dual loading of an anticancer drug and superparamagnetic nanoparticles in these vesicles, at very high loading contents, allows enhancing MRI contrast and controlling drug release kinetics by varying environmental conditions (pH, T) or by using a magnetic hyperthermia effect.

O objetivo deste trabalho foi produzir poli-3-hidroxibutirato (P3HB) e poli-3-hidroxibutirato-co-3-hidroxivalerato (PHB-co-3HV), polímeros biodegradáveis, utilizando hidrolisado hemicelulósico, rico em xilose, de bagaço de cana-de-açúcar. O estudo dos fluxos metabólicos de xilose in silico indicou que, através do redirecionamento do metabolismo, é possível aumentar o rendimento P3HB a partir de xilose de 0.25 g g-1 para 0.40 g g-1. Obtiveram-se mutantes no sistema repressão catabólica nos quais se verificaram consumo simultâneo de carboidratos e redução do tempo de consumo dos açúcares. Porém, diferenças de fluxos de carbono resultaram em menores valores de crescimento e produção de PH3B em relação às linhagens parentais. Um programa de bioprospecção destacou Burkholderia sp. F24, em experimentos em biorreator obteve-se 25.04 g l-1 de biomassa, 49.31% de acúmulo de P3HB na massa seca celular, alcançando uma produtividade de 0.28 g l-1 h-1. Além disso, foi possível controlar a fração molar de 3HV na síntese PHB-3HV em F24 utilizando xilose e ácido levulínico. / The aim of this thesis is to produce poly3-hydroxybutyrate (P3HB) and poli-3-hidroxibutirate-co-3-hydroxyvalerate (PHB-co-3HV), biodegradable polymers, using hemicellulosic hydrolysate, rich in xylose, from sugarcane bagasse. Metabolic flux analysis in silico of xylose metabolism indicated that, though metabolism redirection is possible to increase P3HB yield from 0.25 g g-1 to 0.40 g g-1. It was observed simultaneous consumption of sugars and reduction of time necessary to exhaust of all sugars in the media culture in mutants with catabolite repression partially abolished. However, differences in carbon flux resulted in lower growth and P3HB production in comparison to the parental strain. A bioprospecting program selected Burkholderia sp. F24, in experiments in bioreactor it reached 25.04 g l-1, 49.31% of P3HB accumulation of the dry cell mass and 0.28 g l-1 h-1 of productivity. Moreover, it was possible to modulate to molar fraction of 3HV in PHB-co-3HV biosyntheses with Burkholderia sp. F24 using xylose and levulinic acid.

Os polihidroxialcanoatos (PHAs) são poliésteres acumulados por microrganismos como material de reserva. O conhecimento das vias bioquímicas e enzimas envolvidas na biossíntese e degradação dos PHAs é uma importante ferramenta para auxiliar na produção industrial. A linhagem Burkholderia cepacia IPT64 é capaz de acumular uma blenda composta de P(3HB) e P(3H4PE) a partir de sacarose. Este trabalho está focado em duas das principais enzimas envolvidas na biossíntese de PHAs: a b-cetotiolase (phaA) e a PHA sintase (phaC). A primeira está associada à especificidade pelo substrato, e a segunda é considerada a enzima chave na síntese de PHAs. Neste trabalho a linhagem mutante phaC foi avaliada quanto à atividade enzimática de PHB sintase, que se constatou ter sido perdida. A presença de mais de uma tiolase no genoma de B. cepacia foi detectada. A inativação do gene phaABc identificado anteriormente, bloqueou totalmente a síntese de P(3HB), e não promoveu o aumento da quantidade total de polímero. Este resultado indica que a tiolase identificada é responsável direta do acúmulo de P(3HB). Outra indicação é que não há uma competição das vias de síntese dos dois polímeros P(3HB) e P(3H4PE), já que não houve alteração na quantidade de P(3H4PE) acumulado, mesmo quando P(3HB) deixou de ser acumulado. / The polyhydroxyalkanoates (PHAs) are polyesters accumulated by microorganisms as storage compounds. Knowing the biochemistry pathway and enzymes involved in the biosynthesis and degradation of PHAs is an important tool to help industrial production. The Burkholderia cepacia IPT64 strain is able to accumulate a blend of P(3HB) and P(3H4PE) from sucrose. The focus of this work is on the two main enzymes involved in PHA biosynthesis: the b-ketothiolase (phaA) and the PHA synthase (phaC). The first one is associated with substrate specificity, and the second one is considered the key enzyme in PHA synthesis. In this work a mutant strain phaC was evaluated on its PHB synthase enzymatic activity, that was discovered to have been lost. The presence of other thiolases in the B. cepacia genome was detected. The inactivation of phaABc gene identified previously, blocked totally the P(3HB) synthesis, and didnt increase the polymer content. This result indicates that the identified thiolase is directly responsible for P(3HB) accumulation. Another indication is that the synthesis pathways of the two polymers, P(3HB) and P(3H4PE), dont compete with each other, because the content of P(3H4PE) was not altered, even when the P(3HB) was not accumulated.

O objetivo deste trabalho foi produzir poli-3-hidroxibutirato (P3HB) e poli-3-hidroxibutirato-co-3-hidroxivalerato (PHB-co-3HV), polímeros biodegradáveis, utilizando hidrolisado hemicelulósico, rico em xilose, de bagaço de cana-de-açúcar. O estudo dos fluxos metabólicos de xilose in silico indicou que, através do redirecionamento do metabolismo, é possível aumentar o rendimento P3HB a partir de xilose de 0.25 g g-1 para 0.40 g g-1. Obtiveram-se mutantes no sistema repressão catabólica nos quais se verificaram consumo simultâneo de carboidratos e redução do tempo de consumo dos açúcares. Porém, diferenças de fluxos de carbono resultaram em menores valores de crescimento e produção de PH3B em relação às linhagens parentais. Um programa de bioprospecção destacou Burkholderia sp. F24, em experimentos em biorreator obteve-se 25.04 g l-1 de biomassa, 49.31% de acúmulo de P3HB na massa seca celular, alcançando uma produtividade de 0.28 g l-1 h-1. Além disso, foi possível controlar a fração molar de 3HV na síntese PHB-3HV em F24 utilizando xilose e ácido levulínico. / The aim of this thesis is to produce poly3-hydroxybutyrate (P3HB) and poli-3-hidroxibutirate-co-3-hydroxyvalerate (PHB-co-3HV), biodegradable polymers, using hemicellulosic hydrolysate, rich in xylose, from sugarcane bagasse. Metabolic flux analysis in silico of xylose metabolism indicated that, though metabolism redirection is possible to increase P3HB yield from 0.25 g g-1 to 0.40 g g-1. It was observed simultaneous consumption of sugars and reduction of time necessary to exhaust of all sugars in the media culture in mutants with catabolite repression partially abolished. However, differences in carbon flux resulted in lower growth and P3HB production in comparison to the parental strain. A bioprospecting program selected Burkholderia sp. F24, in experiments in bioreactor it reached 25.04 g l-1, 49.31% of P3HB accumulation of the dry cell mass and 0.28 g l-1 h-1 of productivity. Moreover, it was possible to modulate to molar fraction of 3HV in PHB-co-3HV biosyntheses with Burkholderia sp. F24 using xylose and levulinic acid.

O copolímero biodegradável poli(3-hidroxibutirato-co-3-hidroxihexanoato) P(3HB-co-3HHx) é um polihidroxialcanoato (PHA) que apresenta várias aplicações. A bactéria Burkholderia sacchari acumula P(3HB-co-2mol%3HHx), a partir de glicose e ácido hexanoico. Com o objetivo de obter P(3HB-co-3HHx) com diferentes teores de 3HHx por B. sacchari, foram construídas linhagens recombinantes, contendo genes do operon phaPCJ de Aeromonas spp. Os recombinantes produziram P(3HB-co-3HHx), a partir de ácidos hexanoico, láurico e linoleico, com teores de 3HHx entre 1,88-18 mol%. Experimentos em biorreator com o recombinante, alimentada na fase de acúmulo por glicose 140 g/L e ácido hexanoico entre 0-45 g/L, resultaram copolímeros com composições variando de 0 a 20 mol% de 3HHx. Os copolímeros assim produzidos foram extraídos e analisados quanto às propriedades físicas. A análise de fluxos metabólicos indicou que a produção de PHA pode ser aumentada com mudanças no metabolismo central e deleção/superexpressão de genes. / The biodegradable copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HHx) is a polyhydroxyalkanoate (PHA) presenting various applications. The bacterium Burkholderia sacchari accumulated P(3HB-co-2mol%3HHx) from glucose and hexanoic acid. In order to obtain P(3HB-co-3HHx) with different 3HHx amounts by B. sacchari, recombinant strains containing phaPCJ operon genes from Aeromonas spp were constructed. Recombinant strains produced P(3HB-co-3HHx) from hexanoic, lauric and linoleic acids, with contents of 3HHx ranging from 1.88 to 18 mol%. Experiments with the recombinant in bioreactor, fed in the accumulation phase by glucose 140 g.l-1and hexanoic acid 0-45 g.l-1, resulted in copolymers with compositions ranging from 0 to 20 mol% of 3HHx. The copolymers produced were extracted and analyzed for physical properties. The metabolic flux analysis indicated that PHA production can be increased by modifying the central metabolism and deleting/ overexpressing genes.

No description available.

En aquest treball es descriu el disseny, síntesi i caracterització de diferents biomaterials i la seva possible aplicació en biomedicina. S'ha fet especial èmfasi a ressaltar la importància que tenen l'estructura, la composició i l'activitat superficial en les propietats finals del biomaterial. Així, s'ha desenvolupat un biomaterial biodegradable i elastomèric, altament porós, amb una gran interconnectivitat que permet la difusió de nutrients i de cèl•lules, un cop sigui implantat. La viabilitat d'aquest material s'ha demostrat tant en assaigs in vitro com in vivo. La síntesi del mateix s'ha realitzat mitjançant calefacció assistida per microones, cosa que ha permès una reacció més ràpida sense necessitat de catalitzadors i evitant les etapes posteriors de purificació. S'ha desenvolupat també un material biodegradable i injectable a temperatures de 45°C, que, en refredar fins als 37°C, s'endureix i pot emprar com a suport estructural al mateix temps que actua com a alliberador de fàrmacs. L'aplicació d'aquest material com ciment ossi millora els problemes de biocompatibilitat, tant de composició, com mecànica, dels actuals productes en el mercat, obrint una nova via d'aplicació d'aquest tipus de materials. D'altra banda, el compost desenvolupat s'ha assajat en aplicacions bioadhesives, on ha demostrat uns excel•lents resultats en la unió de teixit intestinal. Finalment, s'ha aconseguit desenvolupar una superfície amb capacitat de reprogramar cèl•lules amb el simple contacte entre la cèl•lula i la superfície. Amb aquesta tecnologia, anomenada cell reprograming surface (CRS), s'han obtingut cèl•lules pluripotents induïdes (iPS) d'una forma molt més eficient i ràpida que els mètodes habituals de treball. / En este trabajo se describe el diseño, síntesis y caracterización de diferentes biomateriales y su posible aplicación en biomedicina. Se ha hecho especial énfasis en resaltar la importancia que tienen la estructura, la composición y la actividad superficial en las propiedades finales del biomaterial. Así, se ha desarrollado un biomaterial biodegradable y elastomérico, altamente poroso, con una gran interconectividad que permite la difusión de nutrientes y de células, una vez sea implantado. La viabilidad de este material se ha demostrado tanto en ensayos in vitro como in vivo. La síntesis del mismo se ha realizado mediante calefacción asistida por microondas, lo que ha permitido una reacción más rápida sin necesidad de catalizadores y evitando las etapas posteriores de purificación. Se ha desarrollado también un material biodegradable e inyectable a temperaturas de 45°C, que, al enfriarse hasta los 37°C, se endurece y puede emplearse como soporte estructural a la vez que actúa como liberador de fármacos. La aplicación de dicho material como cemento óseo mejora los problemas de biocompatibilidad, tanto de composición como mecánica, de los actuales productos en el mercado, abriendo una nueva vía de aplicación de este tipo de materiales. Por otro lado, el compuesto desarrollado se ha ensayado en aplicaciones bioadhesivas, donde ha demostrado unos excelentes resultados en la unión de tejido intestinal. Por último, se ha conseguido desarrollar una superficie con capacidad de reprogramar células con el simple contacto entre la célula y la superficie. Con esta tecnología, denominada cell reprograming surface (CRS), se han obtenido células pluripotentes inducidas (iPS) de una forma mucho más eficiente y rápida que los métodos habituales de trabajo. / This paper describes the design, synthesis and characterization of different biomaterials and their possible applications in biomedicine, highlighting the importance of the structure, composition and surface activity in the final properties of the biomaterial. Thus, a biodegradable and elastomeric biomaterial, highly porous, with a strong interconnectivity that allows diffusion of nutrients and cells, once it is implanted has been developed. The feasibility of this material has been demonstrated both in vitro and in vivo assays. The synthesis has been performed using microwave-assisted heating, which has allowed a faster reaction without catalysts and avoiding the later stages of purification. It has also been developed a biodegradable and injectable material, at temperatures of 45°C, which, when cooled to 37°C, it hardens and can be used as structural support while acting as drug delivery. The application of such material as bone cement avoid biocompatibility problems, both in composition and mechanicas, of the current products on the market, opening a new way of application of such material. On the other hand, the compound has been tested in bioadhesive applications, where it has shown excellent results in the union of intestinal tissue. Finally, a surface with ability to reprogram cells with simple contact between the cell and the surface has been developed. With this technology, called cell reprograming surface (CRS), induced pluripotent cells (iPS) have been obtained in a much more efficient and faster way than the usual methods.

Combinatorial techniques, which overcome limitations of actual models of material research permitting to effectively address this large amount of variables, are utilized in this work to prepare combinatorial libraries of the blend of the biodegradable polymers Poly(e-caprolactone) and Poly(lactic acid). These libraries present continuous composition and temperature gradients in an orthogonal fashion that permit to obtain multiple surface morphologies with controllable microstructures due to the blends low critical solution phase behavior (LCST). The goal of this study is to investigate the effect of surface morphology (surface chemical patterning and surface topography) on cell behavior. The varied surface topography of the libraries is used as a valuable tool that permits to assay the interaction between MC3T3-E1 cells and hundreds of different values of critical surface properties, namely, surface roughness and microstructure size. The outcome of this tool is a rapid screening of the effect of surface topography on cell behavior that is orders of magnitude faster than the standard 1-sample for 1 measurement techniques. The results obtained show that cells are very sensitive to surface topography, and that the final effect of surface properties on cell function is intimately related with the stage of the cell developmental process. Meaning that, for example, areas with optimal characteristics to elicit enhancement of cell attachment is not necessarily the same that promotes cell proliferation. This study imparts an improved understanding of an often neglected factor in biomaterials performance: surface morphology (particularly surface topography). The results provide a new insight into the importance of taking into consideration both chemistry and physical surface features for superior biomaterial design.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.