Assisted reproductive technologies have become a common method used to treat infertility. These techniques have advanced quickly since the first birth of an in vitro fertilization (IVF) baby in 1978, at the Royal Oldham Hospital in the United Kingdom. Currently, IVF with or without intracytoplasmic sperm injection, is used throughout the world to achieve oocyte retrieval, fertilization, implantation of an embryo, clinical pregnancy, ongoing clinical pregnancy, and a live-born infant. The rationale for selecting one type of fertility treatment over another is multifactorial: the confirmed or unconfirmed cause of infertility, the age of the gamete donor and the recipient, the availability of the type of treatment, and the cost associated with the treatment. The ultimate goal of any fertility treatment is to achieve a successful pregnancy that results in a healthy infant. However, the literature is equivocal on the effects of fertility treatment cycles on the health outcomes of infants and mothers. Presently, there are thirty-six fertility treatment centres across Canada, eighteen of which reside in Ontario. A national, comprehensive database of assisted reproductive technology treatment cycles (Canadian Assisted Reproductive Technologies Register (CARTR) Plus) began collecting data in 2013, and has made the research objectives of this doctoral thesis feasible. Before this data collection system, population-wide studies involving fertility treatments were not possible in Canada. Two understudied issues associated with IVF are the impact of fertility treatments on the maternal serum screening markers used in prenatal screening programs to identify fetal aneuploidies; and the association between fertility treatments and adverse perinatal outcomes, such as preeclampsia and stillbirth. Given the increasing number of women who are using fertility treatments to conceive, it is imperative that studies investigating the association with adverse outcomes are conducted. As the science supporting fertility treatment procedure has advanced, so has prenatal screening. One of the first screening tests that are performed for newly pregnant women, including women who conceived following IVF, is maternal serum screening. The first objective of this doctoral thesis was to systematically review the literature on the association between IVF treatment and maternal serum screening marker levels and nuchal translucency (NT) thickness. After the search and screening of the literature there were 40 studies that were included in this systematic review. A decrease in pregnancy-associated plasma protein A (PAPP-A) and an increase in total human chorionic gonadotropin (hCG) was consistently reported for IVF pregnancies. However, since the levels of the other maternal serum screening markers reported also varied we were unable to generalize about the differences between prenatal screening results in the IVF population. These results led to investigating maternal serum screening marker levels among IVF patients in Ontario, Canada. The second objective of this thesis was three-fold: 1) to investigate the accuracy of IVF identification on the Ontario prenatal screening record, relative to reference standard on the CARTR Plus database; 2) to compare the prenatal screening markers in IVF versus non-IVF pregnancies in the population of Ontario; and 3) to propose updated IVF adjustment factors for prenatal screening in the Ontario population, based on the more accurate coding for IVF status in the CARTR Plus database. Significant differences between IVF and non-IVF groups, based on both the prenatal screening requisition information and CARTR Plus information, were found among the ethnicity adjusted mean multiple of the median (MoM)s for several prenatal screening markers: alpha-fetoprotein (AFP), PAPP-A, unconjugated estriol (µE3), first trimester hCG, total hCG, and dimeric inhibin A (DIA). When we developed the proposed adjustment factors for all CARTR Plus identified pregnancies we found that for PAPP-A, total hCG, and µE3 the mean adjusted marker MoMs were significantly closer to 1.00, as compared to the prenatal screening adjusted or the unadjusted mean marker MoMs. Currently, there is no adjustment made to the other maternal serum screening markers and NT measurement. The third objective was to examine the effect of type of infertility on placental-mediated adverse outcomes (preeclampsia, intrauterine growth restriction, placental abruption, and stillbirth). Type of infertility was classified as male factor (sperm count, poor sperm motility, and abnormal sperm morphology), female factor (ovulation disorders, tubal infertility, and uterine or cervical causes), and unexplained infertility. No significant associations were found between type of conception and the composite outcome, as well as each individual primary outcome. Similarly, the type of infertility was not associated with the composite outcome or any of the individual primary outcomes, except for female factor infertility, which was associated with increased probability of placental abruption. Overall, the results from this doctoral thesis suggest that there are substantial differences seen in maternal serum screening marker MoMs among women who use IVF to conceive, suggesting that appropriate adjustment factors should be employed to ensure accurate results for determining the risk of Down syndrome and trisomy 18. Additionally, although the literature has shown an association between fertility treatment and placental-mediated adverse outcomes no significant associations were found in the population of Ontario. Further studies should be performed to confirm the results of these observational studies.

3-[3-(7-chloro-quinolin-4-yl amino) phenyl]-1-(4-methoxy-phenyl) prop-2-enone citrate “CITme” is a substituted quinoline derivative, synthesized as a potential antitumour agent. The aim of this study was to investigate CITme with regard to antitumour activity, toxicity and pharmacokinetics. In vitro screening for neoplastic cytotoxic effects using standard cell culture techniques revealed cytotoxic activity against HeLa, DU-145, MCF-7, Jurkat and CoLo 320 human derived cancer cell lines at low concentrations. Toxicity was significantly less in either normal resting and stimulated lymphocytes or primary chicken fibroblast cells. CITme showed highest cytotoxic specificity for DU-145 with an IC50 of 11 μM compared to 38.2 ìM for lymphocytes after a three-day incubation period and 2.5 μM, after a seven-day incubation period. CITme exhibited poor solubility in aqueous solutions and this had to be addressed prior to in vivo acute toxicity studies being performed. CITme was found to be insoluble in many biocompatible and acceptable formulating solvents. 2-methyl pyrrolidone and Cremophor EL showed promise as potential solvents but the solubility proved to be too low to enable therapeutic CITme dosing while avoiding solvent/carrier toxicity. An oral microcrystalline cellulose suspension, two combinations of 2-methyl pyrrolidone with plasma protein complexation and a propheroid formulation were also tested. These were tested in pilot acute toxicity studies using BALB/c mice or Sprague Dawley rats. No plasma concentrations were observed with the cellulose suspension and both the 2-methyl pyrrolidone formulations elicited toxic responses in vehicle control and experimental groups despite working well below the published LD50 of 2-methyl pyrrolidone. A unique formulation developed by the Department of Pharmacy of NWU referred to as propheroid drug delivery system, provided sufficient solubility for acute and chronic toxicity studies of CITme. In vivo acute and chronic toxicity study of the propheroid delivery system formulation alone and the CITme propheroid at 30 mg/kg/day and 60 mg/kg body weight (acute toxicity study), and 10mg/kg/day (chronic toxicity study) demonstrated safety after seven and forty-five (six weeks) days administration by oral gavage. The potential anti-tumour activity of CITme could not be determined in a nude mouse tumour model, using the most susceptible in vitro cell line, DU-145 due to the low success rate of tumour induction and the long lag period required for the development of subcutaneous prostate tumours after transplantation of either a DU-145 cell suspension or DU-145 tumour tissue blocks. An LC-MS/MS method was developed to separate and quantify CITme from plasma and organ homogenates. The LC-MS/MS method was used to determine the pharmacokinetic parameters of I.V. administered CITme. A pharmacokinetic study of CITme in mice indicated that CITme is absorbed after an oral administration and exhibits a very rapid distribution (metabolism) and/or elimination after IV administration. CITme could be found at relatively high concentrations 24 hours after oral dosing and to a smaller extent 10 minutes after I.V. administration in both the liver and kidney tissues. Despite the fact that the efficacy and oral pharmacokinetics properties of CITme could not be determined, this study has proved that CITme is a selective and potent anti-tumour agent in vitro with low toxicity both in vitro and in vivo and therefore this compound warrants further scientific evaluation. / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

Myrtaceae possui mais de 3000 espécies sendo Acca sp, Eucalyptus sp, Myrcianthes sp, Myrcia sp, Psidium sp, os gêneros mais representativos pela importância ambiental, econômica, farmacêutica, entre outras. O presente estudo teve como objetivo contribuir no aprimoramento das técnicas de propagação de Psidium cattleianum Sab e Eugenia brasiliensis Lam por estaquia e semeadura em condições in vivo e in vitro. Para P. cattleianum, na propagação in vivo foram testados: Doses de AIB na presença ou ausência de BAP no enraizamento de estacas herbáceas, e indução ao enraizamento de estacas de folha; na propagação in vitro foi testado, para organogênese: Efeito do acido giberélico, do tempo de imersão em NaOCl (2 %) e água 55°C por 10 min na germinação das sementes; Efeito da composição do meio de cultura e da relação entre concentrações de BAP:ANA no desenvolvimento de miniestacas. Para calogênese, para desinfestação de explantes na fase de introdução, foram testados: Pré-tratamento com solução de tiofanato-metílico (49 mg L-1 i.a) misturado com ampicilina (250 mg L-1); efeito do tempo de imersão e do pH da solução de NaOCl. Para desdiferenciação foi testado: Doses de cinetina na indução de calogênese em lâmina foliar e doses de 2,4-D em porções de hipocótilo. Para E. brasiliensis , in vivo, foram testados o efeito da concentração de AIB na indução de enraizamento de estacas e in vitro foi testado o efeito da concentração de AG3 no estabelecimento de miniestacas. Os principais resultados permitem inferir que as duas espécies são passíveis de se propagar por estaquia, sendo que para E. brasiliensis a técnica deve ser aprimorada para aumentar o percentual de enraizamento. Na propagação in vitro de P. cattleianum o meio WPM foi mais adequado para o estabelecimento de miniestacas. A eficiência do NaOCl na desinfestação de sementes e explantes para calogênese é afetada pela concentração da solução, tempo de contato e pH da solução. A desdiferenciação celular e a indução de calo em araçazeiro diferem em função do tipo de explante e concentração de cinetina. Análise histológica evidenciou a participação da região do periciclo vascular e borda dos explantes na calogênese e a diferença na população fenotípica nos explantes foliares. / Myrtaceae has over 3000 species being Acca sp, Eucalyptus sp, Myrcianthes sp, Myrcia sp, Psidium sp, the most representative genus for environmental, economic, pharmaceutical importance, among others. The present study aimed to contribute to the enhancement of propagation techniques of Psidium cattleianum Sab. and Eugenia brasiliensis Lam. by cuttings and sowing conditions in vivo and in vitro. For P. cattleianum, propagation in vivo were tested: IBA doses in the presence or absence of BAP on rooting herbaceous and inducing rooting of leaf; in vitro propagation was tested for organogenesis: Effect of gibberellic acid, immersion time in NaOCl (2%) and water (55 ° C for 10 min) on seed germination; Effect of culture medium composition and culture concentration ratio BAP: ANA on developing shoots. For callus induction, explants disinfection stage of introduction were tested: Pre-treatment with a solution of thiophanate-methyl (49 mg L-1 a.i) mixed with ampicilin (250 mg L-1); effect of immersion time and pH of NaOCl solution. For dedifferentiation were tested: doses of kinetin to induce callus formation in leaf blade and doses of 2,4-D in portions hypocotyl. In vivo for E. brasiliensis we tested the effect of IBA concentration in inducing rooting; in vitro was tested and the effect of GA3 concentration in establishing cuttings. The main results infer that the two species are likely to propagate by cuttings, and to E. brasiliensis technique should be enhanced to increase the percentage of rooting. For in vitro propagation of P. cattleianum the WPM was more suitable for the establishment of cutings. The efficacy of NaOCl in seed disinfection and explants for callus formation is affected by the solution concentration, dwell time and solution pH. The cellular dedifferentiation and callus induction in strawberry guava differ depending on the explant type and concentration of kinetin. Histological analysis showed the pericycle and vascular edge participation of explants on callus formation and phenotypic difference in population in leaf explants.

Ingeniera Civil en Biotecnología / Las antocianinas del maqui son una materia prima de gran interés para la industria alimenticia y nutracéutica, debido al valor agregado que le confieren sus distintas propiedades farmacológicas y a la condición de endemismo de la especie vegetal. Por ello, en el presente trabajo se estudian diversas estrategias de producción in vitro de las antocianinas de Aristotelia chilensis y se concibe un proceso con alcances a escala comercial, buscando satisfacer parte de la creciente demanda por estos compuestos, prescindiendo de la recolección de los frutos del maqui. En cultivos de callo, se evaluaron cinco factores de crecimiento: concentración de sacarosa (3 y 6%), medio basal (MS y B5), luminosidad (L y O), fuente de explante (C, E y H) y hormonas (mezclas a, b, c y d); determinándose que, entre las condiciones de cultivo estudiadas, los tratamientos 3B5.E.L.a, 6MS.C.L.b, 3MS.E.O.b y 6B5.C.O.a presentaron la mayor velocidad de crecimiento celular; mientras que 3B5.C.L.d y 3MS.C.L.d destacaron por su alto contenido de antocianinas. Además, se observó que los callos morados podían seguir produciendo estos compuestos, aún en ausencia de las hormonas que inducían su síntesis, alcanzando aún una acumulación más alta que en presencia de estos químicos. En función de lo anterior, fue posible diseñar e implementar una estrategia de producción de antocianinas en suspensiones celulares de maqui. Este proceso contempló tres fases de cultivo: para aumentar la biomasa (Fase A), para inducir la acumulación de los metabolitos secundarios (Fase B) y para eliminar los residuos de la hormona 2,4-D (Fase C). En consecuencia, se evaluaron dos condiciones de cultivo para la Fase A, en luz (6MS.C.L.b) y oscuridad (6B5.C.O.a); mientras que para las Fase B y C se utilizaron los mismos factores que en 3MS.C.L.d, con y sin hormonas, respectivamente. Sin embargo, la acumulación de antocianinas en los cultivos en suspensión fue deficiente en comparación a la obtenida en callos. Adicionalmente, en la estrategia de cultivo con Fase A en luz, se estudió el efecto de dos elicitores (peróxido de hidrógeno y ácido clorhídrico) y un precursor (fenilalanina); no obstante, ninguno de estos factores favoreció una mayor acumulación de los metabolitos de interés. Finalmente, se concibió un proceso de producción in vitro de antocianinas, con alcances a escala comercial, basado en la implementación de un sistema de inmersión temporal para el cultivo de callos de maqui, el cual incluye el desarrollo de las tres fases de cultivo (A, B y C), anteriormente explicadas, en un periodo total de 40 semanas. De acuerdo a una evaluación técnico-económica, no se puede descartar que esta estrategia sea rentable, sin embargo no es técnicamente factible para altos niveles de producción y por lo tanto, no sería atractiva comercialmente; ya que únicamente es posible generar cantidades de antocianinas del orden de 100 mg por batch, equivalente al contenido en 12 g de fruto de maqui.

Memoria para optar al Título Profesional de Médico Veterinario / El factor de crecimiento diferencial 9 (GDF-9) y la proteína morfogénica ósea 15 (BMP-15) regulan en diferentes especies procesos esenciales para la maduración del ovocito, tales como la expansión del cúmulo. Este estudio evaluó en caninos la expresión génica de GDF-9 y BMP-15 en las células del cúmulo (CCs) obtenidas de complejos cúmulos ovocitos (COCs) a través del ciclo estral en relación a la maduración in vitro (IVM) de los ovocitos y la expansión de estas células. Las CCs se obtuvieron de COCs de folículos antrales de anestro, proestro, estro y diestro, previo y después de la IVM, expandidas y no expandidas. Para lograr mayor cantidad de CCs, antes y después de la IVM, estas se cultivaron in vitro por 48 h en medio Dulbecco’s Modified Eagle’s Medium (DMEM) con suero fetal bovino. El desarrollo meiótico luego de la IVM se evaluó mediante microscopía de fluorescencia, clasificándolo en vesícula germinal (VG), reinicio meiótico (GVBD), primera metafase (MI) y segunda metafase (MII). La expresión relativa de GDF-9 y BMP-15 en las CCs se evaluó mediante q-PCR. Estos genes se encontraron en todas las etapas del ciclo antes y después de la IVM, con diferencias significativas entre estas en relación a la expansión y maduración de los ovocitos. De acuerdo a los estados del ciclo estral, en las CCs de COCs no madurados, los niveles de GDF-9 fueron mayores (P < 0,05) durante el estro, seguido por el diestro y los menores (P < 0,05) niveles en proestro y anestro. En BMP-15, los mayores (P < 0,05) niveles de mRNA se observaron en estro y diestro. En las CCs expandidas no hubo diferencias en GDF-9 entre las distintas etapas del ciclo, pero en BMP-15 los menores (P < 0,05) niveles de mRNA se observaron en estro. Luego de la maduración, hubo un incremento (P < 0,05) de los transcriptos de ambos genes en las CCs de anestro, proestro y diestro, excepto los niveles de mRNA de BMP-15 durante el diestro. En estro en cambio, hubo una disminución significativa de la expresión de los dos genes luego de la IVM. No se encontraron diferencias significativas en la capacidad de alcanzar el estado de MII al considerar la etapa del ciclo. Sin embargo, los COCs obtenidos de proestro y diestro que expandieron sus CCs pudieron reiniciar la meiosis en mayor (P < 0,05) porcentaje en comparación con las demás etapas del ciclo y los COCs de estro que expandieron sus CCs lograron mayores (P < 0,05) porcentajes de MII en relación a los que no expandieron el cúmulo. En conclusión, GDF-9 y BMP-15 se expresan en las CCs en todas las etapas del ciclo con un patrón diferente de acuerdo a cada una y estarían involucrados en la expansión del cúmulo de la perra, pudiendo estar relacionado con la maduración meiótica in vitro, al menos en aquellos ovocitos en etapa estral. / Differential growth factor 9 (GDF-9) and bone morphogenic protein 15 (BMP-15) regulate essential processes for oocyte maturation in different species, such as cumulus expansion. This study evaluated gene expression of GDF-9 and BMP-15 in canine cumulus cells (CCs) obtained from cumulus-oocyte complexes (COCs) through the estrous cycle in relation to oocyte in vitro maturation (IVM) and the expansion of CCs. The CCs were obtained from COCs of antral follicles from anestrus, proestrus, estrus, diestrus phases, before and after IVM, expanded and non-expanded. To achieve more CCs, before and after IVM, these were cultured in vitro for 48 h in Dulbecco's Medium Modified Eagle's Medium (DMEM) with fetal calf serum. The meiotic development after IVM was evaluated by fluorescence microscopy, classifying it in germinal vesicle (GV), germinal vesicle breakdown (GVBD), first metaphase (MI) and second metaphase (MII). The relative expression of GDF-9 and BMP-15 in the CCs was evaluated by q-PCR. These genes were found in all stages of the reproductive cycle, before and after IVM, with significant differences between them in relation to the expansion and oocytes maturation. According to the estrous cycle stages, CCs from non-matured COCs expressed higher (P < 0.05) levels of GDF-9 mRNA during estrus in comparison with the other phases and the lowest (P < 0.05) levels at proestrus and anestrus. The highest (P < 0.05) levels of BMP-15 transcripts were observed in estrus and diestrus. There were no differences in GDF-9 gene expression comparing expanded CCs among the different phases of the estrous cycle, regarding BMP- 15 transcripts, the lowest (P < 0.05) levels were registered in estrus. After IVM, the level of both genes increased (P < 0.05) in CCs of anestrus, proestrus and diestrus, except BMP-15 during diestrus. In contrast, in estrus GDF-9 transcripts significantly decreased after IVM. Non expanded CCs exhibited the lowest levels of both genes at anestrus and proestrus, whereas at diestrus the levels of GDF-9 in non-matured CCs were lower (P < 0.05) than that of the expanded cells, the levels of BMP-15 were similar. No differences were found in the oocyte ability to reach MII state when comparing the phases of the estrous cycle. However, COCs obtained from proestrus and diestrus that expanded their CCs showed higher (P < 0.05) percentage of meiosis resumption than to the other phases. At estrus expanded CCs achieved higher (P < 0.05) percentages of MII in comparison to those did not expand. In conclusion, GDF-9 and BMP-15 are expressed in the CCs over the estrous cycle with different patterns according each phase and these genes maybe involved in the cumulus expansion in canines, maybe related to the in vitro maturation, at least in oocytes at estrus phase / Financiamiento: Proyecto Fondecyt 1171670

This work investigates ways to prevent and manage hyperglycemia using preventive nutrition. Uncontrolled and chronic hyperglycemia is a global health issue leading to many health problems including diabetes. This thesis details the manipulation of highly retrograded starch particles in order to produce particles that are digested slowly to release glucose at a prolonged and moderate rate to prevent this. The first section of this study utilized acid hydrolysis to alter starch structure and change digestibility. The hydrolysis treatment showed that hydrolysis of native starch prior to particle formation changed the structure in a way that increased digestibility. The second section of this work introduced polyphenols into the particles which only a marginal effect on digestion. Overall the actual process of retrograding and making the particles themselves appeared to create particles that were more resistant to digestion. These could be used in a product to deliver a moderate glycemic response.

Xylooligosaccharides (XOS) are considered to be prebiotics. Prebiotics are defined as non-digestible food ingredients that benefit the host by stimulating the growth and activity of a limited number of bacteria, such as the Bifidobacterium genus, in the colon. Corn fiber separated from distillers dried grains with solubles (DDGS) could be a valuable feedstock for XOS production. The objective of the first chapter was to determine the efficacy for autohydrolysis to produce XOS using fiber separated from DDGS. Fiber was treated with deionized water in a Parr-reactor, at temperatures ranging from 140 to 220 °C to produce XOS. The maximum total yield of XOS in the solution was 18.6 wt% of the corn fiber at 180 °C. The objective of the second chapter was to evaluate and compare the prebiotic effect of XOS produced by autohydrolysis of DDGS fiber (XOS-D) with other substrates (FOS, commercial XOS (XOS-C), xylose, glucose and inulin) on intestinal bacteria, B. adolescentis, B. breve and Lactobacillus brevis. Bacterial growth on XOS-C was comparable with growth on FOS and inulin. XOS-D promoted bacterial growth more than that of control. Prebiotic potential of XOS produced from corn fiber was confirmed. The objective of third chapter is to determine the yield of XOS from corn fiber separated from ground corn flour (FC) and DDGS (FD) at different autohydrolysis temperatures and hold-times. The conditions for maximum XOS production for FD and FC were 180 °C with 20 min hold-time and 190 °C with 10 min hold-time, respectively. The fourth chapter focuses on production of XOS by enzymatic hydrolysis method for XOS production. Endo-1-4-xylanase enzyme was ineffective for corn fiber as well as corn fiber gum (CFG), despite evaluating a multitude of pretreatment methods and processing conditions. We have proposed use of Multifect Pectinase PE and Multifect Xylanase enzymes, based on work from other researchers. For commercial applications such as food industries, XOS would need to be isolated from liquor. The fifth chapter of this study focuses on literature review of purification methods used in XOS purification.

Die Implantation eines Medizinprodukts in den menschlichen Körper ruft eine Immunreaktion hervor, die zur fibrösen Einkapselung führen kann. Makrophagen in direktem Kontakt mit der Oberfläche des Implantats erfassen sensorisch den Fremdkörper und übersetzten das Signal in die Freisetzung zahlreicher löslicher Mediatoren. Das generierte Entzündungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erhöhung der Matrixsyntheserate in der Wundumgebung führen. Eine dichte fibröse Kapsel um ein Medizinprodukt beeinträchtigt den Ersatz von Körperstrukturen, das Unterstützen physiologischer Körperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterstützt werden. Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begründet die Komplexi-tät der ablaufenden Fremdkörperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verfügbarkeit validierter, verlässlicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen während einer Fremdkörperreaktion abbildet und sich zur Testung über längere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch können in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine höhere Übertragbarkeit auf den Menschen aufweisen. Zusätzlich adressiert die Präferenz zu in vitro Testmethoden den Aspekt „Reduzierung“ der 3R-Prinzipien „Replacement, Reduction, Refinement“ (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begründeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium für die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellulärer Matrix in der Wundumgebung stellen wichtige Teilprozesse während einer Fremdkörperreaktion dar. Für beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von löslichen Mediatoren durch materialadhärente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden. Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialständige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberflächenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix ermöglichte die Übersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Darüber hinaus konnte in einem dreidimensionalen Fremdkörpermodell die komplexe dreidimensionale Struktur der extrazellulären Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdkörpermodelle ermöglichten die Testung von Biomaterialien über längere Testphasen und können in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen ermöglichen, (II) die Identifizierung verlässlicher Testbedingungen unterstützen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verlässliche Unterscheidungskapazität zwischen Biomaterialien aufweist. / The implantation of a medical product into the human body induces an immune reaction, which may lead to its fibrous encapsulation. Macrophages in direct contact to the surface sense the foreign body and translate the signal in the secretion of multiple soluble mediators. This generated inflammatory milieu modulates the healing reaction, may induce the accumulation of fibroblasts and lead in the wound microenvironment to an increased matrix synthesis rate. A dense fibrous capsule surrounding a medical product is able to impair the replacement of body structures, the support of physiological body functions as well as the efficiency of a medical therapy. To identify potential biomaterial candidates with optimal characteristics an evidence-based decision making process is necessary and furthermore affords the support by appropriate test procedures. To study local effects after implantation of biomaterials, the complexity of the foreign body reaction justifies the application of animal models as gold standard. The integration of in vitro test procedures into standardized test strategies often fails by the availability of validated, reliable and reproducible methods. According to that there is no standardized test procedure, which resembles the three-dimensional tissue structures during a foreign body reaction and is suited for longer contact phases in between blood components and biomaterials. In vitro tests are often more cost and time efficient and show as well by applying human cells a high transferability on human beings. Additionally the preference to in vitro test procedures addresses the “reduction” aspect of the Russel and Burch’s (1959) 3R-principles “replace-ment, reduction and refinement” to a conscious and reasoned use of animal models in science. Aim of this research project was the development of human in vitro model systems, which resemble the contact to blood components and the reaction of the surrounding soft tissue following implantation of a biomaterial. Reference materials, whose tissue integration after implantation in animals or humans is described, were applied for the developed model systems as validation criterion. The accumulation of cells and the synthesis of extracellular matrix in the surrounding wound are relevant sub processes during a foreign body reaction. In an indirect cell-based model system the influence of the cell-mediated conditioning initiated by the material-induced and macrophage-mediated liberation of soluble mediators was shown on both sub processes the aligned migration of fibroblasts as well as the remodeling of a three-dimensional tissue model. Additionally, the cytokine secretion profile by material-adherent macrophages was characterized under different test conditions such as the contamination with LPS, the surface treatment with human plasma and the presence of IL-4. The following comparative statistical modelling allowed a transformation of the generated complex multi-factorial data matrix to a biomaterial ranking. The here developed test procedure was suitable for the validation of in vitro test conditions as well as the evaluation of the reference biomaterials. Last, by the combination of different cells and matrix structures the complex three-dimensional structure of the extracellular matrix in a wound was biomimetically reconstructed. Those novel three-dimensional foreign body models enabled the testing of biomaterials over longer test phases and might be applied in following studies to investigate dynamic processes. Summarizing in this research project three different test strategies were developed, which (I) enable the evaluation of sub processes, (II) support the identification of reliable test conditions and (III) biomimetically reconstruct a wound tissue. Most important is, that a three-dimensional tissue model was biomimetically developed, which showed a reliable discriminatory capacity in between biomaterials.

Cancer remains after cardiovascular diseases the leading cause of death worldwide and an estimated 8.2 million people died of it in 2012. By 2030, 13 million cancer deaths are expected due to the growth and ageing of the population. Hereof, colorectal cancer (CRC) is the third most common cancer in men and the second in women with a wide geographical variation across the world. Usually, CRC begins as a non-cancerous growth leading to an adenomatous polyp, or adenoma, arising from glandular cells. Since research has brought about better understanding of the mechanisms of cancer development, novel treatments such as targeted therapy have emerged in the past decades. Despite that, up to 95% of anticancer drugs tested in clinical phase I trials do not attain a market authorisation and hence these high attrition rates remain a key challenge for the pharmaceutical industry, making drug development processes enormously costly and inefficient. Therefore, new preclinical in vitro models which can predict drug responses in vivo more precisely are urgently needed. Tissue engineering not only provides the possibility of creating artificial three-dimensional (3D) in vitro tissues, such as functional organs, but also enables the investigation of drug responses in pathological tissue models, that is, in 3D cancer models which are superior to conventional two-dimensional (2D) cell cultures on petri dishes and can overcome the limitations of animal models, thereby reducing the need for preclinical in vivo models. In this thesis, novel 3D CRC models on the basis of a decellularised intestinal matrix were established. In the first part, it could be shown that the cell line SW480 exhibited different characteristics when grown in a 3D environment from those in conventional 2D culture. While the cells showed a mesenchymal phenotype in 2D culture, they displayed a more pronounced epithelial character in the 3D model. By adding stromal cells (fibroblasts), the cancer cells changed their growth pattern and built tumour-like structures together with the fibroblasts, thereby remodelling the natural mucosal structures of the scaffold. Additionally, the established 3D tumour model was used as a test system for treatment with standard chemotherapeutic 5-fluorouracil (5-FU). The second part of the thesis focused on the establishment of a 3D in vitro test system for targeted therapy. The US Food and Drug Administration has already approved of a number of drugs for targeted therapy of specific types of cancer. For instance, the small molecule vemurafenib (PLX4032, Zelboraf™) which demonstrated impressive response rates of 50–80% in melanoma patients with a mutation of the rapidly accelerated fibrosarcoma oncogene type B (BRAF) kinase which belongs to the mitogen active protein kinase (MAPK) signalling pathway. However, only 5% of CRC patients harbouring the same BRAF mutation respond to treatment with vemurafenib. An explanation for this unresponsiveness could be a feedback activation of the upstream EGFR, reactivating the MAPK pathway which sustains a proliferative signalling. To test this hypothesis, the two early passage cell lines HROC24 and HROC87, both presenting the mutation BRAF V600E but differing in other mutations, were used and their drug response to vemurafenib and/or gefitinib was assessed in conventional 2D cell culture and compared to the more advanced 3D model. Under 3D culture conditions, both cell lines showed a reduction of the proliferation rate only in the combination therapy approach. Furthermore, no significant differences between the various treatment approaches and the untreated control regarding apoptosis rate and viability for both cell lines could be found in the 3D tumour model which conferred an enhanced chemoresistance to the cancer cells. Because of the observed unresponsiveness to BRAF inhibition by vemurafenib as can be seen in the clinic for patients with BRAF mutations in CRC, the cell line HROC87 was used for further xenografting experiments and analysis of activation changes in the MAPK signalling pathway. It could be shown that the cells presented a reactivation of Akt in the 3D model when treated with both inhibitors, suggesting an escape mechanism for apoptosis which was not present in cells cultured under conventional 2D conditions. Moreover, the cells exhibited an activation of the hepatocyte growth factor receptor (HGFR, c-Met) in 2D and 3D culture, but this was not detectable in the xenograft model. This shows the limitations of in vivo models. The results suggest another feedback activation loop than that to the EGFR which might not primarily be involved in the resistance mechanism. This reflects the before mentioned high attrition rates in the preclinical drug testing. / Krebs ist nach Herz- und Kreislauferkrankungen die führende Todesursache weltweit und 2012 starben daran geschätzt 8,2 Millionen Menschen. Für das Jahr 2030 werden 13 Millionen Krebstote erwartet, was auf das Bevölkerungswachstum und deren Überalterung zurückzuführen ist. Dabei ist das kolorektale Karzinom (engl. colorectal cancer, CRC) der dritthäufigste Krebs bei Männern und der zweithäufigste bei Frauen. Für gewöhnlich entwickelt sich CRC aus einem nicht-kanzerösen Wachstum, das zu einem adenomatösen Polyp bzw. Adenom führt, welches aus Drüsenzellen hervorgeht. Da die Forschung in den vergangenen Jahrzehnten ein besseres Verständnis für die Mechanistik der Krebsentstehung hervorgebracht hat, entstanden neuartige Behandlungsformen, wie die zielgerichtete Krebstherapie. Hohe Versagensraten, welche den Medikamentenentwicklungsprozess sehr kostenaufwendig und ineffizient machen, bleiben eine entscheidende Herausforderung für die pharmazeutische Industrie. Deshalb werden dringend neue präklinische in vitro Modelle, die bessere in vivo Wirkungsvorhersagen liefern, benötigt. Das Tissue Engineering bietet die Möglichkeit künstliche dreidimensionale (3D) in vitro Gewebe herzustellen, z.B. funktionelle Organe, aber es ermöglicht auch, die Reaktion auf ein Medikament in pathologischen Gewebemodellen, wie beispielsweise Krebsmodelle, zu untersuchen. Diese sind der konventionellen zweidimensionalen (2D) Zellkultur in Petrischalen überlegen und können die begrenzten Möglichkeiten von Tiermodellen erweitern, was zudem die Notwendigkeit für präklinische in vivo Modelle vermindert. In der vorliegenden Arbeit wurden neuartige 3D CRC Modelle auf Basis einer dezellularisierten intestinalen Matrix entwickelt. Im ersten Teil konnte gezeigt werden, dass die Zelllinie SW480 verschiedene Charakteristika bezüglich des Wachstums in der konventionellen 2D Zellkultur oder der 3D Umgebung aufwies. Im Gegensatz zu den mesenchymalen Eigenschaften der Zellen in der 2D Zellkultur, zeigten sie im 3D Modell einen betonteren epithelialen Charakter. Durch das Hinzufügen von Fibroblasten änderten die Krebszellen ihr Wachstumsverhalten und sie bildeten zusammen tumorartige Strukturen aus, wobei die natürlichen Strukturen der Darmmatrix, Krypten und Villi, umgebaut wurden. Zusätzlich wurde das entwickelte 3D Tumormodell als Testsystem für das Standardchemotherapeutikum 5-Fluorouracil (5-FU) herangezogen. Der zweite Teil der Dissertation konzentrierte sich auf die Entwicklung eines 3D in vitro Testsystems für die zielgerichtete Behandlung. Es gibt schon eine Reihe von der US Food and Drug Administration zugelassenen Medikamente für die zielgerichtete Behandlung spezifischer Tumorentitäten, wie z.B. Vemurafenib (PLX4032, Zelboraf™), das eindrucksvolle Ansprechraten von 50–80% bei Melanompatienten mit BRAF-Mutation erzielt. Trotzdem sprechen nur 5% der CRC-Patienten mit der gleichen BRAF-Mutation auf die Behandlung mit Vemurafenib an. Gründe für diese Unempfindlichkeit könnte eine Rückkoppelung zum aufwärtsgelegenen EGFR sein, der das Signal zur Proliferation aufrecht erhält. Um diese Hypothese zu überprüfen, wurden die zwei Zelllinien HROC24 und HROC87, die beide die BRAF V600E-Mutation tragen aber sich in anderen Mutationen unterscheiden, mit Vemurafenib und/oder Gefitinib behandelt und das Ansprechen auf die Substanzen in der herkömmlichen 2D Zellkultur sowie im fortschrittlicheren 3D Modell verglichen. In 3D Kulturbedingungen zeigten beide Zelllinien eine Senkung der Proliferation nur im Kombinationstherapie-Ansatz. Außerdem wurden bei den 3D Modellen keine signifikanten Unterschiede zwischen den verschiedenen Behandlungsansätzen und der unbehandelten Kontrolle, hinsichtlich der Apoptoserate und Viabilität, gefunden. Das deutet auf eine erhöhte Chemoresistenz der Krebszellen in der 3D Umgebung hin. Wegen der vorhandenen Unempfindlichkeit der Zelllinie HROC87 gegenüber der BRAF-Inhibierung mit Vemurafenib, wie es auch in der Klinik im Fall von Patienten mit BRAF-Mutation des CRC beobachtet werden kann, wurden diese Zellen für weitere Xenograft-Experimente und Analysen von Aktivierungsunterschieden im MAPK-Signaltransduktionsweg herangezogen. Weiterhin zeigten die Zellen eine Aktivierung des „hepatocyte growth factor receptor“ (HGFR, c-Met) in 2D und 3D Zellkultur, der jedoch nicht im Xenograft-Modell zu sehen war, was die limitierte Übertragbarkeit von Ergebnissen des Tiermodells auf den Menschen verdeutlicht. Dies spiegelt wiederum die obenstehend erwähnten hohen Versagensraten in der präklinischen Medikamententestung wider. Zusammengefasst kann das Tissue Engineering Möglichkeiten zur Herstellung und Entwicklung neuartiger 3D Testsysteme bieten, welche besser die in vivo Situation abbilden. Für eine Medikamententestung in Übereinstimmung mit personalisierter Medizin eröffnet das 3D Tumormodell vielversprechende Wege, welche in Zukunft das präklinische Screening verbessern sowie die hohen Versagensraten und Tierversuche vermindern könnten.

Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models. / Aktuelle präklinische Modelle zur Bewertung neuartiger Therapien für eine verbesserte Heilung um- fassen sowohl in vitro als auch in vivo Methoden. Ethische Bedenken im Zusammenhang mit der Ver- wendung von Tieren sowie die schlechte physiologische Übersetzung zwischen tierischer und mensch- licher Hautwundheilung bezeichnen In-vitro-Modelle jedoch als hochrelevante und vielversprechende Plattformen für die Heilungsforschung. Während die aktuellen in vitro 3D-Hautmodelle ein reifes Ge- webe mit heilenden Eigenschaften rekapitulieren, stellen sie dennoch eine Vereinfachung der in vivo- Bedingungen dar, bei denen beispielsweise die nach der Wundbildung entstehende Entzündungsreak- tion den Beitrag von Immunzellen beinhaltet. Makrophagen gehören zu den Hauptverursachern der Entzündungsreaktion und regulieren ihren Verlauf durch ihre Plastizität. Daher könnte ihre Implemen- tierung in die in vitro Haut die physiologische Relevanz der Modelle deutlich erhöhen. Da bisher kein volldickes, immunkompetentes Hautmodell mit Makrophagen berichtet wurde, wurden hier die für eine erfolgreiche Dreifach-Cokultur von Fibroblasten, Keratinozyten und Makrophagen notwendigen Parameter untersucht. Zuerst wurden die Zellquelle und die Kultur zeitlich festgelegt, aber auch eine Implementierungsstrategie für Makrophagen festgelegt. Die Implementierung von Makrophagen in das Hautmodell konzentrierte sich auf die Minimierung der Kultivierungszeit, um die Lebensfähigkeit und den Phänotyp der Immunzellen zu erhalten, da die Umgebung einen großen Einfluss auf die Zell- polarisation und Zytokinproduktion hat. Zu diesem Zweck wurde die Integration von Makrophagen in 3D-Gelen vor der Kombination mit Hautmodellen ausgewählt, um die in vivo-Umgebung besser nach- ahmen zu können. Eingebettet in Kollagenhydrogele zeigten Makrophagen eine homogene Zellvertei- lung im Gel, die die Zelllebensfähigkeit bewahrt, auf Reize reagiert und durch die Matrix wandert, die alle bei der Beteiligung von Makrophagen an der Entzündungsreaktion benötigt werden. Nachdem festgestellt worden war, wie Makrophagen in Hautmodelle eingeführt werden können, wurden ver- schiedene Kulturmedien hinsichtlich ihrer Auswirkungen auf Primärfibroblasten, Keratinozyten und Makrophagen untersucht, um eine geeignete Medienzusammensetzung für die Kultur immunkompe- tenter Haut zu identifizieren. Die vorliegende Arbeit bestätigte, dass jeder Zelltyp eine andere Supple- mentkombination zur Aufrechterhaltung der Funktionsmerkmale benötigt und zeigte erstmals, dass Medien, die eine reife Hautstruktur fördern und aufrechterhalten, negative Auswirkungen auf die pri- mären Makrophagen haben. Hautdifferenzierungsmedien wirkten sich negativ auf die Makrophagen in Bezug auf Lebensfähigkeit, Morphologie, Fähigkeit, auf pro- und antiinflammatorische Reize zu rea- gieren und durch ein Kollagengel zu wandern aus. Die Kombination aus verwundeten Hautäquivalen- ten und makrophagenhaltigen Gelen bestätigte, dass das Kulturmedium die Teilnahme der Makro- phage an der Entzündungsreaktion, die nach der Wunde auftritt, hemmt. Die beschriebene Makrophagen-Einschlussmethode zur immunkompetenten Hautbildung ist ein vielversprechender An- satz zur Generierung relevanterer Hautmodelle. Eine weitere Optimierung des Co-Kulturmediums wird es möglicherweise ermöglichen, eine physiologische Entzündungsreaktion nachzuahmen und die Aus- wirkungen neuartiger Medikamente zur verbesserten Heilung auf verbesserte In-vitro-Modelle zu be- werten.