Дисертації з теми "BIOTECHNOLOGICAL APPROACHES"

The production of new rose cultivars by sexual crossing is problematic and time consuming due to sexual incompatibility. the failure of seeds to genninate. and to a limited gene pool. Biotechnology provides an obvious alternative for the creation of genetic novelty in rose. This thesis focuses on the development of novel approaches, based on embryo rescue, pollen cryopreservation, protoplast and transformation technologies. A reproducible embryo rescue technique was developed in which embryos were excised and genninated on agar solidified medium containing a basic salt mixture and carbohydrate. The choice of carbohydrate and the growth conditions employed were demonstrated to markedly affect the percentage germination and subsequent plantlet development. This technique was used to greatly increase the production of F, hybrid progeny when compared to conventional germination methods. The failure of sexual crosses between several English rose cultivars was shown to be due to a combination of low pollen viability and to the operation of a pollen-style incompatibility mechanism (probably of the gametophytic self-incompatibility type). Degree of flower opening and method of pollen dehiscence were shown to significantly affect pollen viability. A technique was developed for the effective cryopreservation of English rose pollen. Using this technique it was possible to store pollen at ultra-low temperatures without any significant loss in viability. Such a technique compared favourably with conventional techniques (refrigeration and freezing) in which a loss in viability over time was demonstrated to occur. In vitro shoot cultures of English rose were established on MS-based media containing BAP. GA3 and NAA following the treatment of explants with an antioxidant solution to negate the effects of phenolic oxidation. The production of callus was shown to be genotype dependant and lacked regeneration potential. Rhizogenic responses were observed in leaf discs of two cultivars however shoot regeneration was not observed. Using a variety of enzyme mixtures it was possible to isolate protoplasts from both In vitro leaf material and from cell suspensions. Both mesophyll and cell suspension derived protoplasts were cultured to a microcallus stage. Plating density, growth regulator concentration and the use of antioxidants were all demonstrated to have a significant effect on the protoplast plating efficiency. Rhizogenesis was achieved from mesophyll protoplast-derived calli. Protoplasts, sometimes labelled with a fluorescent marker, were subjected to both chemical and electrofusion. Using micromanipulation, heterokaryons, formed during electrofusion, were recovered. Such heterokaryons, when cultured. underwent division and formed microcalli which subsequently developed into calli. The hybrid nature of such calli were conftrmed by isozyme analysis, determination of ploidy level and RAPD analysis. The introduction of a plasmid containing a gus marker gene into zygotic embryos of English rose was shown to be possible. This was achieved by microprojectile-mediated DNA delivery using a laboratory built electrical discharge device. The efficiency of this technique was influenced by the concentration of microprojectiles and DNA used. And by firing distance and choice of DNA construct. The relevance of this study and its applications, in the context of rose breeding are discussed.

Abstract Biotechnology has become a necessity, not only in research, but also in the culture and breeding of lilies. Various methods in tissue culture and molecular breeding have been applied to the production of commercially important lily species and cultivars. However, scientific research data of such species and varieties that have potential in the northern climate is scarce. In this work, different biotechnological methods were developed and used in the production and culture of a diversity of lily species belonging to different taxonomic groups. The aim was to test and develop further the existing methods in plant biotechnology for the developmental work and the production of novel hardy lily cultivars for northern climates. Most of the plant material was started from seeds, which provided genetic variability and new material for breeding. Different features in seed structure were studied with light microscopy and SEM, and different parameters affecting germination were tested. Several tissue culture protocols were also compared with different species using both solid and liquid media. Molecular biological methods were used in assessing genetic background of traditionally grown lilies. Somatic embryogenesis in callus differentiation of callus cultures was studied, and gene expression behind differentiation processes was analyzed with various molecular biological methods. Particle bombardment system was used in genetic transformation. In addition, protoplast isolation methods from various tissues were tested. The main results indicate that many tissue culture methods can be used in research and in mass production with all tested species. Especially in a large-scale production, temporary immersion system is promising. In addition to the conventional bulb scale material, seeds were found to be a suitable starting material for genetic variability required for production of new cultivars, and in the preservation of natural populations. RAPD techniques proved a suitable method for revealing phylogenetic relations of different lily species and cultivars. Methods in DNA and RNA isolation, cloning and analysis were optimized for lily material. In addition, particle bombardment system was successfully used for genetic transformation of lily callus. In the future, more information is needed to understand better the germination and differentiation processes, focusing especially in the genes, their products and function. In addition, the large and still mostly unknown lily genome is a challenge for research in the future. However, the currently presented results provide good opportunities for further developmental work and research of hardy lily species.

The heart can be considered the most important organ of our body, as it supplies nutrients to all the cells. When affected from injuries or diseases, the heart function is hampered, as the damaged area is substituted by a fibrotic scar instead of functional tissue. Understanding the mechanisms leading to heart failure and finding a cure for cardiac diseases represents a major challenge of modern medicine, since they are the leading cause of death and disability in Western world. Being the heart a vital organ it is difficult to have access to its cells, especially in humans. In order to model it or find therapeutic strategies many approaches and cell sources have been studied. For example cardiac stem cells, skeletal myoblasts, bone marrow-derived cells and peripheral blood mononuclear cells have been tested in pre-clinical and clinical trials, without significant tissue regeneration. Human pluripotent stem cells (hPSC) are thought to be the most promising cell type in the field, thanks to their unlimited capacity of self-renewal and retention of differentiation potency. Induced pluripotent stem cells (iPSC) are pluripotent cells derived through reprogramming from adult cells, easily accessible from patients, like keratinocytes. iPSC can be differentiated to cardiac cells, through stage-specific protocols that reproduce embryonic development, offering a very useful platform for modelling diseases of patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, properly mimicking cardiac tissue is very complex, since not only the correct cardiac cell type has to be reproduced, but also its overall cellular composition, architecture and biophysical functions. In order to study these aspects, we applied biotechnological strategies such as the use of transgenic cell lines for obtaining pure and scalable differentiated cells to be cultured in a 3D scaffold with a perfusion bioreactor. Although it is well known that iPSC can give rise to cardiomyocytes in vitro, not every cell line can be efficiently differentiated. Thus, a cell line-specific differentiation protocol has to be identified and optimized. We finally identified a fast and efficient stage-specific differentiation protocol suitable for the iPSC lines used in this work, derived from human keratinocytes. With this protocol, we can reproducibly obtain close to 50% cardiomyocytes after 15 days of differentiation. One important feature of currently available differentiation protocols is that the target cell type is obtained among a heterogeneous cell population. To track the cardiac population of interest we generated transgenic cell lines where the reporter protein GFP follows the expression of different genes specific for stages of differentiation, such as T (Brachyury) for mesoderm; NKX2.5 for cardiac progenitors; and MHC for cardiomyocytes. Moreover, cardiomyocytes obtained from hPSC using currently available differentiation protocols are typically immature, mostly resembling embryonic or fetal cardiomyocytes, arguably because of the lack of mechanical and electrical stimuli that only a 3D environment can provide. In order to create a piece of tissue in 3D we used a collagen and elastin-based scaffold, to mimic the structural proteins of endogenous extracellular matrix. We also built a perfusion bioreactor to culture the construct. After initial validation with primary cultures of rat neonatal cardiomyocytes, we tested iPSC-derived cardiac cells at different stages of differentiation. While early mesoderm or cardiac progenitors could not survive in our system, iPSC differentiated to cardiomyocytes, could be retained and maintained alive within the scaffold for at least 4 days. In conclusion, in this work we combined biotechnological tools in order to obtain a test platform for studying the mechanisms underlying cardiac differentiation, maturation, as well as providing valuable in vitro systems for disease modelling, drug screening of patient-specific heart muscle cells and cell therapy.
El corazón es el órgano más importante del cuerpo: impulsando la sangre, aporta oxigeno y nutrientes a cada célula del organismo. En caso de fallo cardiaco la función del corazón no puede recuperarse, ya que los cardiomiocitos son reemplazados por una cicatriz fibrosa no funcional. Las enfermedades cardiacas representan la mayor causa de muerte y enfermedad en el mundo occidental y entender los mecanismos de las patologías cardiacas, así como encontrar curas para ellas, es un desafío de primaria importancia para la medicina moderna. Siendo el corazón un órgano vital y difícilmente accesible, resulta imprescindible encontrar una fuente celular alternativa. Las células madre humanas con pluripotencia inducida (iPSC – induced pluripotent stem cells) parecen óptimas, porque se derivan de simples biopsias de piel de pacientes y se pueden diferenciar a cualquier tipo celular, cardiomiocitos incluidos. Aún así, diferenciar el tejido cardiaco es muy complejo: no solamente se debe de reproducir el tipo celular, sino también su composición celular, su arquitectura y sus funciones biofísicas. Para estudiar estos aspectos, por un lado obtuvimos tres líneas celulares de iPSC reporteras de genes específicos de diferentes estadios de diferenciación cardiaca (T para mesodermo, NKX2.5 para progenitores cardiacos y alpha-MHC para cardiomiocitos), y por otro desarrollamos un biorreactor adecuado para el cultivo de células cardiacas en 3D. Utilizamos las líneas transgénicas como herramienta para seleccionar células en diferentes estadios de diferenciación y las co-cultivamos con fibroblastos en un andamio compuesto de colágeno y elastina (imitando la matriz extracelular cardiaca y la composición celular del corazón). En conjunto, este estudio revela que las iPSC pueden ser retenidas y cultivadas en nuestro sistema 3D. Mientras células de mesodermo temprano y progenitores cardiacos no completaron la diferenciación cardiaca, los cardiomiocitos derivados de iPSC con cultivo convencional y cultivados en el biorreactor pudieron ser mantenidos viables en el mismo al menos 4 días. La aproximación experimental aquí presentada representa una base para desarrollar plataformas de estudio in vitro paciente-especificas para modelar enfermedades cardiacas humanas y estudios de fármacos, así como ofrecer una herramienta de estudio de los mecanismos de la diferenciación y maduración cardiacas.

Les génomes mitochondriaux diffèrent dans leur structure, leur taille et leurs processus fonctionnels. Leur expression complexe est difficile à disséquer. Les maladies dues à des mutations dans l'ADN mitochondrial sont très répandues et la génétique des mitochondries a une grande importance agronomique. Il y a donc un fort besoin de manipuler le système génétique de ces organelles mais aucune approche n'a abouti à une méthodologie de transformation mitochondriale chez les mammifères et les plantes. Les principaux problèmes sont la transfection des organelles et le maintien de l'ADN transfecté. Les mitochondries isolées peuvent cependant importer de l'ADN. Sur la base de ce mécanisme, nous avons exploré des stratégies pour maintenir l'ADN exogène dans les organelles. Dans les mitochondries humaines, la recombinaison est rare. Nous avons ainsi tenté d'élaborer des constructions capables de se comporter comme des réplicons autonomes. Chez les plantes, la recombinaison homologue façonne le génome mitochondrial et nous avons réussi à intégrer une séquence-rapportrice dans l'ADN génomique des organelles. La compétence pour l'import d'ADN est susceptible d'être exploitable in vivo. Nous avons utilisé des vésicules mitochondriotropiques pour délivrer nos constructions à proximité des mitochondries dans des cellules humaines ou végétales. Suivant une autre stratégie, notre équipe a montré qu'un ARN passager associé à un mime d'ARNt et exprimé à partir d'un transgène nucléaire est importé dans les mitochondries des cellules végétales. Nous avons tenté d'analyser la fonctionnalité de l'ARN passager dans les mitochondries par des tests d'édition in vitro, in organello et in vivo
Mitochondrial genomes from various organisms differ in their structure, size and functional processes. Their complex expression is difficult to dissect. Diseases due to mutations in the mitochondrial DNA are widespread and mitochondrial genetics have a high agronomic relevance. There are thus strong needs to manipulate the mitochondrial genetic system in mammals and plants, but none of the attempted approaches has led to a mitochondrial transformation methodology in these organisms. The main problems to solve are the transfection of the organelles and the maintenance of the transfected DNA. Earlier experiments showed that isolated mitochondria can import DNA. Based on this mechanism, we have explored distinct strategies to achieve maintenance of exogenous DNA in human and plant organelles. In human mitochondria, recombination is a rare event. We have thus attempted to build and test constructs able to behave as autonomous replicons. In plants, homologous recombination is believed to shape the mitochondrial genome and we succeeded in integrating a reporter sequence into the organelle genomic DNA. The DNA import competence might be exploitable in vivo. We therefore used currently developed mitochondriotropic vesicles (DQAsomes, liposomes) to try and deliver our constructs to the vicinity of the mitochondria in human or plant cells. In a further, distinct strategy, our group showed that a passenger RNA associated with a tRNA mimic and expressed from a nuclear transgene is imported into the mitochondria of the transformed plant cells. We have tried to analyse the functionality of the passenger RNA in the mitochondria through in vitro, in organello and in vivo editing tests

[ES] En el primer capítulo, se caracterizó una colección de seis accesiones de berenjenas, 21 accesiones de 12 especies silvestres y 45 híbridos interespecíficos de berenjena con especies silvestres utilizando 27 descriptores morfológicos y 20 descriptores morfométricos de frutos basados en la herramienta fenómica Tomato Analyzer. Observamos diferencias significativas entre los tres grupos, con híbridos que muestran valores intermedios para la mayoría de los descriptores. Las especies silvestres mostraron una amplia diversidad para ampliar la base genética de la berenjena. En el segundo capítulo, el mismo material se caracterizó para el contenido en compuestos fenólicos del fruto, el color de la pulpa de la fruta y los caracteres relacionados con el pardeamiento. Mientras que el ácido fenólico predominante en la berenjena cultivada fue el ácido clorogénico (> 65.0%), en los parientes silvestres fue de menos del 50% del área del pico del cromatograma HPLC. Las variedades cultivadas presentaron un color de carne más claro y baja actividad de polifenol oxidasa (PPO). Los híbridos interespecíficos fueron intermedios para todos los caracteres estudiados. En el tercer capítulo, realizamos un estudio genético Linea por Probador (L × P) utilizando dos líneas cultivadas, una con citoplasma oriental y otra occidental con cuatro probadores que representan tres especies silvestres: S. insanum, S.anguivi y S . lichtensteinii. Además, evaluamos el material para 3 descriptores bioquímicos, 12 morfológicos y 8 de Tomato Analyzer. Encontramos diferencias significativas para los 23 caracteres estudiados. Los valores más altos para la componente SCA se determinaron en comparación con la componente GCA. En general, los probadores del genepool secundarios fueron mejores para la mejora de los caracteres bioquímicos, mientras que las especies de genepool primarias fueron mejores para los caracteres morfológicos. En el cuarto capítulo, para obtener información sobre la genética de caracteres importantes en la berenjena, realizamos el primer estudio genético detallado de la berenjena con 10 genotipos diversos de berenjena, entre ellos una accesión de S. insanum, mientras que el resto fueron variedades tradicionales con forma del fruto muy diversa. Cuando se cruzaron en un diseño de medio dialelo, los 10 padres produjeron un conjunto de 45 híbridos. Evaluamos padres e híbridos para 14 descriptores de características morfológicas y 14 características morfométricas del fruto. También determinamos las distancias genéticas utilizando 7,335 marcadores de SNP polimórficos. Del mismo modo, en el quinto capítulo, estudiamos los compuestos fenólicos del fruto, el color de la pulpa del mismo y los caracteres relacionados con el pardeamiento en el mismo material. Se determinó que la accesión de S. insanum accession INS2 presenta valores altamente significativos para los compuestos fenólicos totales y el contenido de ácido clorogénico. Se encontraron efectos significativos para la aptitud combinatoria específica y general para los caracteres bioquímicos estudiados. De manera similar a lo encontrado anteriormente, la distancia genética entre los padres no fue útil para predecir el comportamiento de los híbridos.Finalmente, en el sexto capítulo, hemos desarrollado un protocolo de agroinfiltración para la expresión transitoria de un gen en el fruto de la berenjena utilizando GUS; Vector pCAMBIA1304. Posteriormente, para probar la utilidad del protocolo, utilizamos la hidroxicinamoil CoA-quinato transferasa (SmHQT) de berenjena, que es la enzima central estudiada para aumentar el contenido de ácido clorogénico, en la construcción del gen con el promotor específico en un vector de transformación de plantas (pBIN19). Además, en nuestro casete, también coexpresamos la proteína P19 del Tomato bushy stunt virus para sobreexpresar la proteína. En esta tesis proporcionamos información sobre los parientes sil
[CAT] En el primer capítol, es va caracteritzar una col·lecció de sis accessions d'albergínies, 21 accessions de 12 espècies silvestres i 45 híbrids interespecífics d'albergínia amb espècies silvestres utilitzant 27 descriptors morfològics i 20 descriptors morfomètrics de fruits basats en l'eina fenómica Tomato Analyzer. Observarem diferències significatives entre els tres grups, amb híbrids que mostren valors intermedis per a la majoria dels descriptors. Les espècies silvestres van mostrar una àmplia diversitat per a ampliar la base genètica de l'albergínia.En el segon capítol, el mateix material es va caracteritzar per al contingut en compostos fenòlics del fruit, el color de la polpa de la fruita i els caràcters relacionats amb el pardejament. Mentre que l'àcid fenòlic predominant en l'albergínia cultivada va ser l'àcid clorogènic (> 65.0%), en els parents silvestres va ser de menys del 50% de l'àrea del pic del cromatograma HPLC. Les varietats cultivades van presentar un color de carn més clar i baixa activitat de polifenol oxidasa (PPO). Els híbrids interespecífics van ser intermedis per a tots els caràcters estudiats. Curiosament, no trobem correlacions significatives entre el contingut de compostos fenòlics totals o àcid clorogènic i el pardejament de la polpa de la fruita. En el tercer capítol, realitzem un estudi genètic Línia per Emprovador (L × P) utilitzant dues línies cultivades, una amb citoplasma oriental i una altra occidental amb quatre emprovadors que representen tres espècies silvestres: S. insanum, S. anguivi i S. lichtensteinii. A més, avaluem el material per a 3 descriptors bioquímics, 12 morfològics i 8 de Tomato Analyzer. Trobem diferències significatives per als 23 caràcters estudiats. Els valors més alts per a la component SCA es van determinar en comparació amb la component GCA. En el quart capítol, per a obtindre informació sobre la genètica de caràcters importants en l'albergínia, realitzem el primer estudi genètic detallat de l'albergínia amb 10 genotips diversos d'albergínia, entre ells una accessió de S. insanum, mentre que la resta van ser varietats tradicionals amb forma del fruit molt diversa. Quan es van creuar en un disseny de mitjà dial·lel, els 10 pares van produir un conjunt de 45 híbrids. Avaluem pares i híbrids per a 14 descriptors de característiques morfològiques i 14 característiques morfomètriques del fruit. També determinem les distàncies genètiques utilitzant 7,335 marcadors de SNP polimòrfics. En l'estudi de l'herència dels caràcters morfològics importants per al desenvolupament d'híbrids en albergínia, es va trobar que la distància genètica entre els pares té un valor limitat per a predir el rendiment dels híbrids en aquest cultiu. Es va determinar que l'accessió de S. insanum accessió INS2 presenta valors altament significatius per als compostos fenòlics totals i el contingut d'àcid clorogènic. Es van trobar efectes significatius per a l'aptitud combinatòria específica i general per als caràcters bioquímics estudiats. De manera similar a l'oposat anteriorment, la distància genètica entre els pares no va ser útil per a predir el comportament dels híbrids. Finalment, en el sisé capítol, hem desenvolupat un protocol d'agroinfiltració per a l'expressió transitòria d'un gen en el fruit de l'albergínia utilitzant GUS; Vector pCAMBIA1304. Posteriorment, per a provar la utilitat del protocol, utilitzem la hidroxicinamoil CoA-quinat transferasa (SmHQT) d'albergínia, que és l'enzim central estudiat per a augmentar el contingut d'àcid clorogènic, en la construcció del gen amb el promotor específic en un vector de transformació de plantes (pBIN19). A més, en el nostre casset, també coexpresem la proteïna P19 del Tomato bushy stunt virus per a sobreexpresar la proteïna. En aquesta tesi proporcionem informació sobre els parents silvestres d'albergínies per a caràcters morfològics i bioq
[EN] In the first chapter, a collection of six eggplant accessions, 21 accessions of 12 wild species (the only primary genepool species S. insanum and 11 secondary genepool species) and 45 interspecific hybrids of eggplant with wild species were characterized using 27 morphological descriptors and 20 fruit morphometric descriptors based on the phenomics tool Tomato Analyzer. We observed significant differences among the three groups with hybrids showing intermediate values for most of the descriptors. The wild species showed an extensive diversity for broadening the genetic base of eggplant. In the second chapter, the same material was characterized for the fruit phenolics, fruit flesh colour and browning related traits. Wild relatives showed greater variation for the fruit phenolics than cultivated eggplant. While, the predominant phenolic acid in cultivated eggplant was chlorogenic acid (>65.0%), in the wild relatives it was less 50% of HPLC chromatogram peak area. Cultivated varieties had lighter flesh colour and low polyphenol oxidase (PPO) activity. The interspecific hybrids were found to be intermediate for all the characters studied. Interestingly, we found no significant correlations between total phenolics or chlorogenic acid contents and fruit flesh browning. In the third chapter, we performed a Line by Tester (L ×T) genetic study using two cultivated lines one with oriental and another with occidental cytoplasm along with four testers representing three wild species namely, S. insanum, S.anguivi, and S. lichtensteinii. Further, we evaluated the material for 3 biochemical, 12 morphological and 8 Tomato Analyzer based descriptors. We found a significant amount of variation for all the 23 traits studied. The higher values for the SCA component were determined as compared to the GCA component. Overall, the secondary genepool testers were better for the biochemical traits improvement whereas, the primary genepool species was better for the morphological traits. In the fourth chapter, in order to gain information regarding the genetics of important traits in eggplant, we performed the first detailed genetic study of eggplant with 10 diverse eggplant genotypes among them an S. insanum accession, while the remaining nine were highly diverse shaped fruits of popular eggplant cultivars. When crossed in a half-diallel matting design 10 parents produced a set of 45 hybrids. We evaluated parents and hybrids for 14 morphological and 14 fruit morphometric traits descriptors. We also determined the genetic distances using 7,335 polymorphic SNP markers. In the study of the genetics of important morphological traits for hybrid development in eggplant, we found that genetic distance among parents had limited value to predict hybrid performance in this crop. Likewise, in the fifth chapter, we studied the fruit phenolics, fruit flesh colour and browning related traits in the same material. We determined that S. insanum accession INS2 displayed values highly significant for the total phenolics and CGA content. Significant specific and general combining ability effects were found for the biochemical traits studied. Similarly, the genetic distance among parents was nonsignificant to predict hybrids performance. Finally, in the sixth chapter, we have developed an agroinfiltration protocol for the transient expression of a gene in the eggplant fruit using GUS bearing; pCAMBIA1304 vector. Thereafter, to prove the usefulness of the protocol, we have used the eggplant hydroxycinnamoyl CoA-quinate transferase (SmHQT), which is the central enzyme studied to increase the chlorogenic acid content, in a gene construct with the specific promoter in a plant transformation vector (pBIN19). Also, in our cassette, we also co-expressed the P19 protein of Tomato bushy stunt virus to overexpress the protein. Overall, in this thesis, we have provided information regarding the wild relatives of eggplants from a morphological and biochemical prospective.
Kaushik, P. (2019). Application of Conventional, Biotechnological and Genomics Approaches for Eggplant (Solanum melongena.L). Breeding with a Focus on Bioactive Phenolics [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/122295
TESIS

L’utilizzo delle cellule iPSC–paziente specifiche costituiscono uno degli strumenti -di modello malattia- utilizzati per studiare i meccanismi biologici e patologici alla base di una specifica malattia. L'obiettivo principale di questa tesi di dottorato è stato quello di generare iPSC da cellule mononucleate del sangue periferico (PBMC) ottenute da pazienti con Sindrome di Cri-du-Chat (CdCS), poi differenziate in cellule staminali mesenchimali indotte (iMSC). CdCS è un malattia genetica causata da una delezione totale o parziale nel braccio corto del cromosoma 5, caratterizzata da un grido acuto, caratteristiche dismorfiche, scarsa crescita e ritardo dello sviluppo. L'aploinsufficienza dei geni situati all'interno di 5p contribuisce al fenotipo. Le cellule PBMC sono state riprogrammate in cellule staminali pluripotenti indotte (iPSC) mediante i fattori Yamanaka Oct4, Sox2, Klf4 e c-Myc utilizzando un vettore di riprogrammazione CytoTune-iPS 2.0 non integrato; le cellule iPSC ottenute sono state caratterizzate per la pluripotenza e la staminalità. Le cellule CdC-iPSC sono state differenziate in iMSC utilizzando MesenCult™-ACF Plus Medium e Culture Kit. Tali cellule CdCs-iMSC sono risultate aderenti alla plastica, positive per i marcatori delle cellule MSC e capaci di differenziarsi in osteociti, adipociti, e condroblasti. Tali proprietà hanno soddisfatto i criteri minimi delle MSC umane proposti dalla Società Internazionale di Terapia Cellulare. Nel periodo trascorso presso l’Università di Santiago De Compostela (Spagna), l’obiettivo dell’attività sperimentale è stato quello di isolare vescicole extracellulari da urina (uEV). L'urina infatti è stata ampiamente studiata per identificare biomarcatori di patologie renali; quindi riflette un quadro olistico dell'intero sistema urinario ed i campioni possono essere raccolti ripetutamente in modo non invasivo. L'analisi delle uEV, isolate appunto dalle urine, può fornire una soluzione interessante per il loro -cargo- (proteine, RNA, lipidi e glicani) che rimane protetto. L'ultracentrifugazione è la tecnica comune per isolare le EV da fluidi biologici. Tuttavia, tale tecnica è laboriosa, dispendiosa in termini di tempo e di solito richiede apparecchiature costose. In questa tesi di dottorato, per isolare le uEV da donatori umani sani, abbiamo utilizzato ExoGAG (NasasBiotech, Santiago de Compostela, Spagna) Kit ed il metodo convenzionale di ultracentrifugazione. Quindi, per esaminare l'internalizzazione delle uEV nelle cellule di topo mIMCD3 e la colocalizzazione delle cilia primarie in colture 2D di tali cellule del dotto di raccolta midollare interno 3 (mIMCD3) di topo, le uEV isolate sono state marcate con la colorazione dei globuli rossi Vybrant™ DiD e le ciglia primarie sono state indagate con anticorpi anti α -Tubulina acetilata ed analisi al microscopio confocale. In particolare abbiamo isolato con successo da donatori sani le uEV che esprimono le tetraspanine di superficie CD63 e marcatori proteici CD81 ed abbiamo valutato il loro contenuto di RNA con Spectrometry Nanodrop e Capillary Tapstation Electhrophoresis. I dati ottenuti dimostrano che sia le uEV isolate con il kit ExoGAG, sia le uEV isolate con ultracentrifugazione non sono state internalizzate dalle cellule mIMCD3 e la microscopia confocale ha rivelato la mancanza di colocalizzazione delle cilia primarie e delle uEV colorate con Vybrant™ DiD Cell-labeling solution. Questi risultati indicano che potrà essere utile utilizzare in questo tipo di esperimenti cellule umane.
Cellular disease modelling using patient-specific iPSCs is one of the tools used to study the biological and pathological mechanisms underlying specific disease. The main objective of this PhD thesis was to generate iPSCs from peripheral blood mononuclear cells (PBMCs) obtained from patients with Cri-du-Chat Syndrome (CdCS) and differentiation into mesenchymal stem cells (MSCs). CdCS is a genetic disease caused by a total or partial deletion in the short arm of chromosome 5, characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. Haploinsufficiency of the genes located within 5p contributed to the phenotype. PBMCs were reprogrammed into iPSCs by introducing the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc using a nonintegrating CytoTune-iPS 2.0 reprogramming vectors and characterized for pluripotency and stemness and their ability to differentiate into the three germ layers. Then CdCs-iPSCs were differentiated into iMSCs using MesenCult™-ACF Plus Medium and Culture Kit. CdCs-iMSCs were plastic adherent, expressed MSC surface markers and could differentiate into osteocytes, adipocytes, and chondroblasts. These properties satisfy the minimal criteria of human MSCs proposed by the International Society of Cellular Therapy. Urine has been tremendously studied to discover biomarkers of distinct renal pathologies; hence it reflects a holistic picture of the entire urinary system. It could be collected repeatedly in a noninvasive manner. The analysis of urinary Extracellular Vesicles, uEVs, encountered in urine provides an attractive solution due to their cargo (Proteins, RNAs, lipids, and Glycans) which remains protected. Ultracentrifugation is the common technique for isolating EVs from biological fluids. However, this technique is laborious, time-consuming, and usually requires expensive equipment. In this PhD thesis, firstly, to isolate uEVs from healthy human donors, we utilized ExoGAG (NasasBiotech, Santiago de Compostela, Spain) Extracellular vesicles purification kit and conventional Ultracentrifugation method. Secondly, to assess uEVs internalization and primary cilia colocalization in 2D culture of mouse inner medullary-collecting duct 3 (mIMCD3) cells, the isolated uEVs were Labelled with Vybrant™ DiD Red Cell stain, and primary cilia were immunologically stained with anti-acetylated α -Tubulin. Confocal microscopy images were obtained for further analysis. We successfully isolated uEVs from healthy donors expressing the surface tetraspanins CD63 and CD81 protein markers and assessed their RNAs contents with Spectrometry Nanodrop and Capillary Tapstation Electrophoresis System. Isolated uEVs with ExoGAG commercial kit and Ultracentrifugation could not be internalized and uptaken by Mouse inner medullary-collecting duct 3 (mIMCD3), Confocal Microscopy images revealed the lack of colocalization of expressed primary cilia and uEVs stained with Vybrant™ DiD Cell-Labelling solution

L’utilizzo delle cellule iPSC–paziente specifiche costituiscono uno degli strumenti -di modello malattia- utilizzati per studiare i meccanismi biologici e patologici alla base di una specifica malattia. L'obiettivo principale di questa tesi di dottorato è stato quello di generare iPSC da cellule mononucleate del sangue periferico (PBMC) ottenute da pazienti con Sindrome di Cri-du-Chat (CdCS), poi differenziate in cellule staminali mesenchimali indotte (iMSC). CdCS è un malattia genetica causata da una delezione totale o parziale nel braccio corto del cromosoma 5, caratterizzata da un grido acuto, caratteristiche dismorfiche, scarsa crescita e ritardo dello sviluppo. L'aploinsufficienza dei geni situati all'interno di 5p contribuisce al fenotipo. Le cellule PBMC sono state riprogrammate in cellule staminali pluripotenti indotte (iPSC) mediante i fattori Yamanaka Oct4, Sox2, Klf4 e c-Myc utilizzando un vettore di riprogrammazione CytoTune-iPS 2.0 non integrato; le cellule iPSC ottenute sono state caratterizzate per la pluripotenza e la staminalità. Le cellule CdC-iPSC sono state differenziate in iMSC utilizzando MesenCult™-ACF Plus Medium e Culture Kit. Tali cellule CdCs-iMSC sono risultate aderenti alla plastica, positive per i marcatori delle cellule MSC e capaci di differenziarsi in osteociti, adipociti, e condroblasti. Tali proprietà hanno soddisfatto i criteri minimi delle MSC umane proposti dalla Società Internazionale di Terapia Cellulare. Nel periodo trascorso presso l’Università di Santiago De Compostela (Spagna), l’obiettivo dell’attività sperimentale è stato quello di isolare vescicole extracellulari da urina (uEV). L'urina infatti è stata ampiamente studiata per identificare biomarcatori di patologie renali; quindi riflette un quadro olistico dell'intero sistema urinario ed i campioni possono essere raccolti ripetutamente in modo non invasivo. L'analisi delle uEV, isolate appunto dalle urine, può fornire una soluzione interessante per il loro -cargo- (proteine, RNA, lipidi e glicani) che rimane protetto. L'ultracentrifugazione è la tecnica comune per isolare le EV da fluidi biologici. Tuttavia, tale tecnica è laboriosa, dispendiosa in termini di tempo e di solito richiede apparecchiature costose. In questa tesi di dottorato, per isolare le uEV da donatori umani sani, abbiamo utilizzato ExoGAG (NasasBiotech, Santiago de Compostela, Spagna) Kit ed il metodo convenzionale di ultracentrifugazione. Quindi, per esaminare l'internalizzazione delle uEV nelle cellule di topo mIMCD3 e la colocalizzazione delle cilia primarie in colture 2D di tali cellule del dotto di raccolta midollare interno 3 (mIMCD3) di topo, le uEV isolate sono state marcate con la colorazione dei globuli rossi Vybrant™ DiD e le ciglia primarie sono state indagate con anticorpi anti α -Tubulina acetilata ed analisi al microscopio confocale. In particolare abbiamo isolato con successo da donatori sani le uEV che esprimono le tetraspanine di superficie CD63 e marcatori proteici CD81 ed abbiamo valutato il loro contenuto di RNA con Spectrometry Nanodrop e Capillary Tapstation Electhrophoresis. I dati ottenuti dimostrano che sia le uEV isolate con il kit ExoGAG, sia le uEV isolate con ultracentrifugazione non sono state internalizzate dalle cellule mIMCD3 e la microscopia confocale ha rivelato la mancanza di colocalizzazione delle cilia primarie e delle uEV colorate con Vybrant™ DiD Cell-labeling solution. Questi risultati indicano che potrà essere utile utilizzare in questo tipo di esperimenti cellule umane.
Cellular disease modelling using patient-specific iPSCs is one of the tools used to study the biological and pathological mechanisms underlying specific disease. The main objective of this PhD thesis was to generate iPSCs from peripheral blood mononuclear cells (PBMCs) obtained from patients with Cri-du-Chat Syndrome (CdCS) and differentiation into mesenchymal stem cells (MSCs). CdCS is a genetic disease caused by a total or partial deletion in the short arm of chromosome 5, characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. Haploinsufficiency of the genes located within 5p contributed to the phenotype. PBMCs were reprogrammed into iPSCs by introducing the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc using a nonintegrating CytoTune-iPS 2.0 reprogramming vectors and characterized for pluripotency and stemness and their ability to differentiate into the three germ layers. Then CdCs-iPSCs were differentiated into iMSCs using MesenCult™-ACF Plus Medium and Culture Kit. CdCs-iMSCs were plastic adherent, expressed MSC surface markers and could differentiate into osteocytes, adipocytes, and chondroblasts. These properties satisfy the minimal criteria of human MSCs proposed by the International Society of Cellular Therapy. Urine has been tremendously studied to discover biomarkers of distinct renal pathologies; hence it reflects a holistic picture of the entire urinary system. It could be collected repeatedly in a noninvasive manner. The analysis of urinary Extracellular Vesicles, uEVs, encountered in urine provides an attractive solution due to their cargo (Proteins, RNAs, lipids, and Glycans) which remains protected. Ultracentrifugation is the common technique for isolating EVs from biological fluids. However, this technique is laborious, time-consuming, and usually requires expensive equipment. In this PhD thesis, firstly, to isolate uEVs from healthy human donors, we utilized ExoGAG (NasasBiotech, Santiago de Compostela, Spain) Extracellular vesicles purification kit and conventional Ultracentrifugation method. Secondly, to assess uEVs internalization and primary cilia colocalization in 2D culture of mouse inner medullary-collecting duct 3 (mIMCD3) cells, the isolated uEVs were Labelled with Vybrant™ DiD Red Cell stain, and primary cilia were immunologically stained with anti-acetylated α -Tubulin. Confocal microscopy images were obtained for further analysis. We successfully isolated uEVs from healthy donors expressing the surface tetraspanins CD63 and CD81 protein markers and assessed their RNAs contents with Spectrometry Nanodrop and Capillary Tapstation Electrophoresis System. Isolated uEVs with ExoGAG commercial kit and Ultracentrifugation could not be internalized and uptaken by Mouse inner medullary-collecting duct 3 (mIMCD3), Confocal Microscopy images revealed the lack of colocalization of expressed primary cilia and uEVs stained with Vybrant™ DiD Cell-Labelling solution

L’utilizzo delle cellule iPSC–paziente specifiche costituiscono uno degli strumenti -di modello malattia- utilizzati per studiare i meccanismi biologici e patologici alla base di una specifica malattia. L'obiettivo principale di questa tesi di dottorato è stato quello di generare iPSC da cellule mononucleate del sangue periferico (PBMC) ottenute da pazienti con Sindrome di Cri-du-Chat (CdCS), poi differenziate in cellule staminali mesenchimali indotte (iMSC). CdCS è un malattia genetica causata da una delezione totale o parziale nel braccio corto del cromosoma 5, caratterizzata da un grido acuto, caratteristiche dismorfiche, scarsa crescita e ritardo dello sviluppo. L'aploinsufficienza dei geni situati all'interno di 5p contribuisce al fenotipo. Le cellule PBMC sono state riprogrammate in cellule staminali pluripotenti indotte (iPSC) mediante i fattori Yamanaka Oct4, Sox2, Klf4 e c-Myc utilizzando un vettore di riprogrammazione CytoTune-iPS 2.0 non integrato; le cellule iPSC ottenute sono state caratterizzate per la pluripotenza e la staminalità. Le cellule CdC-iPSC sono state differenziate in iMSC utilizzando MesenCult™-ACF Plus Medium e Culture Kit. Tali cellule CdCs-iMSC sono risultate aderenti alla plastica, positive per i marcatori delle cellule MSC e capaci di differenziarsi in osteociti, adipociti, e condroblasti. Tali proprietà hanno soddisfatto i criteri minimi delle MSC umane proposti dalla Società Internazionale di Terapia Cellulare. Nel periodo trascorso presso l’Università di Santiago De Compostela (Spagna), l’obiettivo dell’attività sperimentale è stato quello di isolare vescicole extracellulari da urina (uEV). L'urina infatti è stata ampiamente studiata per identificare biomarcatori di patologie renali; quindi riflette un quadro olistico dell'intero sistema urinario ed i campioni possono essere raccolti ripetutamente in modo non invasivo. L'analisi delle uEV, isolate appunto dalle urine, può fornire una soluzione interessante per il loro -cargo- (proteine, RNA, lipidi e glicani) che rimane protetto. L'ultracentrifugazione è la tecnica comune per isolare le EV da fluidi biologici. Tuttavia, tale tecnica è laboriosa, dispendiosa in termini di tempo e di solito richiede apparecchiature costose. In questa tesi di dottorato, per isolare le uEV da donatori umani sani, abbiamo utilizzato ExoGAG (NasasBiotech, Santiago de Compostela, Spagna) Kit ed il metodo convenzionale di ultracentrifugazione. Quindi, per esaminare l'internalizzazione delle uEV nelle cellule di topo mIMCD3 e la colocalizzazione delle cilia primarie in colture 2D di tali cellule del dotto di raccolta midollare interno 3 (mIMCD3) di topo, le uEV isolate sono state marcate con la colorazione dei globuli rossi Vybrant™ DiD e le ciglia primarie sono state indagate con anticorpi anti α -Tubulina acetilata ed analisi al microscopio confocale. In particolare abbiamo isolato con successo da donatori sani le uEV che esprimono le tetraspanine di superficie CD63 e marcatori proteici CD81 ed abbiamo valutato il loro contenuto di RNA con Spectrometry Nanodrop e Capillary Tapstation Electhrophoresis. I dati ottenuti dimostrano che sia le uEV isolate con il kit ExoGAG, sia le uEV isolate con ultracentrifugazione non sono state internalizzate dalle cellule mIMCD3 e la microscopia confocale ha rivelato la mancanza di colocalizzazione delle cilia primarie e delle uEV colorate con Vybrant™ DiD Cell-labeling solution. Questi risultati indicano che potrà essere utile utilizzare in questo tipo di esperimenti cellule umane.
Cellular disease modelling using patient-specific iPSCs is one of the tools used to study the biological and pathological mechanisms underlying specific disease. The main objective of this PhD thesis was to generate iPSCs from peripheral blood mononuclear cells (PBMCs) obtained from patients with Cri-du-Chat Syndrome (CdCS) and differentiation into mesenchymal stem cells (MSCs). CdCS is a genetic disease caused by a total or partial deletion in the short arm of chromosome 5, characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. Haploinsufficiency of the genes located within 5p contributed to the phenotype. PBMCs were reprogrammed into iPSCs by introducing the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc using a nonintegrating CytoTune-iPS 2.0 reprogramming vectors and characterized for pluripotency and stemness and their ability to differentiate into the three germ layers. Then CdCs-iPSCs were differentiated into iMSCs using MesenCult™-ACF Plus Medium and Culture Kit. CdCs-iMSCs were plastic adherent, expressed MSC surface markers and could differentiate into osteocytes, adipocytes, and chondroblasts. These properties satisfy the minimal criteria of human MSCs proposed by the International Society of Cellular Therapy. Urine has been tremendously studied to discover biomarkers of distinct renal pathologies; hence it reflects a holistic picture of the entire urinary system. It could be collected repeatedly in a noninvasive manner. The analysis of urinary Extracellular Vesicles, uEVs, encountered in urine provides an attractive solution due to their cargo (Proteins, RNAs, lipids, and Glycans) which remains protected. Ultracentrifugation is the common technique for isolating EVs from biological fluids. However, this technique is laborious, time-consuming, and usually requires expensive equipment. In this PhD thesis, firstly, to isolate uEVs from healthy human donors, we utilized ExoGAG (NasasBiotech, Santiago de Compostela, Spain) Extracellular vesicles purification kit and conventional Ultracentrifugation method. Secondly, to assess uEVs internalization and primary cilia colocalization in 2D culture of mouse inner medullary-collecting duct 3 (mIMCD3) cells, the isolated uEVs were Labelled with Vybrant™ DiD Red Cell stain, and primary cilia were immunologically stained with anti-acetylated α -Tubulin. Confocal microscopy images were obtained for further analysis. We successfully isolated uEVs from healthy donors expressing the surface tetraspanins CD63 and CD81 protein markers and assessed their RNAs contents with Spectrometry Nanodrop and Capillary Tapstation Electrophoresis System. Isolated uEVs with ExoGAG commercial kit and Ultracentrifugation could not be internalized and uptaken by Mouse inner medullary-collecting duct 3 (mIMCD3), Confocal Microscopy images revealed the lack of colocalization of expressed primary cilia and uEVs stained with Vybrant™ DiD Cell-Labelling solution

Computer-based techniques have become especially important in molecular biology, since they often represent the only viable way to understand some phenomena at atomic and molecular level. The complexity of biological systems, which usually needs to be analyzed with different levels of accuracy, requires the application of different approaches. Computational methodologies applied to biotechnologies allow a molecular comprehension of biological systems at different levels of depth. Quantum mechanics (QM) ab-initio techniques allow the study of enzymes and organometallic models at sub-atomic levels keeping into account electronic effects on stereochemistry and chemical reactivity. We set to study [FeFe]-hydrogenases, enzymes able to both produce and oxidize H2 at high rate. The study was focused to better elucidate some redox states of the cofactor during catalysis. The principal aim of this work was to take advantage of hydrogenases biomimetic complexes to gain further inside on the catalysis of the enzyme, and pinpoint the structural and stereo-electronic features necessary to improve the efficiency of the synthetic models. H2 is a desirable fuel but the usage of this gas is somehow problematic due to its physical properties, leading to safety concerns and low energy density. A possible way to overcome these problems is to store H2 in safe and valued added chemicals. In this perspective we studied the catalytic mechanism of the first iron-containing synthetic complex able to catayze the chemical storage of H2 and CO2, converting it into HCOOH. QM methodologies were also used in a project in collaboration with the Dept. of Forensic Medicine at the University of Verona, aimed at the use of carbohydrate deficient transferrin (Tf) as marker of alcohol abuse. Tf is the protein deputed for the iron transport in the blood stream. Low glycosylated forms are known to be associated to alcohol abuse. Different spectroscopies were useful tools to discover the binding site of terbium and the best experimental conditions for terbium-Tf binding. To get more information about the active site, we optimized a method that allowed us to determine the molecular structure of the metal environment through QM computational techniques. Docking techniques to study small-ligand protein binding are useful methods to predict the binding mode of a molecule to a receptor, in order to understand its mechanism of action and improve its activity. Here, we focused on different pharmacological targets involved in different pathological mechanisms to understand how the ligand is able to interact with the receptor and exert its pharmacological effect, and how to ameliorate its structure to increase the specificity. Since the evolution of the human species exists a struggle for survival between host and pathogens, with measure and countermeasure to respectively infect and defend against infections. Positively selected sites on protein genes are the result of evolutionary pressure on certain aminoacidic residues that could be fundamental for host and pathogen infections. Protein-protein docking is a useful tool, together with computational stability analysis, to understand how residues variations modify the binding among different proteins in the immune system and how the proteins stability is affected. In conclusion, the choice of the computational methods is what determines the level of the description of the molecular system. The study of biotechnologically relevant system with computational techniques is a powerful tool to gain insight into molecular properties that are otherwise not explorable by experimental techniques.

As just described in the introductory chapter, the world of nanotechnology has been in great expansion in recent decades for many different reasons. First of all, it was the unique physical and chemical properties of these materials that enabled some applications, for example in medicine (CNTs are used for bone scaffolds, vascular stents, neuron growth and regeneration (Donaldson, et al., 2012); graphene showed good sensing properties for biological molecules, such as glucose, catecholamine neurotransmitter (Alwarappan et al. 2009), which were initially impossible. Moreover, as has just been pointed out, the global market for nanotechnology was expected to exceed a value of $125 billion by 2024, not including the case of graphene, whose market was estimated at 1.3 billion by 2023, with an annual growth rate of almost 47% compared to 2018 (De Marchi et al. 2018). The innovative power emanating from these new technologies and their predictable and incisive presence in many health and economic sectors, as well as the paucity of data on their toxicity, have led us to design an efficient approach to assess the toxicity of nanoparticles. Based on our own application project in biotechnology, we considered it fundamental to approach this application with a safe-by-design approach and evaluate the toxicity of the materials used in our application. The toxicity of different types of carbon nanomaterials was evaluated using two different cell models (MeT-5A and/or C6 cells). The selection was made considering the possible target of toxicity of the materials and the possible mode of exposure. The nanomaterials tested were: CNTs (different types with different dimensions - short and long; nanomaterial in fibrous form), N-doped graphene (2D carbon nanomaterial), carbon black (nonfibrous form). Cells were also exposed to a non-nanofibrous positive control (asbestos fibers). We used a multistage approach based on four levels of toxicity testing. The first tier was based on toxicity tests (Alamar blue, crystal violet, and LDH assay). The protocols had to be modified somewhat to adapt them to the use of nanoparticles. At this level, it is possible to define the range of action of nanomaterials and to study the effects of exposure on cell viability and metabolism. At the second level, it’s toxicogenomic analysis. This type of test allows a more comprehensive and complete view of what type of response the cells show to exposure to the materials. Moreover, toxicogenomics being a no-hypothesis driven approach, allows an unbiased investigation on the functional effecst of a given materials. As a third and fourth level, we looked at genotoxicity and ROS formation, which can be highly interconnected. Overall, this multi-level approach is the basis to build up an Integrated Approach to Testing and Assessment (IATA) of carbon nanomaterial.

Optimization of the culture conditions for the biomass production of the alga Phaeodactylum tricornutum was investigated in relation to chlorophyllase activity. To obtain the highest total chlorophyllase activity, the biomass of P. tricornutum was harvested after a period of 7 days of incubation during which the incubation temperature was maintained at 18°C for 18 h during the day and 10°C for 6 h during the night. During culture incubation, illumination was provided by fluorescent lamps projecting an incident intensity of 330 mumol/m2.s and the pH of the culture was maintained at 8.4, adjusted by a stream flow of CO 2. The hydrolytic activity of a partially purified chlorophyllase extract, obtained from the fresh biomass of P. tricornutum, was investigated in an aqueous/miscible organic solvent system containing refined bleached deodorized (RBD) canola oil, and chlorophyll or pheophytin as substrate. The effect of a wide range of oil contents, chlorophyll and pheophytin concentrations, acetone concentrations, incubation temperatures and agitation speeds on the enzyme activity was studied. The optimum reaction conditions for chlorophyllase biocatalysis were determined to consist of 20% oil, 10% acetone and a 200 rpm agitation speed with optimum temperatures and substrate concentrations of 35°C and 12.6 muM for chlorophyll, and 30°C and 9.3 muM for pheophytin. The presence of RBD canola oil showed an inhibitory effect on chlorophyllase activity whereas acetone acted as an activator at low concentrations and an inhibitor at higher ones. Moreover, chlorophyllase showed a limited affinity towards pheophytin as substrate compared to that obtained for chlorophyll. Selected samples of crude commercial canola oil were analyzed for their green pigment content using high-performance liquid chromatography and chlorophyllase biocatalysis was investigated on eight varieties of crude commercial canola oil. The chlorophyllase activity was lower in th

Doctora en Ciencias de la Ingeniería, Mención Química
Productos biotecnológicos, como los biofarmacéuticos entre otros, son productos cuyas tecnologías de producción están en constante desarrollo. Adicionalmente, sus escalas de producción son pequeñas haciendo de las plantas batch las más apropiadas para su producción. En particular, plantas batch multi-producto permiten la producción de una variedad de productos biotecnológicos con varias etapas en común. Una forma de modelar el diseño de plantas batch multi-producto es mediante el enfoque basado en optimización que fue estudiado por primera vez para este tipo de plantas por Robinson y Lonkar, quienes estudiaron el diseño de este tipo de plantas dimensionando los equipos que la conforman. Pese a los múltiples avances en el área, los que incluyen decisiones como la duplicación de unidades, la disposición de tanques de almacenamiento intermedio, programación de la producción y consideración ambientales, entre otras mejores, aún existe una falta de trabajos donde este tipo de enfoques es aplicado en plantas reales. En este trabajo se estudia una reformulación Entera-Mixta Lineal (MILP) del problema Entero-Mixto No-Lineal (MINLP) que resulta al plantear el modelo para el diseño de una planta biotecnológica batch multi-producto. En un primer paso se estudia una reformulación MILP que permite modelar el diseño de una planta utilizando tamaños de equipos en un conjunto continuo y una selección de hosts en un conjunto discreto de opciones. Esta reformulación hace uso de técnicas avanzadas de reformulación, probando ser escalable y confiable para su aplicación en casos reales. En un segundo paso, la reformulación MILP original fue modificada para la inclusión de una selección de equipos, tanto en un conjunto discreto, como en uno continuo, dando un enfoque más realista para poder modelar una planta biotecnológica batch multi-producto; donde unidades como los reactores pueden ser construidos de acuerdo con las necesidades del cliente, sin embargo, unidades como las columnas cromatográficas sólo están disponibles en tamaños discretos dados por el proveedor. Información de procesos reales que formaban parte de una planta batch multi-producto real permitieron la determinación de los parámetros del modelo y una comparación entre las distintas lineas de producción versus la planta real mostraron que este tipo de modelos puede permitir grandes ahorros en los costos de los principales equipos de la planta. Finalmente, como el enfoque estudiado utiliza software de modelación y optimización, el modelo es más amigable para quienes puedan utilizarlo en la práctica. Sin embargo niveles más bajos de implementación podrían mejorar los tiempos de resolución permitiendo la inclusión de formulaciones más complejas, como por ejemplo, la inclusión de costos u objetivos de producción variables.

Background: Currently, the development of microbial strains for biotechnological production of chemicals and materials can be improved by using a rational metabolicengineering that may involve genetic engineering and/or systems biology techniques. Elementary ux mode analysis (EFM) and Flux balance analysis (FBA) are the twomost commonly used methods for probing the microbial network system properties for metabolic engineering purposes. EFM can be used to identify all possible pathways. However, combinatorial explosion of the number of EFMs obtained during EFM analysis, especially for large reaction networks, hinders the use of EFM data fordeveloping gene knockout strategies. The objective of this project was to identify interesting target products and design `proof of principle' Saccharomyces cerevisiaestrains capable of overproducing a target product; in this case lysine was chosen. Methods: EFMs were calculated for a reaction network from S. cerevisiae. In order to make sense of the large EFM solution space, a novel approach based on com-putational reduction and clustering of EFM datasets into subsets was developed,which aided the prediction of knockouts for lysine production. A Pattern analysismethod, based on regular expression matching, was also developed to interpret the EFM data. FBA frameworks, OptKnock and GDLS, were used to design in silcoproduction strains based on genome-scale models of yeast. Double and triple S. cerevisiae lysine producing strains were constructed using a PCR-based deletion method. Absolute and relative metabolome measurements for lysine and other metabolites in the single and double mutants were achieved using GC-TOF-MS.Results: The new computational and clustering methodology aided significantly the EFM-based in silico design of S. cerevisiae strains for enhanced yield of lysine andother value chemicals. Ethanol and lysine overproducing in silico strains were also developed by OptKnock and GDLS. Remarkably, the production strains with singledeletions, lsc2 and glt1, excreted into the medium five times the amount of lysine than the control strain. Five S. cerevisiae double mutant strains were successfullyconstructed. Two-fold increase in flux towards lysine production was demonstrated by S. cerevisiae double mutant M1, while both S. cerevisiae double mutants M4 andM5 showed about four-fold increase in lysine production. Conclusion: The general modelling and data reduction approaches developed here contributed in obviating the enormous problems associated with trying to obtainthe EFMs from large reaction network models and interpreting the resulting of large number of EFMs. EFM analysis aided the development of single and double S.cerevisiae mutant strains, capable of increased yield of lysine. The computational method was validated by construction of strains that are able to produce several foldmore lysine than the original strain.

Lo scopo di questo lavoro è stato quello di avviare uno studio sul ruolo di geni appartenenti ai TERMINAL FLOWER 1 (TFL1), and FLOWERING LOCUS T (FTx ,) mediante il loro trasferimento con tecniche di ingegneria genetica in due cultivar ottoploidi di fragola, Calypso (rifiorente) a Sveva (giorno breve), così da confermare le ipotesi basate sui risultati ottenuti in studi di validazione genetica condotti nella fragola diploide Fragaria vesca (Koskela, 2012). Studi di rigenerazione preliminari sono stati effettuati così da mettere a punto il miglior substrato di rigenerazione delle cultivar di fragola impiegate per le prove di trasformazione. Per la cultivar Calypso è stato identificato come più efficiente il substrato MS addizionato con TDZ 0.5 mg L-1 e 0.02 mg L-1 of 2,4 D. Mentre per la cultivar Sveva risultati positive si sono ottenuti utilizzando il substrato MS addizionato con 3 mg L-1 of BA e 0.2 mg L-1 of IBA. Al fine di disporre protocolli utili anche per la trasformazione del mirtillo sono state realizzate anche prove di rigenerazione da tessuti fogliari di Duke, un nota cultivar di mirtillo. I risultati ottenuti hanno permesso di sviluppare due strategie di rigenerazione utilizzando diversi fitoregolatori (benziladenina, thidiazuron e 2iP) mediante due approcci di organogenesi diretta da foglia e indiretta da bulk meristematici. Per ogni approccio sono stati identificati i substrati più interessanti da utilizzare per la proliferazione di germogli (WPM + 2 mg L-1 di zeatina) e per la rigenerazione diretta WPM + Zeatina 2 mg L-1, indiretta 0.5 mg L-1. Sebbene siano stati ottenuti risultati interessanti ed applicabili anche per la trasformazione genetica del mirtillo, il lavoro è però continuato solo sulle cultivar di fragola. In fragola sono state condotte delle prove di trasformazione genetica utilizzando la tecnica di trasformazione mediata da Agrobacterium tumefaciens così da ottenere linee transgeniche da sottoporre ad analisi di espressione dei geni oggetto di studio. I geni utilizzati per gli esperimenti di trasformazione sono stati il FvKSN e il Fvksn, rispettivamente il controllo e il gene con una delezione di 2 pb responsabile del carattere di rifiorenza nella fragola diploide. Riguardo ai promotori della fioritura FTx i diversi FT1, FT2 e FT3, sono stati studiati con lo scopo di capire il ruolo nel “flowering pathways” della fragola ottoploide. Le prove di trasformazione hanno permesso di ottenere line transgeniche di Sveva FTx utilizzando il protocollo suggerito da Mezzetti et al., (2007) e ottimizzato con questo lavoro di tesi (Cappelletti, 2014). Variazioni sono state portate per quanto riguarda il substrato di rigenerazione e soprattutto riguardo il metodo di selezione combinando la tradizionale tecnica di selezione con antibiotico (iterative e non-iterativa) con un lavoro di individuazione e monitoraggio del segnale GFP (fluorescenza), così da rendere efficiente la selezione e ridurre la percentuale di chimere ed escape, molto elevata nelle prime prove condotte.
The aim of work was to validate TERMINAL FLOWER 1 (TFL1), and FLOWERING LOCUS T (FTx ,) genes, in octoploid strawberry cultivars Calypso (everbearing) and Sveva (short day) in order to confirm hypotesis based on results achived in validation studies in diploid strawberry Fragaria vesca. Agrobacterium-mediated transformation technique was used to obtain transgenic lines of the different genes studied. Genes used in transformations trials were FvKSN and Fvksn, as control and with 2 bp deletion confering everbearing habits in diploid strawberry regarding TFL1 gene family, respectively. For FTx, floral activator FT1, FT2 and FT3 genes were used in order to understand their role in flowering pathways depending of the variety genotypes used, everbearing or short day. Preliminary regeneration tests were conducted and positive results was achived culturing Calypso cultivar in a medium supplemented with TDZ 0.5 mg L-1 and 0.02 mg L-1 of 2,4 D. while best regeneration efficiency was obtained culturing Sveva leaves in a medium supplemented with 3 mg L-1 of BA and 0.2 mg L-1 of IBA. Transformation trials were carried out and transgenic lines of Sveva FTx were obtained following and adopting protocol suggested by (Mezzetti, 2007) and currently updated during this activity (Cappelletti, 2014). Several adjusting were carried out regarding type of regeneration media and selection methods combining traditional antibiotics selection methods and green fluorescence protein (GFP) detecting and monitoring method in order to reduce chimaerism and escape plants, improve selection efficiency using two methods of stringent selection (iterative and non-iterative method). Parallel studies were carried out on blueberry regeneration techniques using different growth regulators (benzyladenine, thidiazuron and 2iP) and different regeneretion techniques: direct and indirect organogenesis using leaf and callus as starting material.

ABSTRACT Microbial enzymes have been used for decades in a number of processes in the industrial, biomedical, environmental, and agro-food sectors. The dehairing phase of the leather tanning process is currently mediated by sulphur/sulphides chemicals, requiring expensive and complex procedures for water depollution and increasing safety risks for workers. This project is aimed at the development of a microbially-driven process for hair removal based on secreted enzymes. The first phase of the project consisted in the isolation of microorganisms naturally present on raw hides (cow skin) and displaying dehairing ability. A collection of 52 pure bacterial isolates was first screened for proteolytic activity (25 positive isolates) and then for their ability to grow on minimal media (10 positive isolates). The genome of these 10 isolates was sequenced and the supernatants containing secreted enzymes (and potential other metabolites) were tested for enzymatic activity and dehairing capacity at a laboratory scale. The secretome analysis reported the presence of more than 200 secreted proteins for each isolate and showed an increase in the release of hydrolytic enzymes during growth on minimal media. Further selection among the 10 isolates was based on proteomic analysis, pilot scale dehairing tests and yields after the downstream process The 4 isolates, selected on the base of the unhairing ability, secretome analysis and downstream yields, were subjected to further characterization to choose the most promising for the desired activity. Isolate 1Dm15, selected for the dehairing ability demonstrated at pilot scale, was grown in bioreactor and once the parameters were defined a scale-up of the process was performed. In conclusion, in this work we identified a Bacillus sp. strain able to grow on minimal media and secrete a pool of enzymes active in the dehairing of hides. This microbially driven process shows promising application in the industrial practice substituting the use of reducing agents.

ABSTRACT Microbial enzymes have been used for decades in a number of processes in the industrial, biomedical, environmental, and agro-food sectors. The dehairing phase of the leather tanning process is currently mediated by sulphur/sulphides chemicals, requiring expensive and complex procedures for water depollution and increasing safety risks for workers. This project is aimed at the development of a microbially-driven process for hair removal based on secreted enzymes. The first phase of the project consisted in the isolation of microorganisms naturally present on raw hides (cow skin) and displaying dehairing ability. A collection of 52 pure bacterial isolates was first screened for proteolytic activity (25 positive isolates) and then for their ability to grow on minimal media (10 positive isolates). The genome of these 10 isolates was sequenced and the supernatants containing secreted enzymes (and potential other metabolites) were tested for enzymatic activity and dehairing capacity at a laboratory scale. The secretome analysis reported the presence of more than 200 secreted proteins for each isolate and showed an increase in the release of hydrolytic enzymes during growth on minimal media. Further selection among the 10 isolates was based on proteomic analysis, pilot scale dehairing tests and yields after the downstream process The 4 isolates, selected on the base of the unhairing ability, secretome analysis and downstream yields, were subjected to further characterization to choose the most promising for the desired activity. Isolate 1Dm15, selected for the dehairing ability demonstrated at pilot scale, was grown in bioreactor and once the parameters were defined a scale-up of the process was performed. In conclusion, in this work we identified a Bacillus sp. strain able to grow on minimal media and secrete a pool of enzymes active in the dehairing of hides. This microbially driven process shows promising application in the industrial practice substituting the use of reducing agents.

Sorghum (Sorghum bicolor L., Moench) is the fifth most important cereal crop in the world and represents an important source of food, feed and energy in several countries. Recently, there has been an increasing interest in sorghum cultivation worldwide, since it is relatively more drought- and heat-tolerant than other cereal crops, and it is better suited for the predicted consequences of global warming. In Africa and Asia, sorghum is primarily used as food for more than 500 million people, while in the Americas and Australia, it is used mainly as a maize-substitute in livestock feed. In the United States, sorghum is also being used in the production of ethanol. In view of its diverse utility, sorghum offers a large number of target traits that could be modified to meet the required applications. In this work, we have used different genetic engineering approaches to address two important issues in sorghum: seed quality and nitrogen use efficiency. First, we examined the temporal and spatial activity of a rice glutelin gene (GluA-2) promoter, in transgenic sorghum. Results from quantitative and histochemical GUS assays, as well as from transcript analyses, showed that this promoter is highly active during the middle stages of sorghum seed development and that it controls transgene expression specifically in the seed endosperm. This means that the GluA-2 promoter can serve as a useful tool in introducing novel traits into sorghum seed in order to improve the quality of this important cereal. Furthermore, we investigated the effects of cytosolic glutamine synthetase (GS1) and alanine aminotransferase (AlaAT) gene overexpression on nitrogen metabolism and plant growth in sorghum. T_(2) generation plants transformed with a sorghum GS1 gene (Gln1) driven by the maize ubiquitin promoter exhibited enhanced grain yield and biomass accumulation under optimal nitrogen levels.

Demand for medicinal plants has drastically enhanced due to the presence of therapeutically important compounds and is continuously rising in national and international markets. Exploring elite accession among numerous accessions, collected from different locations is an alternative approach to satisfy the increase demand of medicinal plants. Biotechnological approaches are reliable source for production of therapeutically important compound. It also provides long-term utilization of plants. Plumbago zeylanica, a pharmaceutically important medicinal plant has been explored in the present study. In vitro shoot culture was established for five accessions of Plumbago zeylanica. Four different plant tissue culture media, three different carbon sources and three different nitrogen sources were tested for five accessions of P. zeylanica to evaluate optimum culture condition based on growth. The accession which showed maximum shoots number was chosen for further investigation. Accession-based study of in vitro shoot culture showed that accession number IC 524441 is one of the elite accessions and chosen for further studies. Adventitious root suspension culture was explored for enhanced production of plumbagin. Optimization of adventitious root suspension culture showed that highest plumbagin production was obtained in ½ strength MS liquid media having 3% sucrose with 2 g/L of inoculum density. Further elicitation with yeast extract (150 mg/L) increases threefold plumbagin production as compared to control one and highest plumbagin production was 90.96±0.51 µg/mL. Further cell suspension culture was also explored for enhanced production of plumbagin. Optimization of cell suspension culture showed that MS medium having 1 mg/L NAA with 3 g/L inoculum density and 150mg/l yeast extract at pH 5.8 was optimal for plumbagin production. Maximum plumbagin production was enhanced up to 3.3 times as compared to control one and maximum amount of plumbagin production was 83.30±0.18 µg/mL. Biochemical analysis of thirteen different accessions of Plumbago zeylanica were performed where concentration of therapeutically important compounds such as total plumbagin, total flavonoids content, total phenol content, total tannin content and antioxidant activity were evaluated and results showed that IC-524441 is an elite accession. Same thirteen accessions were assessed for genetic diversity analysis using CBDP and SCoT marker. Genetically diverse accessions can be utilized by plant breeders for the generation of elite accessions which have high quantity of pharmaceutically important secondary metabolites. Further residual plant material of P. zeylanica was used for silver nanoparticles synthesis and its application in antibacterial and dye degradation were investigated. Biochar was also derived from residual shoot and root culture and its application in removal of cadmium and chromium were studied. Role of plumbagin against different cancer receptor using in silico method was also evaluated. The ligand plumbagin was docked against three different cancer receptors i.e. COX-2, EGFR and TACE to evaluate its potential effect on different cancer. In-silico studies showed that plumbagin is a potential therapeutic agent for treatment of cancer and same can be established through in vivo studies and clinical trials to confirm its efficiency in patients.

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2014
Thymus caespititius is an aromatic plant from the NW Iberian Peninsula and from the Azores and Madeira archipelagos. In this species chemical polymorphism in the essential oils has been described, with seven well defined chemotypes, namely thymol, carvacrol, α-terpineol, sabinene, and the mixed chemotypes thymol/sabinene, thymol/carvacrol and thymol/sabinene/carvacrol. T. caespititius essential oils already proved to have antimicrobial, antioxidant activity and acetylcholinesterase inhibition, and more recently they were shown to have high nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus, namely the carvacrol and thymol rich oils. Since this species is not used commercially, the oils diversity observed results only from natural evolution; however, the molecular mechanisms underlying such chemical diversity are not yet clear. In this work were used molecular and biotechnological approaches to understand the basis for chemical diversity of T. caespititius’ essential oils. Five T. caespititius in vitro cultures from plants with different chemotypes were established, allowing having enough plant material for the following studies. This advantageous in vitro system avoids plant material collection from the natural habitat and the controlled growth conditions reduce the sources of variability known to affect essential oil composition. Terpenes are the main group components identified in T. caespititius essential oils. However the molecular basis of their synthesis has not yet been fully understood. Given this, the genes encoding enzymes of monoterpene biosynthesis were searched and identified in the five established in vitro genotypes and latter their expression level was assessed. It has been shown that biotic (pathogens, herbivores) and abiotic (temperature, light quality, nutrients type and availability) factors could influence the essential oil composition as well as the genes involved in their biosynthesis. So, nutrient composition and Botrytis cinerea fungal extracts were assayed in in vitro cultures to study the biosynthesis and accumulation of terpenes in this aromatic species. Collectively, it was demonstrated that the conditions established in this work also step up the possibility of using T. caespititius shoot cultures as an experimental model to investigate the terpene biosynthesis and accumulation in plants. The large biomass increase obtained in vitro advance the possibility of using large-scale in vitro production of desirable secondary metabolites. The terpene synthase genes identified and characterized here were a small contribution to understanding how these genes evolved, are regulated and function in Lamiaceae
Thymus caespititius é uma planta aromática nativa do NO da Península Ibérica e dos arquipélagos dos Açores e da Madeira. Nos óleos essenciais desta espécie foi encontrado polimorfismo químico, tendo sido definidos sete quimiotipos, nomeadamente timol, carvacrol, α-terpineol, sabineno, timol/sabineno, timol/carvacrol e timol/sabineno/carvacrol. Em estudos anteriores, os óleos essenciais de T. caespititius revelaram ter actividade antimicrobiana, antioxidante, anticolinesterásica e mais recentemente mostraram elevada actividade nematicida contra o nemátode da madeira do pinheiro (Bursaphelenchus xylophilus), principalmente os óleos ricos em carvacrol e timol. Uma vez que esta espécie de tomilhos não está disponível comercialmente, a diversidade observada nos óleos visível mesmo em plantas cultivadas nas mesmas condições ambientais são resultado de evolução natural e não de selecção. Contudo os mecanismos moleculares envolvidos nesta diversidade química não são ainda claros. Neste trabalho, foram utilizadas abordagens moleculares e biotecnológicas de modo a compreender e esclarecer a diversidade química dos óleos essenciais de T. caespititius. A utilização de culturas in vitro em estudos de produção de óleos essenciais tem sido explorada em várias espécies aromáticas. A utilização deste sistema é vantajoso, pois não só evita a recolha de material vegetal do seu habitat natural, como também permite, pelas condições de crescimento controladas, reduzir as fontes de variabilidade que afectam a composição dos óleos essenciais. No presente trabalho de investigação, cinco genótipos foram estabelecidos in vitro, três com os quimiotipos carvacrol (C), carvacrol/timol (CT) e sabineno/carvacrol (SC) e dois resultantes de uma planta mãe com o quimiotipo α-terpineol (G1 e G2). Os óleos essenciais destes genótipos foram avaliados em culturas de rebentos em proliferação (6 a 24 repicagens após o estabelecimento in vitro) e comparados com os das respectivas plantas de campo. Os óleos essenciais foram isolados por hidrodestilação e analisados por GC e GC-MS. As culturas in vitro mantiveram estável a composição dos óleos essenciais durante os dois anos de análise. As culturas resultantes de gemas axilares (C, CT e SC) produziram óleo qualitativamente semelhante ao das plantas correspondentes em campo, apesar de pequenas diferenças quantitativas. O genótipo C revelou um perfil químico semelhante ao da planta de campo, sendo o carvacrol (20-69% nos rebentos e 71% na planta de campo) o componente maioritário dos óleos, seguido pelo acetato de carvacrilo (6-40% nas culturas in vitro e 4% na planta de campo). Para o genótipo CT, os componentes maioritários detectados na planta de campo foram o carvacrol (42%) e o timol (23%); enquanto nas culturas in vitro os componentes maioritários detectados foram o carvacrol (16-28%), o timol (17-25%), o acetato de carvacrilo (11-23%) e o acetato de timilo (9-15%). No genótipo SC, os componentes dominantes dos óleos essenciais tanto das plantas de campo como das culturas in vitro foram o carvacrol (11-28%), o sabineno (18-49%), e o timol (8-12%). As culturas in vitro resultantes de sementes (G1 e G2) apresentaram perfis de óleos essenciais diferentes dos da planta mãe (quimiotipo α-terpineol), esta diferença pode dever-se à variabilidade genética das sementes resultantes de cruzamentos abertos. Para G1 e G2 foi impossível determinar o seu quimiotipo. Nestes genótipos foram detectados baixos níveis de α-terpineol e o inverso foi observado para o timol, o carvacrol metil éter, o acetato de timilo e o carvacrol. O aumento de biomassa obtido com as culturas in vitro permitiu estudar a biossíntese dos terpenos e a sua regulação sob factores bióticos e abióticos. Os óleos essenciais de T. caespititius são uma mistura complexa de compostos, na sua grande maioria de natureza terpénica. No entanto, as bases moleculares da biossíntese destes compostos ainda não foram completamente esclarecidas. Posto isto, utilizaram-se as culturas in vitro de T. caespititius para procurar os genes envolvidos na biossíntese dos terpenos, nomeadamente os genes das terpeno sintase (TPS). Foram identificados e caracterizados três genes Tctps (Tctps2, Tctps4 e Tctps5) neste sistema; dois dos quais, Tctps2 e Tctps5, haviam já sido identificados em plantas de campo. O gene Tctps5 foi apenas detectado nos genótipos G1 e G2. Com o alinhamento dos três genes Tctps detectou-se que o Tctps4 não possuía péptido-sinal ou este era muito pequeno (3-4 aa) em comparação com Tctps2 e Tctps5 (46-47 aa). A grelha de leitura deste gene (1665 bp) é também menor que a dos genes Tctps2 (1794 bp) e Tctps5 (1806 bp). O alinhamento das sequências de aminoácidos das três terpeno sintases revelou que estas partilhavam 77% de semelhança. Tendo em conta a informação disponível das terpeno sintases de espécies da família Lamiaceae, o tamanho do gene Tctps4 sugeria que provavelmente este gene codificava uma sesquiterpeno sintase. Contudo, os ensaios enzimáticos das Tctps com o substrato GPP revelaram que tanto Tctps2 como Tctps4 codificavam para a γ-terpineno sintase, enquanto Tctps5 codificava para uma α-terpineol sintase. Estas proteínas não revelaram qualquer actividade sesquiterpénica em ensaios com FPP como substracto. Os ensaios enzimáticos com os extractos proteicos foram realizados a três temperaturas diferentes, 4º, 21º e 42ºC. Apesar destes dados serem apenas qualitativos, a temperatura não pareceu afectar a actividade da Tctps4, enquanto a 4ºC a actividade de Tctps2 isolada do genótipo C foi muito reduzida, sendo retomada com o aumento da temperatura. Nos restantes ensaios com Tctps2 não foram detectadas grandes diferenças de actividade. Quanto à Tctps5 pareceu existir uma variação no produto relacionada com a temperatura. Em baixas temperaturas o produto maioritário formado foi o limoneno, enquanto a 21ºC e 42ºC formou-se predominantemente o α-terpineol. A existência de dois isogenes para γ-terpineno sintase, sugere duplicação de genes durante o processo evolutivo, seguido de mutações levando à diferenciação dos dois genes, não comprometendo, no entanto, a actividade da proteína. É de salientar que nas plantas de T. caespititius analisadas, o gene Tctps4 encontrava-se menos expresso que o Tctps2. Nas plântulas in vitro foi detectada uma elevada acumulação de transcritos dos genes Tctps, ao contrário do verificado nas raízes. Este resultado era expectável, uma vez que, a síntese e acumulação dos terpenos em Lamiaceae ocorrem em células especializadas, como os tricomas glandulares existentes na parte aérea. A quantidade de γ-terpineno encontrada nos óleos essenciais de T. caespititius foi reduzida, o que poderá dever-se à sua rápida conversão em carvacrol e timol, dois dos componentes maioritários dos óleos desta espécie. Os estudos de mutagénese dirigida em Tctps2 demonstraram que o resíduo Arg-505 (R505G) é essencial para a estabilidade e/ou correcto enrolamento da proteína e a alteração desse aa por um aa hidrofóbico não-polar (Gly) quebrou essa estabilidade. A inserção de duas outras mutações neste gene (D405G e A521V) não revelou efeito na actividade da proteína. Ao longo dos anos, tem-se verificado que tanto os óleos essenciais como os genes envolvidos na sua biossíntese são influenciados por factores bióticos (agentes patogénicos, herbívoros) e abióticos (temperatura, qualidade da luz, tipo e disponibilidade de nutrientes). Por este motivo foram feitos ensaios nas culturas in vitro de T. caespititius para avaliar o efeito de diversos factores na biossíntese e acumulação dos terpenos. Os cinco genótipos estabelecidos in vitro foram colocados a crescer em dois meios de cultura, MS e SH, de forma a testar o efeito da composição de nutrientes. Nas culturas crescidas em SH observou-se maior rendimento de óleo (0.3-3.4%) do que nas culturas crescidas em MS (0.3-1.2%). Os óleos essenciais das plantas crescidas nas duas condições foram qualitativamente semelhantes. Contudo, observaram-se algumas diferenças quantitativas, principalmente nos compostos com o grupo acetato, acetato de timilo e acetato de carvacrilo, cuja quantidade relativa diminuiu nos rebentos crescidos em SH. Os componentes maioritários dos óleos isolados do genótipo C foram o carvacrol (25-60%) e o acetate de carvacrilo (9-45%). As plantas crescidas em SH continham menos acetato de carvacrilo (9-13%) do que as plantas desenvolvidas em MS (23-45%). Quanto aos óleos do genótipo CT, detectou-se elevada percentagem de carvarol (13-28%), timol (14-23%), acetato de carvacrilo (10-26%) e acetato de timilo (8-18%). Nestes óleos também se observou uma diminuição da quantidade relativa nos compostos com o grupo acetato. Nas culturas SC, os componentes maioritários dos óleos foram o sabineno (35-45%), o carvacrol (11-19%) e o timol (8-11%). Já nos óleos essenciais dos genótipos G1 e G2, os componentes identificados em maior concentração foram o p-cimeno (7-34%), o γ-terpineno (4-21%), o timol (4-18%) e o carvacrol metil éter (4-17%). Nas plantas crescidas em SH observou-se uma diminuição na quantidade relativa de carvacrol metil éter (4-8%), sendo o inverso observado no p-cimeno (10-34%) e no γ-terpineno (6-21%). Em G2, também se observou diminuição na quantidade de timol (4-7%). A acumulação dos transcritos das terpeno sintase (Tctps2 e Tctps4) foi superior nas culturas in vitro desenvolvidas em SH, excepto no genótipo SC. Em G1 e G2, o gene Tctps4 apresentou-se menos expresso que nos restantes genótipos. Quanto ao gene Tctps5 a sua expressão foi apenas detectada em G1 e G2; culturas onde se observou maior quantidade de α-terpineol. Entre os dois meios de cultura testados observam-se várias diferenças na sua composição nutricional, sendo a concentração de azoto e a razão NH4 +/NO3- as mais notórias. Estes poderão estar relacionados com as diferenças observadas na composição dos óleos essenciais e na expressão dos genes das terpeno sintase. No entanto, será necessário confirmar esta hipótese, testando diferentes concentrações de azoto e razões NH4 +/NO3- em MS. Os extractos fúngicos de Botrytis cinerea não afectaram qualitativamente nem quantitativamente os óleos de CT, no entanto em SC, observou-se diminuição do conteúdo de sabineno e aumento dos compostos com actividade antifúngica, carvacrol e timol. A expressão dos genes Tctps2 e Tctps4, avaliada ao longo de 30 dias não revelou diferenças entre controlo e stress (presença de extracto de B. cinerea). No genótipo SC, os extractos fúngicos aumentaram cerca de 6x a expressão dos genes Tctps nas primeiras horas de tratamento. Enquanto em CT apenas se observou um ligeiro aumento (aproximadamente 2.8x). Nos restantes pontos de análise observou-se diminuição da regulação destes genes (cerca de 1.5-15.7x nas culturas CT e 1.0-3.5x nas culturas SC), o que pode estar relacionado com a translocação dos recursos energéticos para actividades vitais da planta. Neste trabalho foi possível demonstrar que as culturas in vitro de T. caespititius são um bom modelo experimental para a investigação da biossíntese e acumulação de terpenos. A identificação e caracterização dos genes das terpeno sintase neste trabalho permitem contribuir para a compreensão da função, regulação e evolução destes na família Lamiaceae.
Fundação para a Ciência e a Tecnologia (FCT)

Tomato (Solanum lycopersicum) belongs to the nightshade family Solanaceae. The goals of public and private tomato breeding programs are focusing on nutritional quality currently. Tomatoes represent a major contribution to nutrition worldwide and a reservoir of diverse antioxidant molecules, such as ascorbic acid, vitamin E, carotenoids, flavonoids and phenolic acids. Among abiotic stresses, drought is by far the leading environmental stress in agriculture, most crop plants, including tomato, are sensitive to drought stress (DS) throughout the ontogeny of the plant, from seed germination to harvest. Substantial genetic variation for Drought Tolerance (DT) exists within the cultivated tomato, as well as in its related wild species. This study was carried out to identify genomic regions involved in the control of fruit quality traits in tomato, headed to select new genotypes fitting the increasingly high public demand for a sustainable agriculture asking for limited levels of energy inputs, such as mainly the water supply. We identified Quantitative Trait Loci leading (QTLs) to an higher fruit nutritional quality and to an increased drought tolerance. The transcriptomic analysis allowed us to identify candidate genes involved in the pathway of main secondary metabolites. We developed a breeding program to transfer wild dissected QTLs controlling quality traits, such as the content in soluble solids and AsA into advanced breeding tomato lines selected for displaying high agronomic performances.

Tese de doutoramento em Programa Doutoral em Biologia de Plantas
Since the development of Bordeaux mixture in the late 1800’s, copper-based fungicides have been widely used against grapevine (Vitis vinifera L.) diseases, mainly in organic but also in conventional viticulture. Although they initially seemed to improve plant growth in unproductive lands, their intensive use has raised concerns regarding toxicity to plants and soil contamination, and research has emerged to characterize copper effects on grapevine physiology. However, at grape berry level the effects of plant exposure to copper remain largely unexplored. In the present study molecular, biochemical and biotechnological approaches were combined to investigate the mechanisms mediating copper transport and compartmentation in grape cells and the consequences of altered copper status in grape berry composition and wine quality. Toxicological assessments in grape cell suspensions (CSB, Cabernet Sauvignon Berry) showed that copper reduced the growth and cell viability in a dose-dependent manner above 100 μM and was accumulated in specific metal ion sinks. Copper transport across the plasma membrane of grape cells was characterized with the copper-sensitive probe Phen Green SK, and results showed that copper uptake was mediated by a high capacity saturable transport system (Km = 583 μM; Vmax = 177 x10-6 %ΔF min-1 protoplast-1), that was regulated by copper availability in the culture medium. The compartmentation of copper in the vacuole of grape cells was studied by fluorescence microscopy and spectrofluorimetry. The pH-sensitive fluorescent probe ACMA showed the involvement of H+-dependent copper transport across the tonoplast, which was dependent on the transmembrane pH gradient generated by both V-H+-ATPase and V-H+-PPase, but apparently not regulated by copper availability in the culture medium. Eight putative COPT/Ctr-type Vitis vinifera copper transporters were identified in the grapevine genome, and expression studies in CSB suspensions showed that VvCTr1 and VvCTr8 were distinctly affected by CuSO4 availability. The in silico analysis of all VvCTr members showed a close phylogenetic relationship to COPT proteins from Arabidopsis and rice, and typical features of Ctr transporters, including the presence of three transmembrane domains. In a field trial, the expression of each VvCTr was studied by qPCR in grape berries and leaves cv. “Vinhão” throughout the fructification season. In plants treated with a triazol-based fungicide, transcripts of VvCTr1 and VvCTr8 were the most abundant in leaves and berries, respectively, while VvCTr4, VvCTr5 and VvCTr7 were the least expressed. The application of Bordeaux mixture induced a transcriptional reprogramming of VvCTr gene expression depending on the developmental stage, which was associated to increased copper levels in grape berries (33.3 μg g-1 DW in the mature phase – washed berries). Further characterization of Ctr-mediated transport was achieved with the functional characterization of VvCTr1. Fusions with fluorescent proteins demonstrated that VvCTr1 monomers are self-interacting and the protein is located to the vacuolar membrane. The expression of VvCTr1 in yeast ctrΔ strains and Arabidopsis copt5 seedlings allowed the validation of its involvement in intracellular copper transport. The presence of VvCTr1 transcripts in grapevine roots, stem and leaves further supported its essential role in copper homeostasis. The effect of Bordeaux mixture on the metabolomics profile of grape berries and leaves cv. “Vinhão” was studied throughout fruit development by GC-TOF-MS, UV-visible spectrophotometry. While copper-treated berries accumulated 7 to 14-fold more copper than control fruits, the levels of sugars, essentially fructose, organic acids and flavan-3-ols were modified at specific developmental stages. A 40% decrease of free natural amino acids including arginine and proline was observed in mature fruits, together with a decrease in mineral nitrogen and protein content. This shift in grape berry composition is likely to have implications in fruit and wine quality. The effect of Bordeaux mixture on the volatile composition of wines cv. “Vinhão” was evaluated by Liquid-Liquid Microextraction GC-FID and SPE GC-Ion Trap-MS analyses. Laboratory-scale vinifications were performed, and a significant delay in the degradation of reducing sugars was observed in musts from copper-treated grapes, which contained 12.6 mg L-1 copper at the end of fermentation. Increased copper levels promoted a decrease in the levels of higher alcohols, including isoamyl alcohol, aldehydes, including acetaldehyde, esters of organic acids, lactones, volatile phenols and ketones, while increasing the levels of acetates, volatile fatty acids, fatty acid ethyl esters and terpenes, namely linalool. Besides its importance at a scientific standpoint, the present study contributed to elucidate the effect of copper-based fungicides in grape berry composition and wine quality.
Desde o desenvolvimento da calda bordalesa no final do século XIX, os fungicidas à base de cobre têm sido amplamente usados para combater doenças da videira (Vitis vinifera L.), especialmente na agricultura biológica. Diversos estudos têm sido direcionados para avaliar o impacto da aplicação de cobre na fisiologia da videira, embora permaneça pouco estudado o seu efeito ao nível do fruto. No presente trabalho foram combinadas abordagens moleculares, bioquímicas e de carácter biotecnológico para investigar os mecanismos envolvidos no transporte e compartimentação do cobre nas células do bago de uva, bem como os efeitos da alteração dos níveis de cobre na composição do bago e na qualidade do vinho. A sonda fluorescente Phen Green SK foi utilizada para estudar o transporte de cobre através da membrana plasmática de células CSB (Cabernet Sauvignon Berry). Os resultados mostraram que a entrada de cobre é mediada por um sistema de transporte saturável de elevada capacidade, regulado pela disponibilidade de cobre no meio de cultura. Os resultados obtidos com a sonda fluorescente ACMA, sensível ao pH, mostraram que o movimento do cobre através da membrana vacuolar é mediado por um sistema de antiporte com H+, dependente do gradiente de H+ gerado pelas bombas V-H+-ATPase e V-H+-PPase. A análise do genoma da videira permitiu identificar oito genes codificantes de transportadores de cobre do tipo COPT/Ctr (VvCTr1-VvCTr8). Estudos in silico mostraram um elevado grau de semelhança entre as sequências aminoacídicas dos VvCTr e dos COPT de Arabidopsis e de Oryza sativa, bem como a presença de motivos típicos dos Ctrs. O trabalho subsequente incluiu ensaios de campo que possibilitaram o estudo do efeito da aplicação da calda bordalesa na expressão dos VvCTrs em bagos e em folhas de videira da variedade Vinhão. Os resultados de qPCR mostraram que em plantas tratadas unicamente com fungicidas à base de triazol, os genes VvCTr1 e VvCTr8 foram os mais expressos em folhas e bagos, respectivamente, enquanto os genes VvCTr4, VvCTr5 e VvCTr7 foram os menos expressos. Em plantas tratadas com calda bordalesa ocorreu uma reprogramação da expressão dos VvCTrs, cujo padrão foi dependente do estado de desenvolvimento do fruto. O tratamento com a calda bordalesa provocou aumentos de 7 a 14 vezes dos níveis de cobre em bagos lavados. Os estudos prosseguiram com o intuito de se caracterizar funcionalmente os transportadores de cobre da videira. Neste âmbito, o gene VvCTr1 foi clonado a partir de cDNA de bago e a sua função como transportador intracelular de cobre foi demonstrada por complementação de mutantes de levedura (ctr1Δctr3Δctr2Δ e ctr1Δctr3Δ) e de Arabidopsis (copt5). Adicionalmente, estudos de localização subcelular com proteínas de fusão fluorescentes mostraram que os monómeros de VvCTr1 são capazes de interagir entre si e que o transportador se localiza na membrana vacuolar. O efeito da calda bordalesa no metaboloma do bago e de folhas de videira da variedade Vinhão sujeitas ao protoloco de tratamento descrito anteriormente foi também alvo de estudo. A análise dos extratos por “GC-TOF-MS” mostrou que os bagos de plantas tratadas com cobre sofreram alterações metabólicas, traduzidas por níveis diferentes de açúcares, de aminoácidos, de ácidos orgânicos e de alguns metabolitos secundários em fases específicas do desenvolvimento. Em particular, em bagos maduros de plantas sujeitas ao tratamento com cobre o conteúdo em aminoácidos livres sofreu uma redução de 40%, acompanhada por uma redução menos significativa dos níveis de proteína total e de azoto mineral. No presente trabalho foi ainda estudado o efeito dos níveis de cobre no mosto - resultantes da aplicação de calda bordalesa -, na constituição volátil do vinho. Para o efeito, foram efectuadas vinificações à escala laboratorial, tendo-se observado um atraso significativo na conversão de açúcares redutores em mostos de bagos tratados com cobre. Os níveis elevados de cobre (12.6 mg L-1) promoveram uma diminuição do conteúdo em álcoois, aldeídos, estéres de ácidos orgânicos, lactonas, fenóis voláteis e cetonas. Em paralelo, foi observado um aumento dos níveis de acetatos, ácidos gordos voláteis, ésteres etílicos de ácidos gordos e terpenos. O presente estudo revestiu-se de uma dimensão fundamental e aplicada e contribuiu para se compreenderem melhor os efeitos dos fungicidas à base de cobre na constituição química do bago de uva e no perfil aromático do vinho.
Fundação para a Ciência e a Tecnologia - Bolsa de Doutoramento SFRH/BD/64587/2009; Fundação para a Ciência e a Tecnologia – projeto “GrapeBerryFactory: Sugars, acids, phenolics and water on grape development and ripening” (FCOMP-01-0124-FEDER-008760; PTDC/AGR-ALI/100636/2008); Fundação para a Ciência e a Tecnologia - projeto de cooperação científica Portugal-Tunísia (FCT/5955/27/5/2013/S); FEDER/COMPETE - Operational Competitiveness Programme - projeto FCOMP-01-0124-FEDER-022692; Projeto Europeu INNOVINE – “Combining innovation in vineyard management and genetic diversity for a sustainable European viticulture” (FP7-KBBE-2012-6-311775); COST Action FA1106 – QualityFruit, “An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine”; COST Action FA1003 - "East-west collaboration for grapevine diversity exploration and mobilization of adaptive traits for breeding”.

Fire blight, caused by the bacterium Erwinia amylovora (E. amylovora), is one of the most economically important apple (Malus x domestica) pathogens worldwide. Various chemical and biological approaches can be applied to deal with the disease, but none of these is decisive. Such strategies are also prohibited in many countries due to their potential impact on human health and environment. To date, the most efficient strategy for controlling E. amylovora is thus to breed resistant/tolerant apple cultivars by manipulating one or multiple plant genes, which are associated with resistance or susceptibility to the disease. Within this context, classical breeding or genetic engineering can be applied. While conventional breeding is still considered a time-consuming and laborious process, genetic engineering methodologies represent rapid, precise and powerful alternatives to insert the desired trait into the crop of interest. In this thesis, we exploit different biotechnological approaches on the one hand to improve fire blight resistance trait by knocking-out a known susceptibility gene and on the other hand to investigate potential disease-related key genes. At first, we develop a CRISPR/Cas9-FLP/FRT-based gene editing system, mediated by Agrobacterium tumefaciens, to knock-out the fire blight susceptibility gene MdDIPM4 and generate apple (‘Gala’ and ‘Golden Delicious’) cultivars with reduced susceptibility to the disease and a minimal trace of exogenous DNA. Several transgenic lines were screened by sequencing to identify mutations in MdDIPM4. An editing efficiency of 75% was observed. Candidate lines showing loss-of-function mutation were inoculated with E. amylovora and a significant reduction (of about 40%) in disease symptoms was observed compared to wild-type plants. No CRISPR/Cas9 off-targeting activity was detected in five potential off-target regions. Thus, with the aim of removing the ‘entire’ T-DNA in those lines with reduced susceptibility to the pathogen, the FLP/FRT system was induced and the excision of the T-DNA was validated. This work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT-based editing system for the production of ‘clean’ fire blight resistant apple cultivars Secondly, we investigate the apple miRNA MdmiR285N which is predicted to play a key role in the post-transcriptional regulation of 35 RNA transcripts coding for different disease resistance proteins. A complex network of potential transcriptional regulatory elements involved in plant growth and development, and in response to different hormones and stress conditions has been identified in MdmiR285N promoter in both apple and the model plant species Arabidopsis thaliana. Moreover, Spatio-temporal expression of MdmiR285N has been assessed in plants at physiological growth conditions and in response to bacterial pathogens. Our results suggest that MdmiR285N is a multifunctional microRNA which may control different processes, such as biotic stress response, plant growth and development. In parallel, a methodological work has been carried out for a precise and rapid characterization of the transgenic apple lines produced. A quantitative, rapid and cost-effective method has been developed, based on real-time PCR to quantify the copy number of nptII marker gene in apple lines and to evaluate its elimination after the activation of the recombinase system. This method may be valuable for those institutions committed to tracing ‘gmo’ apple products.

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas
Catharanthus roseus (L.) G. Don produces an impressive number of specialized metabolites, over 130 terpenoid indole alkaloids (TIAs), among which the anticancer vinblastine (VLB) and vincristine (VCR). VLB and VCR were the first natural drugs used in cancer therapy, still holding an important position in the treatment of cancer nowadays. VLB and VCR accumulate in very low levels in C. roseus leaves that remain the only source of the anticancer TIAs, since efforts to obtain their entire chemical synthesis have been unfruitful. An intense research has been carried out over the last decades in C. roseus to unveil the biosynthesis of TIAs pathway, however, the characterization of several biosynthetic steps is still missing, and the membrane transport mechanisms are basically uncharacterized despite their importance for TIA accumulation. Previous studies, in our group, allowed the identification of a number of very strong candidates to important functions within the TIA pathway, including MATE1, a strong candidate to TIA vacuolar accumulation, and CYP2, a strong candidate to two of the last steps of VLB and VCR biosynthesis. These genes are strong candidates for the possible manipulation of TIA levels in order to improve their accumulation. The work developed here includes the sub-cloning of each gene into a plant binary vector followed by the genetic transformation of C. roseus hypocotyls using Agrobaterium tumefaciens. The induction of transgenic callus was optimized, leading to the successful establishment of transgenic callus lines confirmed by performing a Gus assay. The regeneration into a whole plant was actively pursued by changing an array of conditions but remained unsuccessful. Complementarily, downregulation of MATE1 expression was carried out using Virus Inducing Silencing Gene (VIGS), by sub-cloning a specific MATE1 fragment into a viral based plant vector followed by Agrobacterium mediated transformation of C. roseus plants. The putatively silenced plants showed significantly lower levels of the major alkaloids accumulated in C. roseus leaves, strongly supporting a role of MATE1 in their transtonoplast transport. Overall, the work developed gives a significant contribution to the possibility of performing future biotechnological improvements of C. roseus for increased levels of the anticancer VLB and VCR.
Catharanthus roseus (L.) G. Don produz um número impressionante de metabolitos secundários, mais de 130 alcalóides terpenóides indólicos (TIA), entre os quais, os anticancerígenos vinblastina (VLB) e vincristina (VCR). VLB e VCR foram os primeiros medicamentos de origem natural a serem utilizados no tratamento do cancro, mantendo ainda hoje uma posição relevante na terapia do cancro. VLB e VCR acumulam-se em níveis muito baixos nas folhas de C. roseus, que permanecem como a única fonte dos anticancerígenos, uma vez que, os esforços para os obter integralmente por síntese química têm sido infrutíferos. Ao longo das últimas décadas, C. roseus tem sido intensamente investigada com o intuito de desvendar a via de síntese dos TIA, no entanto, várias etapas ainda estão por caracterizar assim como os mecanismos de transporte membranar, apesar da sua importância para a acumulação dos TIAs. Estudos anteriores, no nosso grupo, possibilitaram a identificação de genes candidatos a funções importantes dentro da via dos TIAs, incluindo MATE1 um forte candidato ao transporte dos TIAs para o vacúolo, e CYP2, um forte candidato a intervir num dos dois últimos passos da biossíntese de VLB e VCR. Estes genes são fortes candidatos para uma eventual manipulação dos níveis de TIA, a fim de aumentar a sua acumulação. O presente trabalho inclui a sub-clonagem de cada um dos genes num vetor binário seguido pela transformação genética dos hipocótilos de C. roseus usando Agrobacterium tumefaciens. A indução de callus transgénico foi otimizada, alcançando-se com sucesso linhas de calli transgénicas, confirmadas através da realização de um ensaio de GUS. A regeneração para obter a planta foi arduamente perseguida alterando-se uma série de condições, mas até ao momento não foi alcançada. Complementarmente, o silenciamento da expressão do gene MATE1 foi levado a cabo usando Vírus Indutor de silenciamento do gene (VIGS), através da sub-clonagem de um fragmento específico de MATE1 num vetor viral modificado, seguindo-se a transformação das plantas mediada por Agrobacterium. As plantas putativamente silenciadas apresentaram níveis significativamente mais baixos dos principais alcalóides acumulados em folhas de C. roseus, reforçando fortemente o papel do MATE1 no seu transporte. No geral, o trabalho desenvolvido contribui significativamente para a possibilidade de no futuro realizarem-se melhorias biotecnológicas em C. roseus com vista a aumentar os níveis dos anticancerígenos VLB e VLC.

Thesis submitted in fulfilment of the requirements for the degree of Ph.D. in Chemical and Biological Engineering
The increase of biodiesel production results in an accumulation of crude glycerol, the main byproduct from biodiesel industry. To take advantage of the glycerol surplus, many biotechnological processes are being studied. Crude glycerol can be used as carbon source to produce citric acid by Yarrowia lipolytica under nitrogen-limited growth conditions. However, other operational and medium conditions directly affect citric acid production. Yield of citric acid depends upon the concurrent production of other organic acids, for instance isocitric acid, which is strongly dependent of the strain used. Although there are some works described in the literature, several factors still need to be completely understood and optimized in the production of citric acid using crude glycerol. To start with, an experimental design, based on Taguchi method was applied to optimize the culture conditions and to evaluate the effect of pH, carbon/nitrogen (C/N) ratio in the medium, oxygen mass transfer rate (OTR) and salts concentration on citric acid production from pure glycerol by two different Y. lipolytica strains (W29 (ATCC 20460) and CBS 2073). OTR and pH were the factors, which had more effect on citric acid production. Moreover, a significant interaction between the factors OTR and salts was observed. The optimal conditions were also validated with crude glycerol and the citric acid production was similar for both strains using this low cost substrate. Since, as shown by the Taguchi approach, a high OTR was crucial for citric acid production, it seemed appropriate to further study this matter. Therefore a model describing oxygen volumetric mass transfer coefficient ( kL a), in a lab-scale stirred tank bioreactor (STR), as a function of operating conditions (stirring and aeration rates) and cellular density in the citric acid bioprocess, was developed. An empirical correlation was established that fit well in a wide range of operating conditions. As a result, it was found that raising kL a from 7 h-1 to 55 h-1 the citric acid concentration increased. On the other hand, the increase of dissolved oxygen concentration (DO) up to 60 % using controlled DO, led to an increase of citric acid concentration, reaching identical concentration as obtained at kL a of 55 h-1. This work demonstrated that kL a is an adequate parameter for the optimization and scale-up of citric acid production from crude glycerol by Y. lipolytica W29. Taking into account that oxygen is a crucial parameter in citric acid production by Y. lipolytica W29 from crude glycerol, a pressurized and an airlift bioreactor, both reactors associated to high mass transfer efficiency, were used for batch cultures. Increasing air pressure from 1 bar to 2 bar led to an improvement of 40 % in citric acid concentration, whereas in the airlift bioreactor, with an increase from 1 vvm to 1.5 vvm of the aeration rate a 30 % enhancement was attained. Both bioreactor types can be used as alternative ways of improving OTR for citric acid production, leading to important operating costs savings due to less power input. The simultaneous production of isocitric acid is the major problem of using Y. lipolytica strains as citric acid producer. In order to isolate improved strains with reduced isocitric/citric acid ratio and/or enhanced citric acid production, Y. lipolytica W29 was treated with ultraviolet (UV)-irradiation and/or ethyl methane sulfonate (EMS). A 76% and 2.2- fold higher concentration yield of citric acid, was obtained with a mutant strain, Y. lipolytica UV/EMS-10, isolated after the combined treatment with UV-irradiation and EMS.
O crescimento na produção de biodiesel tem produzido um aumento da quantidade de glicerol bruto disponível, o principal subproduto da indústria do biodiesel. Vários processos biotecnológicos têm sido estudados como alternativas para poder escoar a quantidade de glicerol bruto disponível. O glicerol bruto pode ser utilizado como fonte de carbono na produção de ácido cítrico pela levedura Yarrowia lipolytica em condições de crescimento com quantidade limitante de azoto. No entanto, outras condições operacionais, o meio de cultura e a estirpe utilizada podem influenciar o perfil de produção de ácido cítrico. Para maximizar a produção de ácido cítrico é muito importante otimizar todos os factores que possam influenciar a sua produção e também entender como esses factores podem afectar o perfil de produção. Inicialmente, foi utilizado um desenho experimental, baseado no método de Taguchi, para otimizar as condições de cultura e avaliar o efeito dos fatores pH, razão carbono/azoto (C/N), taxa de transferência de oxigénio (OTR) e a concentração de sais, na produção de ácido cítrico por duas estirpes diferentes de Y. lipolytica (W29 (ATCC 20460) e CBS 2073) a partir de glicerol puro. Os fatores OTR e pH foram os que mais influenciaram a produção de ácido cítrico. No entanto observou-se um importante efeito de interação entre fatores, principalmente entre OTR e a concentração de sais. As condições ótimas foram também validadas usando glicerol bruto, sendo a produção de ácido cítrico semelhante para ambas as estirpes estudadas. Uma vez que, através do método Taguchi, ficou comprovada a importância crucial da oxigenação, foi decidido aprofundar esta matéria, tendo-se desenvolvido uma correlação empírica explicativa da relação entre o coeficiente volumétrico de transferência de massa de oxigénio ( kL a) num biorreactor de tanque agitado (STR), em função das condições de operação (agitação e taxa de arejamento) e da densidade celular. O aumento do kL a até 55 h-1 resultou num aumento na concentração de ácido cítrico. Por outro lado, utilizando oxigénio dissolvido (DO) constante, o aumento de DO até 60 % de saturação levou a um aumento da concentração de ácido cítrico, tendo-se obtido uma concentração semelhante à conseguida para o kL a de 55 h-1. Neste trabalho demonstra-se que o kL a é um fator adequado de otimização e a ter em conta no aumento de escala da produção de ácido cítrico. Considerando que o oxigénio dissolvido é um parâmetro crucial na produção de ácido cítrico pela Y. lipolytica W29 a partir de glicerol bruto foram utilizados dois bioreatores normalmente associados a elevada eficiência de OTR, um bioreator pressurizado e um airlift. Um aumento da pressão total de ar até 2 bar melhorou em 40 % a concentração e rendimento em ácido cítrico, enquanto que, no biorreator do tipo airlift, um aumento na taxa de arejamento até 1,5 vvm resultou numa melhoria de 30 % para ambos os parâmetros. Ou seja, ambos os biorreatores podem ser usados como alternativa para aumentar OTR na produção de ácido cítrico, levando a importantes poupanças nos custos de operação. A produção simultânea de ácido isocítrico é o principal problema quando se utiliza estirpes de Y. lipolytica na produção de ácido cítrico. Assim, numa tentativa de obter estirpes que apresentem um menor rácio ácido isocítrico/cítrico e/ou uma melhor produção de ácido cítrico, a estirpe Y. lipolytica W29 foi sujeita a uma exposição a irradiação ultra violeta (UV) e/ou um mutagénico químico, metano sulfonato de etilo (EMS). Após o tratamento que combinou a irradiação UV com a exposição a EMS, isolou-se a estirpe mutante Y. lipolytica UV/EMS-10, que apresentou um aumento de 76 % na concentração de ácido cítrico e um rendimento em ácido cítrico 2,2 vezes maior em comparação com a estirpe parental.
À instituição de acolhimento CEB- Centro de Engenharia Biológica da Universidade do Minho pelo acolhimento e por me ter proporcionado todas as condições para realizar este trabalho científico. Ao Projeto “BioInd – Biotechnology and Bioengineering for improved Industrial and Agro- Food processes", REF. NORTE-07-0124-FEDER- 000028, cofinanciado pelo Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER), ao Projecto RECI/BBBEBI/ 0179/2012 (FCOMP-01-0124-FEDER-027462), e à Fundação para a Ciência e Tecnologia pela atribuição da bolsa de doutoramento (SFRH/BD/72621/2010).

Sponges represent the most prolific producers of novel marine bioactive secondary metabolites. In the last years, several drugs derived from marine natural products have appeared in the market, and others are in clinical trials. The aim of my research project was to exploit the unusual and often surprising chemistry of marine sponges, in the frame of the more general purpose of discovering and developing new drugs from natural products. The research work presented in this PhD Thesis was directed to two different aspects of the study of marine secondary metabolites. On one hand, in parallel with the advent of environmental genomics from a drug discovery perspective, the largest part of my research activity focused on the metagenomic analysis of the Caribbean sponge P. simplex, and was aimed at the identification of new genes coding for polyketide synthases (PKSs), the giant enzyme complexes that produce polyketides, a large class of secondary metabolites that include many antibiotic and antitumor compounds. On the other hand, the remaining part of the research described in this PhD Thesis was more related to the “core activity” of natural product chemistry, and directed to the isolation and structure elucidation of new bioactive compounds from different specimens of sponges living in tropical oceans, wonderful sources of unusual molecular architectures to be used as leads and scaffolds for the elaboration of new drugs. Metagenomic investigations on Plakortis simplex (Demospongiae, Homosclerophorida, Plakinidae) was started because the sponge is known for the production of large amounts of polyketide peroxides, of which plakortin is the most abundant. Plakortin is of special interest due to its anti-malarial activity, which is retained also against chloroquine-resistant strains of Plasmodium falciparum. Therefore, a study of the biosynthesis of plakortin was undertaken, with the final aim of its biotechnological production. Most non-aromatic polyketides are synthesized by type I polyketide synthases (type I PKSs), produced in a number of cases by bacterial symbionts. The bacterial origin of plakortin is therefore a reasonable hypothesis, and indeed cell fractionation of P. simplex has shown that plakortin is mainly present in the bacterial cells. Since cultivation of true sponge symbionts failed in most cases, the search for the plakortin genes had to rely on cultivation-independent techniques, such as the study of the sponge metagenome (collective genome of the sponge and its symbionts). While the putative genes implied in plakortin biosynthesis could not be identified, an unexpected result from the metagenomic library screening was the discovery of Swf, a new group of mono-modular type I PKS/FAS (“hybrid polyketide synthase/fatty acid synthase”), which appears to be specifically associated to sponge symbionts. The putative swf operon consists of swfA (FAS/PKS I), swfB (R and ST domains), and swfC (radical SAM). SwfA contains a single PKS module, which builds the backbone of the acyl chain by recruiting iteratively malonyl units according to the substrate determining motif of its AT domain. The domain organization of SwfA is KS-AT-DH-ER-KR-ACP and from this architecture a saturated fatty acyl chain is expected, although a (poly)unsaturated and/or (poly)hydroxylated acyl chain cannot be excluded, because in iterative PKSs the reduction domains can be optionally used during each of the elongation steps. SwfB [composed of R (thioester reductase) and ST (sulfotransferase) domains] and SwfC (a radical SAM), are expected to modify the acyl chain produced by SwfA in unknown ways. As the R and ST domains are contiguous in SwfB, the expected product of elaboration of an acyl chain by SwfB would possibly be an alkyl sulphate or an alkylaminosulphonate: while the R domain can reductively release the assembled chain as a primary alcohol or amine, the ST domain can transfer the sulfate group to the hydroxyl or amino group. SwfC represents a radical SAM enzyme which can catalyze methylation of the substrate through a radical mechanism. Two different examples of the swf cluster were found in the metagenome of P. simplex, PS11G3 and PSA11D7 (PSA11D7 lacks the swfC gene). In addition, PCR amplification of metagenomic DNA from three different and taxonomically distant “high microbial abundance” sponges, Aplysina fulva, Smenospongia aurea and Pseudoceratina crassa, with primers designed for swf , produced amplicons which showed high sequence similarities to the AT domain of swfA. Therefore, the swf cluster is widespread in marine sponges and presumably associated to ubiquitous sponge symbionts. It represents the second group of mono-modular PKS, after the supA family, to be ubiquitously present in marine sponges. Preliminary studies of heterologous expression of swf genes were undertaken with the final aim of characterizing the unknown metabolite produced by the cluster. Activation of the ACP domain of the SwfA protein to its holo-form by co-expression with the phosphopantetheinyl transferase Svp was the first functional proof of swf type genes in marine sponges. Furthermore, applying homologous recombination for expression vector engineering, swfA was clearly expressed at the protein level in E.coli BL21-CodonPlus®(DE3)-RIPL cells by coexpression with the chaperone plasmid pTf16, which encodes for the molecular chaperone Trigger factor aiding the protein folding process. After cloning the whole swf operon into the expression vector pHIS8-Svp by homologous recombination, the new recombinant construct was used for heterologous expression trials of the whole cluster in E. coli BL21-(DE3) BAP1. Methanol extracts of transformants and their culture broths were analysed by LC-HR-ESI-MS, but no compounds which were present in all the transformants and absent in all the negative controls could be detected. In addition, fatty acid composition of transformants and their culture broths was characterized by saponification of the lipid extract and derivatization to fatty acid methyl esters (FAMEs) followed by GC/MS analysis. Even in this case, no new metabolite was detected, suggesting that the swf pathway is not functional in this expression system. As a consequence, the biosynthetic function of the swf cluster remains unknown at present. In parallel, metagenomic investigations conducted using high-throughput sequencing based on massively parallel 454 pyrosequencing led to a comprehensive overview of the polyketide metabolism of P.simplex and its symbionts, shedding light on the existence of novel polyketide synthase pathways potentially involved in bioactive compound biosynthesis. 454 pyrosequencing was performed on complex and heterogeneous PCR products amplified from the metagenomic DNA of P.simplex with degenerate probes targeting ketosynthase and acyltransferase domains of type I PKSs. Next generation sequencing of AT amplicon mixture generated 8995 reads; applying this modern approach, no PKS/FAS other than known SupA and SwfA could be found. Almost 51% of the total reads belonged to the Swf enzymes, while only the 4% was represented by AT belonging to SupA enzymes (the remaining reads appear not to be related to AT domains). On the other hand, 454 pyrosequencing of KS amplicon PCR mixture generated 19333 reads. Besides the expected huge presence of KS forming parts of SupA enzymes (~ 80% of the total reads), BLASTx analyses led to the detection of 8 new KS fragments, not reported in genbank database. All the eight putative KS fragments (which based on phylogenetic analysis appeared to be part of one hybrid NRPS-cis-AT PKS and seven cis-AT PKSs) are significantly different (E values ≥ 10-6) to each other, and BLASTx analysis as well as the rebuilt phylogenetic taxonomy revealed that they are only distantly related to PKSs of characterized function. In addition, phylogenetic analyses suggest that these KS fragments are mainly related to PKSs from Cyanobacteria, Actinomycetes and Myxobacteria, commonly known as precious sources of bioactive polyketides. These fragments may represent important starting points for further research towards the isolation of new PKS genes. The second line of research of my project was directed to the isolation and structure elucidation of new secondary metabolites from two tropical sponges, Chalinula molitba and Plakortis cf. lita. Stereo structure determination of the new compounds was determined by a combination of mass spectrometry, mono- and bidimensional NMR experiments and micro-scale chemical degradation. The analysis of the organic extract of the Caribbean sponge C. molitba led to the identification of chalinulasterol, a new C-24 chlorinated sterol disulfate. On the basis of the structural similarity with the PXR agonist solomonsterol A, the possible role of chalinulasterol as modulator of the pregnane-X receptor activity was evaluated by carrying out a transactivation assay (luciferase assay) on HepG2 cells, a human hepatocarcinoma cell line. Despite the structural similarity, chalinulasterol failed in transactivating PXR. The possibility that chalinulasterol could act as potential PXR antagonist was investigated, thus, also in this case, failed to reverse the induction of luciferase caused by rifaximin. Although negative, these results have an important implication in terms of structure-activity relationship, because they suggest that the sulfate group (absent in chalinulasterol) present in the side chain at position C-24 of solomonsterol A is essential in the ligand-receptor binding and receptor transactivation, confirming a proposed binding model where a clear interaction of the 24-sulfate with the positively charged Lys210 is hypothesized. Chemical analysis of the Indonesian sponge P. lita revealed, as first remarkable result, a secondary metabolite profile (glycolipids, hopanoids, polyketides) that, in spite of the geographical distance, was very similar to that of the Caribbean sponges of the genus Plakortis. Taking into account that secondary metabolites are often of bacterial origin, this indicates that bacterial communities associated to many species of Plakortis sponges are highly specific and consistently conserved in specimens collected in different times and geographical areas, suggesting vertical transmission within their hosts. In addition, among the many known compounds, plakohopanoid, a novel type of hopanoid, was isolated. Plakohopanoid is composed of a C32 hopanoid acyl ester-linked to a mannosyl-myo-inositol unit. It is interesting to note that C32 hopanoic derivatives are commonly considered as geohopanoids, i.e. diagenetic products formed through abiotic degradation of the hopanoids biosynthesized by bacteria (biohopanoids). As a consequence, the presence of plakohopanoid in a marine living organism is worthy of note, because it shows that there is a biosynthetic pathway to C32 hopanoic acids, which therefore should not be classified anymore as sure geohopanoids.

This thesis aims to respond to the urgent need for innovation in plant protection, by studying several potential bacterial targets against which innovative and eco-friendly molecules could be then developed. To this aim, new virulence determinant of Gram-negative phytopathogenic bacteria never studied before will be investigated, the Multi Drugs and Toxic Compounds Extrusion (MATE) pumps and its role in IAA homeostsis in Pseudomonas savastanoi pv. nerii. Moreover, the same approach will expand our research also to Gram-positive bacteria, which have been definitely less investigated until now. Phenotypic and genomic analyses were conducted on a pool of curtobacterium flaccumfaciens strains. The results have provided interesting informations about epidemiological development of curtobacterium flaccumfaciens pv. flaccumfaciens, and have detected hypotetical virulence determinats of this pathogen.

Enhancement of the catalytic performance of various enzymes in the reaction systems requires advanced studies of enzymes' structure-function relationships. The obtained information is used to engineer favourable enzyme variants exhibiting beneficial properties for applications in biotechnology. In the thesis X-ray crystallographic analysis was successfully employed for the structure-functional characterization of the glyceraldehyde dehydrogenase from thermophilic bacterium Thermoplasma acidophilum (TaAlDH). This enzyme is one of the key enzymes in the cell-free system for the production of ethanol or isobutanol from glucose. The second part of the thesis describes optimization by structure-guided engineering of the haloalkane dehalogenase LinB from the soil bacterium Sphingobium japonicum UT26, used for the effective degradation of halogenated environmental pollutants.

博士
國立中興大學
園藝學系所
103
The purposes of this research were to manipulating the in vitro cultivation of Erycina pussila by LEDs illumination, to develop the novel, new flower color/type, and early blooming of Erycina pusilla via transformation of MADS14 (AP1-like) and CHRC genes, and to create new cultivars of Erycina pusilla by EMS and sodium azide mutagenesis and hybridization. The results indicated that compared with the fluorescent light and other LED treatments, RB+W+RGB LEDs significantly increased root and leaf number and plant high of in vitro plantlets of Erycina pusilla. RB+W+RB LEDs significantly increased the values of Fo, Fv, and Fv/Fm of in vitro plantlets after 25 weeks of cultivation, whereas significantly increased values of chlorophyll fluorescent parameters was found in the fluorescent light treatment after 50 weeks of cultivation. These results suggested RB+W+RGB LEDs and RB+W+RB LEDs could promote the quality of in vitro cultivation of Erycina pusilla. Three transformation vectors, p1305.3-CHRC, p1304-CHRC-IN, and p1301-AP1-MADS14 were transferred into the PLBs of the Erycina pusilla via Agrobacterium-mediated gene transformation. The regenerated plantlets were confirmed by PCR, RT-PCR and. Results indicated that the MADS14 gene was present in the genome of transformed Erycina pusilla plants and expressed its mRNA. Changes in the leaf and plant morphology, flower shape and color, blooming time, and multiple flower stalks were founded in the MADS14 transformed Erycina pusilla plants. This is the first report on the ectopic expression of MADS box gene in Erycina pusilla using an efficient and sophisticated gene transformation technology. In general, EMS treatments were more effective in reducing survival percentages and numbers of plant regeneration as compared to those of NaN3 treatments. Changes in profiles of flow cytometric histograms were found in several regenerated plants of EMS and NaN3 treated Erycina pulsilla PLB. In this study, 10 cultivar of Oncidiinae (♀) were crossed with Erycina pusilla (♂), in order to obtain novel Erycina pusilla. Several females tribute Oncidiinae crossed relatively easily with male Erycina pusilla but the reciprocal cross is rare. Hybrid seeds could be produced in Oncsa. Sweet Sugar x Erycina pusilla cross, and germinated under asymbiotic conditions. Changes in plant mophology and profiles of flow cytometric histograms were detected in vegetative leave of F1 plants.

Dissertação de mestrado em Biotechnology
Daily, huge quantities of waste cooking oils (WCO) are produced worldwide. These residues rich in lipids can be used as substrate in several biotechnological processes, such as those based in Yarrowia lipolytica cultures. This yeast species has the special ability to degrade lipid-rich substrates while producing value-added metabolites, such as lipase and microbial lipids of interesting TAGs composition. The effective use of WCO by Y. lipolytica depends on the WCO concentration, the presence of surfactants and pH, among other parameters, thus its optimization is crucial for the development of an industrial process of WCO valorization. Initially, it was intended to study the effect of medium composition (pH, WCO concentration and arabic gum concentration) on lipase and microbial lipids production in experiments carried out in Erlenmeyer flasks using an experimental design based on Taguchi method. The initial pH value of the production medium was the most influential parameter on production of lipase and microbial lipids. The increase of pH value from 5.6 to 7.2 improved lipase production by Y. lipolytica, since lipase activity was 4-fold higher at pH value of 7.2. By contrast, highest microbial lipids accumulation was attained in the experiments performed at pH 5.6. The remaining parameters did not show any influence on lipase production; however, it was observed that gum arabic concentration was an important parameter for the production of microbial lipids by Y. lipolytica W29. The interaction between the parameters WCO concentration and arabic gum concentration was the interaction with most influence on both production of lipase and microbial lipids by yeast. Taguchi also established optimum conditions for the production of lipase and microbial lipids (pH 7.2, WCO 10 g·L-1 and pH 5.6, WCO 30 g·L-1 and arabic gum 5 g·L-1 for lipase and microbial lipids production, respectively). In both cases, the experimental results obtained in the optimum conditions were identical to the expected values. The influence of oxygen mass transfer on lipase and microbial lipids production by Y. lipolytica W29 was also studied. Increasing kLa from 9 h-1 to 93 h-1 improved cell growth as well as protease production. By contrast, values of kLa above 16 h-1 led to a decrease in lipase production. The accumulation of microbial lipids by yeast was also favored at lower kLa values. The fatty acids composition of microbial lipids accumulated by Y. lipolytica cells was mainly linoleic (≥60 %) and oleic (≥30 %) acids, demonstrating the potential of these lipids to be used as food supplements.
Diariamente, são produzidas grandes quantidades de óleos alimentares usados (OAU) em todo mundo. Estes resíduos, ricos em lípidos, podem ser utilizados como substrato em vários processos biotecnológicos e a levedura Yarrowia lipolytica é capaz de degradar compostos ricos em lípidos enquanto produz metabolitos de valor acrescentado, como lipase e lípidos microbianos. A concentração da fonte de carbono, a presença de surfactantes bem como o pH do meio são parâmetros que podem influenciar produção de lipase e lípidos microbianos por Y. lipolytica. A otimização destes parâmetros é essencial para uma máxima produção de lipase e lípidos microbianos. Inicialmente, foi aplicado um desenho experimental baseado no método de Taguchi e estudou-se o efeito dos parâmetros pH, concentração de WCO e de goma arábica na produção de lipase e lípidos microbianos por Y. lipolytica em experiências realizadas em frascos de Erlenmeyer. O valor de pH inicial do meio de produção foi o parâmetro com maior influência na produção de lipase e lípidos microbianos. O aumento do pH favoreceu a produção de lipase, uma vez que 4 vezes mais atividade lipolítica foi observada a pH 7.2. Pelo contrário, uma maior acumulação de lípidos foi conseguida nos meios de produção a pH 5.6. Os restantes parâmetros não demonstraram qualquer influência na produção de lipase; contudo, observou-se que a concentração de goma arábica foi um parâmetro importante para a produção de lípidos microbianos por Y. lipolytica W29. A interação entre os parâmetros concentração de OAU e concentração de goma arábica foi a interação com maior influência na produção de lipase e lípidos microbianos. Taguchi também estabeleceu as condições ótimas para a produção de lipase e lípidos microbianos (pH 7.2, 10 g·L-1 de OAU e pH 5.6, 30 g·L-1 de OAU e 5 g·L-1 de goma arábica para a produção de lipase e lípidos microbianos, respetivamente). Em ambos os casos, os resultados experimentais obtidos nas condições ótimas foram idênticos aos valores esperados. A influência da transferência de oxigénio na produção de lipase e lípidos microbianos em culturas descontínuas de Y. lipolytica W29 foi também estudada. O aumento do kLa de 9 h-1 até 93 h-1 favoreceu o crescimento celular bem como a produção de protease. Por outro lado, valores de kLa acima de 16 h-1 levaram a um decréscimo na produção de lipase. A acumulação de lípidos microbianos pela levedura foi também favorecida a valores kLa mais baixos. A composição, em ácidos gordos, dos lípidos microbianos acumulados pela levedura era, maioritariamente, ácido linoleico (≥60 %) e ácido oleico (≥30 %), demonstrando o potencial destes lípidos para serem utilizados como suplementos alimentares.

碩士
嶺東科技大學
財務金融研究所
99
For measuring operational efficiency, this research focuses on 32 biotech pharmaceutical and biotech medical treatment firms which listed in the stock exchange and the over-the-counter (OTC) market in Taiwan. Following the metafrontier approach methodology of Battese and Rao(2002), the biotech firms are divided into four subgroups: listed biotech pharmaceutical firms, OTC biotech pharmaceutical firms, listed biotech medical treatment firms and OTC biotech medical treatment firms for realizing difference of operation efficiency and technology between subgroups. For evaluating firms’ efficiency and decision factors, fixed asset, numbers of employees and R&D expenses shown in financial report are used as input variables; net income, intangible assets and market value are used as output variables, respectively. Empirical results indicate that the listed and OCT should focus on the scale of outputs to improve their efficiency. Consulting the TGR, the TGR of the listed biotech pharmaceutical firms is the best, and the TGR of the OCT biotech medical treatment firms is the worst.

Tetraselmis sp. CTP4 was selected from a bioprospection screening as a promising candidate for industrial cultivation and exploitation of different biotechnological applications. At lab-scale, several experiments revealed that this strain is highly robust to environmental conditions, has the ability to accumulate significant amounts of lipids under nitrogen depletion, displays also high growth and sedimentation rates. In collaboration with Allmicroalgae (Algafarm, Secil, Leiria, Portugal), experiments at industrial-scale in 100-m3 tubular photobioreactors showed that this strain is able to attain promising areal productivities, remaining a monoalgal culture throughout the whole trial. Thereafter, a low-cost pilot-scale harvesting system enabled the recovery of 97% of the total biomass by natural sedimentation, reducing the harvesting costs by 93%. Biochemical characterization of industrially produced biomass revealed a high content of proteins and dietary fibres as well as interesting levels of chlorophyll, carotenoids and vitamins, whereas microbial pathogens and contaminants analysed were absent from the biomass. Later, upon the development of a biorefinery platform, the wet biomass was extracted with ethanol and fractionated using a liquid-liquid triphasic system, leading to four different streams: non-polar (NP), colloidal (CP) and water (WP) phases as well as the residual biomass (RB) leftover of the ethanolic extraction. The CP was characterized as a source of high value molecules, while the NP, WP and RB were successfully upgraded into biodiesel, bioethanol and biogas, respectively. The RB was also tested as an ingredient for juvenile seabream, showing to be a promising substitute to replace soybean meal in aquafeeds. Overall, Tetraselmis sp. CTP4 was successfully produced at industrial scale, a low-cost harvesting system was established, and the produced biomass had a promising composition suitable for prospective nutritional applications. In addition, the biorefinery approach implemented led to the production of different streams that were effectively upgraded into different bioproducts, namely biofuels and aquaculture feed.
No âmbito de um esforço de bioprospeção de novas microalgas para desenvolvimento biotecnológico realizado pelo grupo MarBiotech (Centro de Ciências do Mar, Universidade do Algarve), a estirpe Tetraselmis sp. CTP4 foi selecionada para o cultivo de biomassa à escala industrial e exploração de diferentes aplicações biotecnológicas. À escala laboratorial, ensaios preliminares revelaram que esta estirpe apresenta elevada taxa de crescimento e robustez para tolerar diversas condições ambientais. Adicionalmente, esta estirpe tem a capacidade de acumular quantidades significativas de lípidos, que podem chegar a 33% do peso seco da biomassa produzida, quando as culturas são submetidas a limitação de azoto no meio de crescimento. Estes ensaios revelaram também que a Tetraselmis sp. CTP4 apresenta uma composição lipídica muito interessante, e que após a conversão da fração de lípidos em biodiesel, as propriedades do biocombustível produzido estão dentro dos parâmetros regulados pelas normativas europeia e americana. Dado o elevado potencial desta microalga, foi estabelecida uma colaboração com a Allmicroalgae (Algafarm, grupo Secil, Leiria, Portugal), a maior unidade de produção de microalgas em sistemas fechados da Europa, para avaliar o crescimento das culturas desta estirpe em condições exteriores, utilizando fotobiorreatores tubulares industriais com um volume de 100 m3. Os ensaios realizados no exterior mostraram que foram necessárias oito semanas para realizar o aumento de escala de uma placa de agar até aos reatores de produção industrial. Durante o aumento de escala, realizou-se a otimização da produção em sistemas tubulares à escala piloto. Esta otimização mostrou melhores produtividades de biomassa quando a velocidade de cultura se encontra entre 0,65 a 1,35 m/s e com o valor de pH de 8,0 para injeção de CO2 na cultura. À escala industrial, verificou-se uma adaptação imediata das culturas aos sistemas de produção tubulares com produtividades areais muito promissoras (10-20 g/m2/d) e elevada eficiência fotossintética (3,5% da irradiância solar total), sendo possível manter uma cultura monoalgal durante todo o período de produção (60 dias). A sequestração de CO2 foi seguida no fotobiorreator de 100 m3, revelando uma eficiência média de mitigação de CO2 de 65% e uma relação de biomassa/carbono de 1,8. Posteriormente, foi desenvolvido um sistema de colheita piloto de baixo custo da biomassa de Tetraselmis sp. CTP4, nas instalações da Algafarm. Para este fim, adaptou-se um tanque cilindro-cónico para recolher a biomassa através da sedimentação natural das células sem custos energéticos. Os ensaios mostraram que após se introduzir a cultura no tanque, as células conseguem sedimentar na parte inferior do tanque após 24 horas, sendo possível recolher o meio de cultura pelas entradas laterais do sistema. Através do processo desenvolvido é possível recuperar 97% da biomassa total por sedimentação natural, sendo que se perdem apenas 3% com a remoção do meio de cultura do sistema. Usando esta abordagem, 93% do volume total da cultura é recuperado do tanque, com um peso seco de 0,07 g/L, o que representa uma redução muito significativa dos custos associados à colheita da biomassa. A restante cultura (7%) é recuperada na forma de uma cultura concentrada e pasta húmida de microalgas com pesos secos aproximados de 20 e 273 g/L, respetivamente. Na fase seguinte, pretendeu-se avaliar o potencial nutricional da biomassa de Tetraselmis sp. CTP4 produzida em fotobiorreatores industriais (100 m3). Para este fim, realizou-se uma avaliação minuciosa da composição bioquímica, microbiológica e toxicológica da biomassa. As análises efetuadas mostraram que a biomassa contém elevadas quantidades de proteína (31,2 g/100 g), fibras alimentares (24,6 g/100 g), glícidos digestíveis (18,1 g/100 g) e cinzas (15,2 g/100 g), mas com baixo conteúdo lipídico (7,04 g/100 g). A biomassa apresentou ainda níveis interessantes de clorofila (3,5 g/100 g), carotenóides (0,61 g/100 g) e vitaminas (por exemplo, 79,2 mg de ácido ascórbico/100 g) e atividade antioxidante. Por outro lado, bactérias patogénicas, metais pesados, cianotoxinas, micotoxinas, hidrocarbonetos aromáticos policíclicos e pesticidas não foram detetados na biomassa produzida. De um modo geral, a biomassa produzida tem uma composição promissora para aplicações nutricionais em humanos e animais. Subsequentemente, pretendeu-se desenvolver um novo método de processamento da biomassa tendo em conta o conceito de biorrefinaria, de modo a rentabilizar ao máximo todos os componentes bioquímicos presentes na biomassa de Tetraselmis sp. CTP4, para produzir diferentes bioprodutos. Neste contexto, realizou-se uma extração com etanol diretamente da biomassa húmida e o extrato resultante foi fracionado usando um sistema trifásico líquido-líquido (LTPS). No final deste processo, a partir da biomassa, obtiveram-se 4 frações distintas, nomeadamente as frações não polar (NP), coloidal (CP) e aquosa (WP), obtidas a partir do extrato etanólico, e a biomassa residual (RB) remanescente da extração etanólica. A fração CP foi caracterizada como fonte de moléculas de valor acrescentado, devido à presença de fosfolípidos e carotenóides, que apresentam uma elevada aplicabilidade para diferentes indústrias. As frações NP, WP e RB foram convertidas com sucesso em diferentes biocombustíveis, nomeadamente, biodiesel, bioetanol e biogás, respetivamente. No final desta dissertação, foi ainda realizado um ensaio em colaboração com a Sparos Lda. para testar a biomassa residual como um ingrediente para rações de aquacultura. Neste contexto, foi realizado um ensaio para avaliar o efeito de uma incorporação de 10% de biomassa residual de Tetraselmis sp. CTP4, em substituição da farinha de soja em juvenis de dourada (Sparus aurata). O ensaio foi realizado durante 61 dias e mostrou que os critérios gerais de desempenho (peso corporal final, índice de crescimento diário, taxa de conversão alimentar e taxa de eficiência proteica), composição corporal total e retenção de nutrientes não foram significativamente afetados pela introdução da biomassa residual. No entanto, a dieta com biomassa residual apresentou valores significativamente superiores nos coeficientes de digestibilidade aparente (ADC) de proteína, energia e fósforo, comparativamente à dieta com farinha de soja. No final, um teste de confinamento agudo mostrou uma resposta de cortisol significativamente menor nos peixes alimentados com a dieta com biomassa residual (120 ± 23 ng/mL) do que naqueles alimentados com a dieta com farinha de soja (160 ± 33 ng/mL). Os resultados gerais mostraram que a biomassa residual de Tetraselmis sp. CTP4 pode reduzir as elevadas necessidades de farinha de soja em alimentos para a aquacultura como atualmente se verifica. Em conclusão, a estirpe Tetraselmis sp. CTP4, foi produzida com sucesso à escala industrial, o sistema de recolha de biomassa de baixo custo foi efetivamente estabelecido e a biomassa produzida revelou um alto potencial para aplicações nutricionais. Além disso, a metodologia desenvolvida para o processamento da biomassa tendo em conta o conceito de biorrefinaria, permitiu a produção de diferentes frações que foram posteriormente convertidas em diferentes bioprodutos, nomeadamente biocombustíveis e rações para aquacultura.
The author acknowledges the Portuguese Foundation for Science and Technology (FCT) for funding the PhD fellowship (Grant SFRH/BD/105541/2014)