Els adenovirus oncolítics són uns nous agents antitumorals molt prometedors gràcies a la seva capacitat d'autoamplificar-se selectivament a la massa tumoral. Els assajos clínics realitzats fins a la data amb una primera generació d'aquests agents han indicat la necessitat d'una millora substancial de la capacitat antitumoral, sense descuidar però els possibles efectes adversos de la replicació als teixits normals. Una de les limitacions més importants d'aquesta teràpia és la incapacitat del virus per travessar les barreres estructurals que imposen les cèl·lules de l'estroma i la matriu extracel·lular del tumor. L'expressió de transgens que destrueixin aquestes barreres és una estratègia que s'ha mostrat molt prometedora per superar aquesta limitació i augmentar així l'activitat antitumoral. Per aconseguir aquest propòsit, cal optimitzar la maquinària d'expressió i el genoma de l'adenovirus oncolític on els transgens seran inserits per compatibilitzar l'expressió del transgen amb el cicle viral de l'adenovirus, i mantenir la mida del genoma dintre del límit que imposa la capacitat d'encapsidació dels adenovirus. En aquest treball, s'explora l'ús de caixes palindròmiques d'unió del factor de transcripció E2F per augmentar la selectivitat i la potència d'adenovirus oncolítics. En un primer virus construït, aquestes caixes van aconseguir augmentar significativament l'activitat antitumoral, tant in vitro com in vivo, però la combinació d'elements genètics presents en aquest virus augmentava molt la mida del genoma i no va permetre la introducció d'un transgen per superar les barreres presents als tumors. En un segon virus, l'optimització dels elements genètics va permetre aconseguir un virus que, gràcies a les caixes d'unió d'E2F, presentava un baix perfil de toxicitat i una gran potència antitumoral, fins i tot superior a la dels adenovirus salvatges. A més, la mida tan reduïda del seu genoma permet una expressió eficient i potent de transgens. Addicionalment, en aquest treball també s'analitzen els possibles beneficis de selectivitat i potència que poden tenir dos modificacions genètiques, la mutació T1 i la fibra RGDK, descrites amb anterioritat pel nostre grup. Tot plegat, aquest treball analitza la millor combinació possible d'elements genètics per aconseguir una bona base que permeti la construcció d'un futur adenovirus potent, selectiu i amb capacitat per destruir les barreres estromals dels tumors, superant així les limitacions més importants de la teràpia adenoviral del càncer i presentant-se com un ferm candidat a ser testat en futurs assajos clínics. / Conditionally Replicating Adenoviruses (CRADs) are novel antitumor agents due to their ability to self-amplify at the tumour site. Clinical trials performed with a first generation of these agents pointed out a critical need of improved antitumor activity, without overlooking concerning adverse effects of replication in normal tissues. However, one of the main limitations of this therapy is the inability of CRADs to overcome the barriers imposed by tumour stroma, including stromal cells and extracellular matrix. Transgene expression is valuable strategy to counteract these limitations and to enhance antitumor activity. For this purpose, the genetic backbone in which the transgene is inserted should be optimized to render transgene expression compatible with the adenovirus replication cycle and to keep genome size within the encapsidation size limit. In this work, we explore the use of palindromic E2F-binding sites to enhance the selectivity and the potency of CRADs. In a first virus, the insertion of these sites in an E2F-1 promoter controlling the expression of the E1A protein resulted in a significant increment in the oncolytic activity, both in vitro and in vivo. However, the unique combination of genetic elements present in this virus hindered the incorporation of transgenes to overcome stromal barriers. In a second virus, the insertion of E2F-binding sites into the endogenous E1A promoter allowed to solve this limitation. The insertion of these sites results in a low systemic toxicity profile in mice. Importantly, the E2F-binding sites also increased the cytotoxicity and the systemic antitumor activity relative to wild-type adenovirus in all cancer models tested. Furthermore, the constrained genome size of this backbone allows an efficient and potent expression of transgenes. In addition to this, in this work we also test the benefits in selectivity and potency of two previous mutations reported by our group, the T1 mutation and the RGDK fibre. All together, this thesis analyzes the best possible combination of genetic elements to achieve an adenoviral backbone that permits the design of a future CRAD, potent, selective and capable of destroy tumour stromal barriers, overcoming the main limitations of cancer virotherapy and constructing a firm candidate to be tested in future clinical trials.

Made available in DSpace on 2014-12-17T15:48:06Z (GMT). No. of bitstreams: 1 RicardoMCSJ.pdf: 1145445 bytes, checksum: 72e5375ae05e7cf3104e5c18ae0c8314 (MD5) Previous issue date: 2006-02-20 / Este trabalho aborda o problema de otimiza??o em braquiterapia de alta taxa de dose no tratamento de pacientes com c?ncer, com vistas ? defini??o do conjunto de tempos de parada. A t?cnica de solu??o adotada foi a Transgen?tica Computacional apoiada pelo m?todo L-BFGS. O algoritmo desenvolvido foi empregado para gerar solu??es n?o denominadas cujas distribui??es de dose fossem capazes de eiminar o c?ncer e ao mesmo tempo preservar as regi?es normais

Regulatorische CD4+CD25+ T-Zellen (Tregs) stellen eine kleine Zellpopulation dar (1-5% der peripheren Blutzellen), die hauptsächlich für die Regulierung von Immunreaktionen verantwortlich ist. In der vorliegenden Arbeit wurden diese Zellen gemeinsam mit Stammzellen syngen und allogen kotransplantiert, um ihren Effekt auf das Anwachsen der Spenderzellen und die Rekonstitution der Hämatopoese nach Ganzkörperbestrahlung zu untersuchen. Es wurden humanisierte dreifach transgene Empfängermäuse (C57Bl/6-TTG) verwendet (human CD4+, murin CD4-, human HLA-DR+), wodurch sowohl bei syngener als auch bei allogener Transplantation eine Unterscheidung zwischen Spender- und Empfängerzellen möglich ist. Zunächst wurden CD4+CD25+ T-Zellen durch Separation aus Milzzellen bzw. Buffy Coats gewonnen und in vitro mittels Durchflusszytometrie und ELISpot charakterisiert. Anschließend fanden syngene und allogene Transplantationen mit einer Laufzeit von 61 Tagen statt. Überleben und Gewicht wurden täglich ermittelt und außerdem wurden wöchentlich Blutbilder erstellt und durchflusszytometrische Chimärismusanalysen (murines und humanes CD4, CD8, MHC (H2Db, H2Kd)) durchgeführt. Durch die magnetische Separation konnte die FoxP3-Expression der murinen Zellen (Transplantat) von 1,6% in der Ausgangspopulation auf 68,5% in der CD4+CD25+ Population gesteigert werden. In den ELISpot-Assays zeigten diese separierten Zellen, wie für Tregs typisch, keine Produktion von Interleukin-2. Nach syngener Transplantation (Spender: wildtyp C57Bl/6) von 2x106 Knochenmarkzellen und 1x106 CD4+CD25+ T-Zellen überlebten 100% der Tiere, wie zu erwarten war. Dabei setzte bei Tregs-kotransplantierten Tieren die Blutbildung nach bestrahlungsbedingter Leukozytopenie aufgrund bisher nicht bekannter Mechanismen früher wieder ein und der Donor-Zell-Chimärismus war an Tag 19 nach Transplantation signifikant höher als in der Kontrollgruppe. Dies zeigt, dass regulatorische T-Zellen im syngenen Transplantationsmodell einen positiven Effekt auf die Akzeptanz bzw. das Anwachsen des Transplantats haben. Dieses Modell entspricht klinisch einer autologen Transplantation. Nach einer knochenmarkzerstörenden Therapie werden dem Patienten eigene Stammzellen reinfundiert, um die Blutbildung und das Immunsystem wieder in Gang zu bringen. Der Zusatz von regulatorischen T-Zellen zum autologen Stammzelltransplantat könnte das Anwachsen der Zellen beschleunigen und die gefährliche Phase der Immunsuppression, in der es häufig zu Sekundärinfektionen kommt, verkürzen. Die Transplantation der gleichen Zahl von allogenen Spenderzellen (wildtyp Balb/c) führte überraschend zum Tod aller dreifach transgenen Empfängertiere. Der Vergleich zu Experimenten mit wildtyp C57Bl/6-Empfängertieren zeigte, dass dreifach transgene Mäuse sehr viel höhere Zellzahlen im Transplantat zum Überleben benötigen (Daten nicht gezeigt). Das Ausbleiben der Blutbildung nach der Bestrahlung führte zu vermindertem Allgemeinbefinden, gestörter Futter- und Wassseraufnahme und Exsikkose bis zum Tod bzw. aus Tierschutzgründen zur Euthanasie. Durch Erhöhung der Zellzahl im Transplantat auf 1x107 Knochenmark + 5x106 Milzzellen überlebten 25% der Mäuse, bei 3x107 Knochenmark + 5x106 Milzzellen waren es 50%. Anders als im syngenen Modell führte die Kotransplantation 1,5x106 allogener CD4+CD25+ T-Zellen zu 3x107 Knochenmark + 5x106 Milzzellen zum Versterben der Tiere. Dies verdeutlicht, dass regulatorische T-Zellen in diesem allogenen Transplantationsmodell das Anwachsen des Transplantats behindern (Transplantatversagen). Hier gilt es zu klären, ob dieser Effekt spezifisch für die gewählten Mausstämme ist und welche Mechanismen für das Transplantatversagen verantwortlich sind. In einem dreifach transgenen Mausmodell konnte ein positiver Effekt von regulatorischen T-Zellen auf die Rekonstitution der Hämatopoese bei syngener Kotransplantation nachgewiesen werden. Im allogenen Transplantationsmodell hingegen führte die Kotransplantation CD4+CD25+ T-Zellen zum Versterben der Empfänger. Der beschriebene und schon publizierte positive Effekt spenderspezifischer Tregs zur Behandlung von Graft versus Host Disease nach allogener Stammzelltransplantation widerspricht diesen Ergebnissen nicht, da es bei diesen Patienten schon zum Engraftment von hämatopoetischen Stammzellen gekommen ist. Dies hat weitreichende Konsequenzen für die therapeutische Anwendung regulatorischer T-Zellen bei hämatologischen Erkrankungen in der Human- und Veterinärmedizin. / Regulatory CD4+CD25+ T cells (Tregs) represent a small cell population (1-5% of peripheral blood cells) mainly responsible for the regulation of the immune system. In the present work, these cells were cotransplanted with syngeneic and allogeneic stem cells in order to analyze the effect of Tregs on the reconstitution of hematopoiesis after total body irradiation. Humanized triple transgenic hosts (C57Bl/6-TTG) (human CD4+, murine CD4-, human HLA-DR+) were applied allowing differentiation of donor and host cells in syngeneic and allogeneic transplantation settings. Murine and human CD4+CD25+ T cells were magnetically separated out of splenocytes or buffy-coats and characterized in vitro by means of flow cytometry and ELISpot. Afterwards syngeneic and allogeneic transplantation experiments were performed for a period of 61 days. Survival and weight were assessed daily and once a week blood parameters and chimerism analyses (murine and human CD4, CD8, MHC (H2Db/ H2Kd)) were carried out. FoxP3 expression increased from 1,6% in the initial murine cell fraction to 68,5% in the separated CD4+CD25+ T cells. ELISpot assays showed the typical lack of interleukin 2 production of Tregs. After syngeneic transplantation (donor: wildtype C57Bl/6) of 2x106 bone marrow cells and 1x106 CD4+CD25+ T cells, 100% of mice survived what was to be expected. Cotransplanted animals showed earlier reconstitution of hematopoiesis after leukocytopenia and significant higher donor-cell-chimerism on day 19 after transplantation. The mechanisms for this positive effect of Tregs in syngeneic transplantation on the engraftment have to be investigated. This model clinically correspond an autologous transplantation where patients are treated with their own stem cells after a myeloablative treatment (chemotherapy or irradiation). The addition of regulatory T cells to the transplant could accelerate the engraftment and shorten the risky period of immunosuppression. Injection of the same numbers of allogeneic cells (donor: wildtype Balb/c) did not preserve hosts from mortality. Compared to experiments with wildtype recipients, results showed that triple transgenic mice need much higher cell numbers in the transplant for survival (data not shown). The failure of hematopoiesis after irradiation led to reduced general condition, disordered ingestion and exsikkosis leading to death respectively to euthanasia for reasons of protection of animals. By scaling up the cell number in the inoculum to 1x107 bone marrow cells + 5x106 splenocytes 25% of mice survived, with 3x107 bone marrow cells + 5x106 splenocytes survival was 50%. In contrast to syngeneic experiments, cotransplantation of 1,5x106 allogeneic CD4+CD25+ T cells and 3x107 bone marrow cells + 5x106 splenocytes did not prevent animals from mortality. In this allogeneic transplantation model Tregs restrain engraftment (graft failure). It has to be clarified if this effect is specific for the utilized mouse strains and which mechanisms are responsible for the graft failure. In the syngeneic triple transgenic mouse model cotransplantation of CD4+CD25+ T cells showed a positive effect on reconstitution of hematopoiesis after irradiation. In the allogeneic setting however cotransplantation of allogeneic regulatory T cells avoided the engraftment of transplanted cells. The described and published effect of donor-specific Tregs for treatment of graft versus host disease after allogeneic transplantation does not contradict the presented results because treated patients already possessed engrafted hematopoietic stem cells. The results have wide consequences for the therapeutic appliance of regulatory T cells in hematological diseases in human and veterinary medicine.

Made available in DSpace on 2014-12-17T15:48:11Z (GMT). No. of bitstreams: 1 LigiaBB.pdf: 1036516 bytes, checksum: 36260a287f3ddf0bc38abbb0ec32b82f (MD5) Previous issue date: 2007-02-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Traveling Purchaser Problem is a variant of the Traveling Salesman Problem, where there is a set of markets and a set of products. Each product is available on a subset of markets and its unit cost depends on the market where it is available. The objective is to buy all the products, departing and returning to a domicile, at the least possible cost defined as the summation of the weights of the edges in the tour and the cost paid to acquire the products. A Transgenetic Algorithm, an evolutionary algorithm with basis on endosymbiosis, is applied to the Capacited and Uncapacited versions of this problem. Evolution in Transgenetic Algorithms is simulated with the interaction and information sharing between populations of individuals from distinct species. The computational results show that this is a very effective approach for the TPP regarding solution quality and runtime. Seventeen and nine new best results are presented for instances of the capacited and uncapacited versions, respectively / O Problema do Caixeiro Comprador ? uma variante do Problema do Caixeiro Viajante, onde existe um conjunto de mercados e um conjunto de produtos. Cada produto est? dispon?vel em um subconjunto de mercados e o pre?o da unidade varia de acordo com o mercado. O objetivo ? comprar todos os produtos, partindo e retornando para o dep?sito, de maneira que a soma do custo da rota e dos produtos seja m?nimo. Um Algoritmo Transgen?tico, algoritmo evolucion?rio com base na endosimbiose, ? utilizado para resolver a vers?o Capacitada e N?o Capacitada desse problema. A evolu??o no algoritmo transgen?tico ? simulada com a intera??o e troca de informa??es entre popula??o de indiv?duos de diferentes esp?cies. Os resultados computacionais mostram que a abordagem ? satisfat?ria para o PCC , tanto na qualidade da solu??o, quanto no tempo de execu??o. Dezessete e nove novas melhores solu??es s?o encontradas para o PCC Capacitado e para o PCC N?o Capacitado, respectivamente

Made available in DSpace on 2014-12-17T15:48:12Z (GMT). No. of bitstreams: 1 CristineCS.pdf: 714473 bytes, checksum: 61f78bdfcd48bd6e64e25178e846e81b (MD5) Previous issue date: 2007-02-08 / Este trabalho apresenta um algoritmo transgen?tico h?brido para a solu??o de um Problema de Configura??o de uma Rede de Distribui??o de G?s Natural. O problema da configura??o dessas redes requer a defini??o de um tra?ado por onde os dutos devem ser colocados para atender aos clientes. ? estudada neste trabalho uma maneira de conectar os clientes em uma rede com arquitetura em forma de ?rvore. O objetivo ? minimizar o custo de constru??o da rede, mesmo que para isso alguns clientes que n?o proporcionam lucros deixem de ser atendidos. Esse problema pode ser formulado computacionalmente atrav?s do Problema de Steiner com Pr?mios. Este ? um problema de otimiza??o combinat?ria da classe dos NP?rduos. Este trabalho apresenta um algoritmo heur?stico para a solu??o do problema. A abordagem utilizada ? chamada de Algoritmos Transgen?ticos, que se enquadram na categoria dos algoritmos evolucion?rios. Para a gera??o de solu??es inicias ? utilizado um algoritmo primaldual, e pathrelinking ? usado como intensificador

The production of haploid sperm is a precondition for sexual reproduction of males. PRDM9 protein is a histone methyltransferase which localizes sites of meiotic recombination in many mammals. Mouse males of the C57BL/6J (B6) strain deficient for Prdm9 (Prdm9-/- ) are sterile, while Prdm9-/- males of PWD/Ph (PWD) strain have reduced sperm count. The comparison of the distribution of trimethylation of histone 3 on lysine 36 (H3K36me3) in genome of Prdm9-/- males of these two strains will help to determine the role of this epigenetic modification on meiotic recombination and fertility of Prdm9-/- males. The second part of this thesis is focused on transgenic males. Male offspring from the first generation of B6 female and PWD male crosses (B6PF1) have reduced fertility parameters due to incompatibility of Prdm9 alleles. The fertility parameters of B6PF1 hybrids carrying CHORI-34-289M8 or RP24-346I22 transgene are even lower. The candidate gene, which participates in the reduction of fertility of the transgenic B6PF1 hybrids, was determined as the proteasome subunit encoding gene Psmb1, because its relative transcription level best correlates with sperm count. The reason of lowered fertility thus might be a defect in proteasome assembly. The investigation of the fitness of transgenic animals is...

Wood is a complex material and the mechanical properties are influencedby a number of structural parameters. The objective of this study has been toinvestigate the relationship between the structure and the mechanical propertiesof hardwood. Two levels were of special interest, viz. the cellular structureand morphology of the wood, and the ultra-structure of the cell wall. In thenext step, it was of interest to examine how the mechanical properties ofhardwood change with spontaneous/induced changes in morphology and/orchemical composition beyond the natural variation found in nature. Together, this constituted the framework and basis for two larger projects,one on European aspen (Populus tremula) and hybrid aspen (Populus tremulax Populus tremuloides), and one on European oak (Quercus robur). Amethodology was developed where the concept of relative density and compositemechanics rules served as two useful tools to assess the properties ofthe cell wall. Tensile testing in the longitudinal direction was combined withchemical examination of the material. This approach made it possible to revealthe mechanical role of the lignin in the cell wall of transgenic aspen trees,and investigate the consequences of holocellulose degradation in archaeologicaloak from the Vasa ship. The study on transgenic aspen showed that a major reduction in lignin inPopulus leads to a small but significant reduction in the longitudinal stiffness.The longitudinal tensile strength was not reduced. The results are explainableby the fact that the load-bearing cellulose in the transgenic aspen retained itscrystallinity, aggregate size, microfibril angle, and absolute content per unitvolume. The results can contribute to the ongoing task of investigating andpinpointing the precise function of lignin in the cell wall of trees. The mechanical property study on Vasa oak showed that the longitudinaltensile strength is severely reduced in several regions of the ship, andthat the reduction correlates with reduced average molecular weight of theholocellulose. This could not have been foreseen without a thorough mechanicaland chemical investigation, since the Vasa wood (with exception fromthe bacterially degraded surface regions) is morphologically intact and witha micro-structure comparable to that of recent oak. The results can be usedin the ongoing task of mapping the condition of the Vasa wood. / QC 20110420

Zusammenfassung Thure Adler Der Streptozotozin-induzierte Diabetes in der transgenen CD4/DR17-Maus Aus dem Institut für Immunologie der Veterinärmedizinischen Fakultät und dem Institut für Klinische Immunologie und Transfusionsmedizin der Medizinischen Fakultät der Universität Leipzig, 80 Seiten, 24 Abbildungen, 20 Tabellen, 217 Literaturangaben Die Verwendung transgener Tiere, die humane Moleküle exprimieren, gewinnt zunehmend an Bedeutung bei der Erforschung der Funktionen solcher Moleküle in Krankheitsprozessen und bei der experimentellen Erprobung neuartiger Therapieverfahren, in denen solche Moleküle die Zielstrukturen darstellen. In der vorliegenden Arbeit wurde die CD4/DR17-Maus, welche das humane CD4- und das DR17-Molekül exprimiert, im MLD-STZ-induzierten Diabetes, einem Tiermodell für den Typ 1 Diabetes, eingesetzt. Die funktionelle Beteiligung der Transgene wurde durch einen Vergleich mit Segreganten untersucht, denen die Transgene teilweise fehlen. Als klinische Parameter sind Blutglukose und Glukosetoleranz erfaßt worden, histopathologisch wurden Insulitis und Insulingehalt der Inselzellen bestimmt. Ferner wurde getestet, ob sich durch Verabreichung von monoklonalen Antikörpern, die gegen das transgene hCD4- oder gegen das CD8-Molekül gerichtet sind, dieser STZ-induzierte Diabetes beeinflussen läßt. Mit Hilfe der durchflußzytometrischen Immunfluoreszenzanalyse von Blutzellen wurde zusätzlich überprüft, ob Veränderungen auf T-Zellen hinsichtlich der Expression der Aktivierungsmarker CD25, CD69 und CD71 während des STZ-induzierten Diabetes auftreten. Es wurde gezeigt, dass die CD4/DR17-transgene Maus nach der Behandlung mit mehrfachen subdiabetogenen Dosen von Streptozotozin eine transiente Hyperglykämie entwickelt, die mit einer verringerten Glukosetoleranz sowie Insulitiden und einem Rückgang des Insulingehaltes in den Langerhans’schen Inseln einhergeht. Vergleiche mit Segreganten zeigen, dass die Expression beider transgener Merkmale zur maximalen Ausprägung einer schwergradigen Insulitis beiträgt. Die Anwendung von monoklonalen Antikörpern gegen das transgene hCD4-Molekül nach Beginn der STZ-Behandlung hat den Diabetes nicht wirkungsvoll verzögert. Dagegen milderte eine Behandlung mit Antikörpern, die gegen das CD8-Molekül gerichtet sind, den Diabetesverlauf. Während des STZ-Diabetes veränderte sich die Expression von Aktivierungsmarkern auf Lymphozyten des peripheren Blutes nicht signifikant. Die Arbeit belegt, dass die CD4/DR17-Maus suszeptibel gegenüber der Induktion eines experimentellen Diabetes mit mehrfachen subdiabetogenen Dosen von Streptozotozin ist. Die transgenen Moleküle hCD4 und DR17 sind dabei am Krankheitsprozeß beteiligt. / Summary Thure Adler The streptozotocin-induced diabetes in the transgenic CD4/DR17 mouse From the Institute of Immunology, Faculty of Veterinary Medicine and the Institute of Clinical Immunology and Transfusion Medicine, Faculty of Medicine, University of Leipzig 80 pages, 24 figures, 20 tables, 217 references Today, transgenic animals that express human molecules are getting important tools in functional studies and experimental, therapeutical attempts, that target these molecules. In this study, the CD4/DR17 mouse expressing the human CD4 and the human DR17 molecules and with a defective murine CD4 gene, was used in the multiple low-dose streptozotocin-induced (MLD-STZ) diabetes model, a model for type 1 diabetes. The functional involvement of the transgenic molecules in the development of the MLD-STZ-diabetes was analysed by comparing CD4/DR17 mice and segregants that lack one or more of the transgenes. The described parameters included the measurement of blood glucose levels and oral glucose tolerance tests, histopathologically grading of insulitis and determination of the content of insulin in pancreatic islets by immunohistological methods. In addition, the model was used to test the potential therapeutic effect of the administration of monoclonal antibodies against hCD4 or CD8. Furthermore, alterations of the expression of the activation markers CD25, CD69 and CD71 during the experimentally induced diabetes has been measured by FACS analysis. The study shows, that CD4/DR17 mice develop a transient hyperglycemia after MLD-STZ treatment, accompanied by a reduced tolerance to oral glucose, insulitis and the reduction of the content of insulin in the pancreatic islets. The full incidence of insulitis requires the expression of both transgenes. The treatment performed with monoclonal antibodies against the transgenic hCD4 after STZ-treatment could not meliorate the diabetic course, while the treatment with anti CD8 antibodies moderated the diabetic process. After STZ-treatment the expression of activation marker of peripheral T-cells did not alter significantly. Thus, the CD4/DR17 mouse is shown to be susceptible to the induction of experimental diabetes with MLD-STZ. The transgenic molecules CD4 and DR17 are involved in the pathogenesis of the disease.

The aim of this study was to analyze number of incorporated T-DNA harbouring pmi and nptII transgenes and expression of the pmi gene in tomato transformants using Southern blot and Northern blot methods. The presence of a functional PMI protein was assesed using PMI-assay with Chlorophenol red dye. Mannose selection of tomato seeds was established and segregation patterns in T1 progeny were studied.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-08T22:40:13Z No. of bitstreams: 1 ThatianaCunhaNavarroDeSouza_TESE.pdf: 4253732 bytes, checksum: b88b33669e4903291d2e3da03d76f832 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-11T22:01:06Z (GMT) No. of bitstreams: 1 ThatianaCunhaNavarroDeSouza_TESE.pdf: 4253732 bytes, checksum: b88b33669e4903291d2e3da03d76f832 (MD5) / Made available in DSpace on 2016-04-11T22:01:06Z (GMT). No. of bitstreams: 1 ThatianaCunhaNavarroDeSouza_TESE.pdf: 4253732 bytes, checksum: b88b33669e4903291d2e3da03d76f832 (MD5) Previous issue date: 2015-03-13 / Um importante problema enfrentado pela ind?stria petrol?fera ? distribuir v?rios produtos derivados de petr?leo atrav?s de polidutos. Tal distribui??o ? feita atrav?s de uma rede composta por refinarias (n?s fonte), parques de armazenagem (n?s intermedi?rios) e terminais (n?s de demanda), interligados por um conjunto de polidutos que transportam petr?leo e derivados entre ?reas adjacentes. Restri??es relativas a limites de armazenamento, tempo de entrega, disponibilidade das fontes, limites de envio e recebimento, entre outras, t?m de ser satisfeitas. Alguns pesquisadores lidam com este problema sob o ponto de vista discreto onde o fluxo na rede ? visto como o envio de bateladas. Geralmente, n?o existem dispositivos de separa??o entre bateladas de produtos diferentes e as perdas devidas ? interface podem ser significativas. Minimizar o tempo de entrega ? um objetivo usual dos engenheiros durante a programa??o do envio de produtos em redes de polidutos. No entanto, os custos devidos ?s perdas geradas nas interfaces n?o podem ser desconsiderados. O custo do envio dos produtos tamb?m depende das despesas de bombeamento as quais s?o, em grande parte, devidas ao custo da energia el?trica. Uma vez que a tarifa industrial de energia el?trica varia ao longo do dia, o bombeamento em diferentes per?odos ter?o diferentes custos. Este trabalho apresenta uma investiga??o experimental de m?todos computacionais desenvolvidos para lidar com o problema do envio de bateladas de derivados de petr?leo considerando a minimiza??o simult?nea de tr?s fun??es objetivo: tempo de entrega, perdas devidas ?s interfaces e custo de energia el?trica. Tal problema ? NP- ?rduo e ser? abordado atrav?s de algoritmos evolucion?rios h?bridos. As hibridiza??es t?m como foco principal os Algoritmos Transgen?ticos e arquiteturas cl?ssicas de algoritmos evolucion?rios multi-objetivo como MOEA/D, NSGA2 e SPEA2. Tr?s arquiteturas denominadas MOTA/D, NSTA e SPETA, s?o aplicadas ao problema. ? apresentado um estudo experimental dos algoritmos propostos onde ? utilizado um conjunto de trinta casos teste. Para analisar os resultados obtidos com os algoritmos s?o empregados indicadores de qualidade Pareto concordantes e testes estat?sticos n?o param?tricos. / An important problem faced by the oil industry is to distribute multiple oil products through pipelines. Distribution is done in a network composed of refineries (source nodes), storage parks (intermediate nodes), and terminals (demand nodes) interconnected by a set of pipelines transporting oil and derivatives between adjacent areas. Constraints related to storage limits, delivery time, sources availability, sending and receiving limits, among others, must be satisfied. Some researchers deal with this problem under a discrete viewpoint in which the flow in the network is seen as batches sending. Usually, there is no separation device between batches of different products and the losses due to interfaces may be significant. Minimizing delivery time is a typical objective adopted by engineers when scheduling products sending in pipeline networks. However, costs incurred due to losses in interfaces cannot be disregarded. The cost also depends on pumping expenses, which are mostly due to the electricity cost. Since industrial electricity tariff varies over the day, pumping at different time periods have different cost. This work presents an experimental investigation of computational methods designed to deal with the problem of distributing oil derivatives in networks considering three minimization objectives simultaneously: delivery time, losses due to interfaces and electricity cost. The problem is NP-hard and is addressed with hybrid evolutionary algorithms. Hybridizations are mainly focused on Transgenetic Algorithms and classical multi-objective evolutionary algorithm architectures such as MOEA/D, NSGA2 and SPEA2. Three architectures named MOTA/D, NSTA and SPETA are applied to the problem. An experimental study compares the algorithms on thirty test cases. To analyse the results obtained with the algorithms Pareto-compliant quality indicators are used and the significance of the results evaluated with non-parametric statistical tests.