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Advances in communicat ions have been unarguably essent ial to enablemodern systems and applicat ions as we know them. Ubiquity has turned into reality, allowing specialised embedded systems to eminent ly grow and spread. That is notably the case of unmanned vehicles which have been creat ively explored on applications that were not as efficient as they currently are, neither as innovative as recent ly accomplished. Therefore, towards the efficient operat ion of either unmanned vehicles and systems they integrate, in addition to communicat ion improvements, it is highly desired that we carefully observe relevant , co-related necessit ies that may lead to the full insert ion of unmanned vehicles to our everyday lives. Moreover, by addressing these demands on integrated solut ions, better resultswill likely be produced. This thesis presentsHAMSTER, theHeAlthy, Mobility and Security based data communication archiTEctuRe for unmanned vehicles, which addresses threemain types of communicat ions: machine-to-machine, machine-to-infrast ructure and internal machine communications. Four addit ional elements on co-related requirements are provided alongside with HAMSTER for more accurate approaches regarding security and safety aspects (SPHERE platform), crit icality analysis (NCI index), energy efficiency (NP plat form) and mobility-oriented ad hoc and infrast ructured communicat ions (NIMBLE platform). Furthermore, three specialised versions are provided: unmanned aerial vehicles (Flying HAMSTER), unmanned ground vehicles (Running HAMSTER) and unmanned surface/ underwater vehicles (Swimming HAMSTER). The architecture validat ion is achieved by case studies on each feature addressed, leading to guidelines on the development of vehicles more likely to meet certificat ion requirements, more efficient and secure communicat ions, assert ive approaches regarding crit icality and green approaches on internal communicat ions. Indeed, results prove the efficiency and effectiveness of HAMSTER architecture and its elements, as well as its flexibility in carrying out different experiments focused on various aspects of communication, which helps researchers and developers to achieve safe and secure communicat ions in unmanned vehicles. / Os avanços na área de comunicações foram indiscutívelmente essenciais para a obtenção de sistemas e aplicações modernos como os o atuais. A computação ubíqua se tornou realidade, permitindo que sistemas embarcados especializados ganhassem espaço e cada vez mais autonomia. Esse é notavelmente o caso de veículos não tripulados que têm sido criativamente explorados em aplicações inovadoras e avançadas. Entretanto, para o funcionamento eficiente desses veículos e sistemas não tripulados, além de melhorias de comunicação, é altamente desejável que as necessidades relevantes co-relacionadas a comunica¸cao sejam cuidadosamente observadas, levando a uma facilitação na inserção de veículos não tripulados em espaços públicos. Além disso, ao abordar essas demandas de modo integrado, as chances de produzir melhores resultados é maior. Esta tese apresenta a HAMSTER, uma arquitetura de comunicação de dados baseada em mobilidade e segurança para veículos não tripulados, que aborda três tipos principais de comunicação: máquina-para- máquina, máquina-para-infraestrutura e comunicações internas. Quatro elementos adicionais co-relacionados são fornecidos juntamente com a arquitetura HAMSTER de modo a prover abordagens mais precisas em relação a aspectos de segurança física e da informação (plataforma SPHERE), análise de criticalidade (índice NCI), eficiência energética (plataforma NP) e comunicações ad hoc e infraestruturadas orientadas a mobilidade (plataforma NIMBLE). Além disso, são fornecidas três versões especializadas: para veículos aéreos não tripulados (Flying HAMSTER), veículos terrestres não tripulados (Running HAMSTER) e veículos submarinos e de superfície não tripulados (Swimming HAMSTER). A validação da arquitetura é obtida por meio de estudos de caso sobre cada recurso abordado, levando a diretrizes sobre o desenvolvimento de veículos mais preparados para atender a requisitos de certificação, comunicação mais eficiente e segura, abordagens assertivas sobre criticidade e abordagens verdes nas comunicações internas. Por fim, os resultados comprovaram a eficiência da arquitetura HAMSTER e os elementos com ela providos, bem como a flexibilidade em realizar experimentos focados em vários aspectos de comunicação, auxiliando na obtenção de comunicações seguras em veículos autônomos.

Previous investigators demonstrated that epididymal white adipose tissue (EWAT) lipectomy suppressed spermatogenesis and caused atrophy of the seminiferous tubules. EWAT lipectomy, however, may disrupt testicular innervation, which reportedly compromises testicular function. To resolve this confound and better clarify the role of EWAT in spermatogenesis, three experimental groups of hamsters were created in which: i.) the superior and inferior spermatic nerves were transected (SSNx) at the testicular level, ii.) EWAT was extirpated (EWATx), and iii.) testicular nerves and EWAT were left intact (SHAM controls). It was hypothesized that transection of the superior and inferior spermatic nerves would disrupt normal spermatogenesis. The findings indicate a significant reduction in spermatogenic activity and marked seminal tubule atrophy within the EWATx testis, as compared to the SSNx and controls testes, which did not differ significantly from each other. From these data, it is concluded that EWAT, and not testicular innervation, is central to normal spermatogenesis.

In this work an intersection between optimization methods and animal cell culture modeling is considered. We present optimization based methods for analyzing and building models of cell culture; models that could be used when designing the environment cells are cultivated in, i.e., medium. Since both the medium and cell line considered are complex, designing a good medium is not straightforward. Developing a model of cell metabolism is a step in facilitating medium design. In order to develop a model of the metabolism the methods presented in this work make use of an underlying metabolic reaction network and extracellular measurements. External substrates and products are connected via the relevant elementary flux modes (EFMs). Modeling from EFMs is generally limited to small networks, because the number of EFMs explodes when the underlying network size increases. The aim of this work is to enable modeling with more complex networks by presenting methods that dynamically identify a subset of the EFMs. In papers A and B we consider a model consisting of the EFMs along with the flux over each mode. In paper A we present how such a model can be decided by an optimization technique named column generation. In paper B the robustness of such a model with respect to measurement errors is considered. We show that a robust version of the underlying optimization problem in paper A can be formed and column generation applied to identify EFMs dynamically. In papers C and D a kinetic macroscopic model is considered. In paper C we show how a kinetic macroscopic model can be constructed from the EFMs. This macroscopic model is created by assuming that the flux along each EFM behaves according to Michaelis-Menten type kinetics. This modeling method has the ability to capture cell behavior in varied types of media, however the size of the underlying network is a limitation. In paper D this limitation is countered by developing an approximation algorithm, that can dynamically identify EFMs for a kinetic model. / I denna avhandling betraktar vi korsningen mellan optimeringsmetoder och modellering av djurcellodling.Vi presenterar optimeringsbaserade metoder för att analysera och bygga modeller av cellkulturer. Dessa modeller kan användas vid konstruktionen av den miljö som cellerna ska odlas i, dvs, medium.Eftersom både mediet och cellinjen är komplexa är det inte okomplicerat att utforma ett bra medium. Att utveckla en modell av cellernas ämnesomsättning är ett steg för att underlätta designen av mediet. För att utveckla en modell av metabolismen kommer de metoder som används i detta arbete att utnyttja ett underliggande metaboliskt reaktions\-nätverk och extracellulära mätningar. Externa substrat och produkter är sammankopplade via de relevanta elementära metaboliska vägarna (EFM).Modellering med hjälp av EFM är i allmänhet begränsad till små nätverk eftersom antalet EFM exploderar när de underliggande nätverket ökar i storlek. Målet med detta arbete är att möjliggöra modellering med mer komplexa nätverk genom att presentera metoder som dynamiskt identifierar en delmängd av EFM. I artikel A och B betraktar vi en modell som består av EFM och ett flöde över varje EFM.I artikel A presenterar vi hur en sådan modell kan bestämmas med hjälp av en optimeringsteknik som kallas kolumngenerering.I artikel A undersöker vi hur robust en sådan modell är med avseende till mätfel. Vi visar att en robust version av det underliggande optimeringsproblemet i artikel A kan konstrueras samt att kolumngenerering kan appliceras för att identifiera EFM dynamiskt. Artikel C och D behandlar en kinetisk makroskopisk modell. Vi visar i artikel C hur en sådan modell kan konstrueras från EFM.Denna makroskopiska modell är skapad genom att anta att flödet genom varje EFM beter sig enligt Michaelis-Menten-typ av kinetik. Denna modelleringsmetod har förmågan att fånga cellernas beteende i olika typer av media, men storleken på nätverket är en begränsning.I artikel D hanterar vi denna begränsing genom att utveckla en approximationsalgoritm som identifierar EFM dynamiskt för en kinetisk modell. / <p>QC 20150827</p>

Main purpose of this study was to increase protein expression in Chinese hamster ovary (CHO) cells by improving protein secretion of translated proteins. The goal was to find signal peptides from the screening of signal peptide libraries for improvement of protein secretion using a CHO-cell express selection system. Biopharmaceutical products, proteins such as monoclonal antibodies (mAbs), are most commonly produced using mammalian expression systems such as the expression in CHO cells. The posttranslational modifications of the proteins being expressed in CHO cells are similar to the expressional modifications in human cells, why the CHO cells are suitable for production of proteins used for human therapy. The expression of proteins in the cell is a complex mechanism, fundamentally depending on the DNA sequences in the cell nucleus. Secretion of translated proteins has been showed to be a bottleneck when improving expression. Secretion is initiated by the signal peptide, a n-terminal prolongation of the protein that is recognized by a signal recognition particle (SRP) when being translated by the ribosome. The sequence and structure of the signal peptide has been proved to affect secretion and altering the signal peptide could improve secretion even when changing signal peptide between different species. Designing variants of the signal peptides and analyzing protein expression might lead to improvements of the construct design and more protein produced from the cells, which would save time, money and material for the producer. To construct plasmids containing the gene of interest (GOI) and different signal peptides, several gene cloning methods were used. The plasmids were amplified using Escherichia coli (E. coli) transformation. The constructs were expressed by transfection into the CHO cell genome, and expression were analyzed using flow cytometry. When analyzing expression of a Fc-fusion protein with 5 different signal peptides, the signal peptide Azurocidin is the one showing highest expression levels in this study. In addition, IgG kapa and Albumin signal peptides did not show as high protein expression levels, even if they were better than the L1d and H5b signal peptides. Since signal peptides are exchangeable between proteins and species, it might be that Azurocidin is improving secretion and protein expression with other proteins than Fc-fusion proteins which would be an interesting aspect for further studies. When altering signal peptides with library sequences, the experimental challenges were crucial for the protein expression results and due to these issues, no library sequence could be seen to conquer others when it comes to protein expression levels. Transfection and cultivation procedures needs to be studied and improved before being able to draw conclusions about which signal peptide library sequences that might improve secretion and increase the protein expression.

Advances in communicat ions have been unarguably essent ial to enablemodern systems and applicat ions as we know them. Ubiquity has turned into reality, allowing specialised embedded systems to eminent ly grow and spread. That is notably the case of unmanned vehicles which have been creat ively explored on applications that were not as efficient as they currently are, neither as innovative as recent ly accomplished. Therefore, towards the efficient operat ion of either unmanned vehicles and systems they integrate, in addition to communicat ion improvements, it is highly desired that we carefully observe relevant , co-related necessit ies that may lead to the full insert ion of unmanned vehicles to our everyday lives. Moreover, by addressing these demands on integrated solut ions, better resultswill likely be produced. This thesis presentsHAMSTER, theHeAlthy, Mobility and Security based data communication archiTEctuRe for unmanned vehicles, which addresses threemain types of communicat ions: machine-to-machine, machine-to-infrast ructure and internal machine communications. Four addit ional elements on co-related requirements are provided alongside with HAMSTER for more accurate approaches regarding security and safety aspects (SPHERE platform), crit icality analysis (NCI index), energy efficiency (NP plat form) and mobility-oriented ad hoc and infrast ructured communicat ions (NIMBLE platform). Furthermore, three specialised versions are provided: unmanned aerial vehicles (Flying HAMSTER), unmanned ground vehicles (Running HAMSTER) and unmanned surface/ underwater vehicles (Swimming HAMSTER). The architecture validat ion is achieved by case studies on each feature addressed, leading to guidelines on the development of vehicles more likely to meet certificat ion requirements, more efficient and secure communicat ions, assert ive approaches regarding crit icality and green approaches on internal communicat ions. Indeed, results prove the efficiency and effectiveness of HAMSTER architecture and its elements, as well as its flexibility in carrying out different experiments focused on various aspects of communication, which helps researchers and developers to achieve safe and secure communicat ions in unmanned vehicles. / Os avanços na área de comunicações foram indiscutívelmente essenciais para a obtenção de sistemas e aplicações modernos como os o atuais. A computação ubíqua se tornou realidade, permitindo que sistemas embarcados especializados ganhassem espaço e cada vez mais autonomia. Esse é notavelmente o caso de veículos não tripulados que têm sido criativamente explorados em aplicações inovadoras e avançadas. Entretanto, para o funcionamento eficiente desses veículos e sistemas não tripulados, além de melhorias de comunicação, é altamente desejável que as necessidades relevantes co-relacionadas a comunica¸cao sejam cuidadosamente observadas, levando a uma facilitação na inserção de veículos não tripulados em espaços públicos. Além disso, ao abordar essas demandas de modo integrado, as chances de produzir melhores resultados é maior. Esta tese apresenta a HAMSTER, uma arquitetura de comunicação de dados baseada em mobilidade e segurança para veículos não tripulados, que aborda três tipos principais de comunicação: máquina-para- máquina, máquina-para-infraestrutura e comunicações internas. Quatro elementos adicionais co-relacionados são fornecidos juntamente com a arquitetura HAMSTER de modo a prover abordagens mais precisas em relação a aspectos de segurança física e da informação (plataforma SPHERE), análise de criticalidade (índice NCI), eficiência energética (plataforma NP) e comunicações ad hoc e infraestruturadas orientadas a mobilidade (plataforma NIMBLE). Além disso, são fornecidas três versões especializadas: para veículos aéreos não tripulados (Flying HAMSTER), veículos terrestres não tripulados (Running HAMSTER) e veículos submarinos e de superfície não tripulados (Swimming HAMSTER). A validação da arquitetura é obtida por meio de estudos de caso sobre cada recurso abordado, levando a diretrizes sobre o desenvolvimento de veículos mais preparados para atender a requisitos de certificação, comunicação mais eficiente e segura, abordagens assertivas sobre criticidade e abordagens verdes nas comunicações internas. Por fim, os resultados comprovaram a eficiência da arquitetura HAMSTER e os elementos com ela providos, bem como a flexibilidade em realizar experimentos focados em vários aspectos de comunicação, auxiliando na obtenção de comunicações seguras em veículos autônomos.

Etwa 15% aller Paare bleiben ungewollt kinderlos. Männliche Faktoren sind in circa einem Drittel der Fälle als ursächlich anzusehen. Jedoch sind die Erfolgsraten der Therapie männlicher Infertilität durch assistierte Reproduktion auch nach über 30 Jahren seit deren Einführung unbefriedigend. Bestehende Spermienaufbereitungsmethoden wie einfaches Waschen, swim up oder die Dichtegradientenzentrifugation basieren auf makroskopisch-funktionellen Parametern wie Motilität und Morphologie. Spezifische Eigenschaften wie etwa eine aktivierte Apoptosesignalkaskade der Spermien werden dabei nicht berücksichtigt. Die wesentlichen Elemente verschiedener Signalwege der aus somatischen Zellen bekannten Apoptose konnten auch am humanen ejakulierten Spermatozoon nachgewiesen werden. Über die (negativen) Auswirkungen der Apoptose auf die männliche Fruchtbarkeit gibt es einen Konsens. Ziel der vorliegenden Arbeit war es, Selektionsmethoden zu entwickeln, welche auf subzellulärer Ebene intakte Spermien mit dem größtmöglichen Fertilisationspotential aus dem Ejakulat extrahieren. In einem ersten Versuchskomplex konnte gezeigt werden, dass durch die Kombination von Dichtegradientenzentrifugation und swim-up (Standardmethoden in Reproduktionskliniken und andrologischen Laboren) zur Aufbereitung der Spermien von subfertilen Patienten eine akzeptable Reduktion der Spermien mit aktivierter Apoptosesignalkaskade erreicht werden kann. Jedoch gaben die großen interindividuellen Unterschiede im Separationseffekt Anlass zur Entwicklung innovativer Untersuchungs- und Separationsmethoden. So wurden unter anderem fluoreszenzbasierte Tests zur Evaluation von Spermiendefekten, wie beispielsweise einer gestörten Integrität des mitochondrialen Membranpotentials, eingeführt. In den Untersuchungen wurde die Praktikabilität dieser neuen Analyseverfahren im Routineeinsatz unter Standardbedingungen getestet und bestätigt. Die innovative Selektionsmethode der Annexin V-MACS Separation basiert auf der Bindung von Annexin V-MicroBeads an apoptotische Spermien, womit eine Subpopulation reifer, motiler und vitaler Spermien mit inaktivierter Apoptosesignalkaskade gezielt angereichert wird. Das Konzept wurde zudem erfolgreich auf ein (Glaswoll-) Festphasen-Filtersystem ohne frei schwimmende Microbeads übertragen. Dadurch gelang die Minimierung eines potentiellen Transmissionsrisikos der Microbeads bei der Anwendung im Rahmen der künstlichen Befruchtung. Den hohen Stellenwert dieser Verfahren belegen die Ergebnisse zweier in-vitro Modelle, an denen erstmalig gezeigt werden konnte, dass durch die Selektion von Spermien mit inaktivierter Apoptose-Signalkaskade höhere Fertilisationsraten erreichbar sind.

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No description available.