Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

Im Rahmen der vorliegenden Dissertation wurde mithilfe eines 4 stündigen Radmarathons die zeitabhängige Freisetzung von zirkulierenden Vorläuferzellen untersucht. Hierzu wurde den 17 gesunden, gut trainierten Probanden während der Prozedur zu festgelegten Zeitpunkten Blut aus einer Venenverweilkanüle entnommen. Daraus wurden mittels Dichtegradient die mononuklearen Zellen isoliert und im Anschluss mittels Durchflusszytometrie die Konzentration der zirkulierenden Progenitorzellen quantifiziert. Es ließ sich ein signifikanter zeitversetzter Anstieg der Konzentration hämatopoietischer Progenitorzellen (CD34pos und CD133pos) wie auch endothelialer Progenitorzellen (CD34/KDRpos und CD133/KDRpos) ermitteln. Die maximale Konzentrationsänderung wurde nach 210 Minuten erreicht. Als Freisetzungsmediatoren wurden in dieser Arbeit die Konzentration von VEGF und IL-6 bestimmt. Auch hier stellte sich ein signifikanter Anstieg der Spiegel dar. Die Konzentrationsänderungen der einzelnen Zellpopulationen waren spätestens 24 Stunden nach Beendigung des Radmarathons nicht mehr nachweisbar. Des Weiteren ging die Arbeit der Frage nach, wie sich die Konzentration von Mikropartkel und endothelialer Mikropartikel als Marker für einen Endothelzellschaden auswirkt. Ein Anstieg der Konzentration reifer (CD146pos) Endothelzellen wurde verzeichnet. Ausgangspunkt dieser Arbeit war die sehr beschränkte und kontroverse Datenlage bezüglich der Konzentrationen von Progenitorzellen freigesetzt durch sportliche Aktivität.

This thesis describes a machine learning method for automatically imitating a particular person's facial expressions in a human-like avatar through a hybrid Particle Swarm Optimization - Tabu Search algorithm. The muscular structures of the facial expressions are measured by Ekman and Friesen's Facial Action Coding System (FACS). Using a neutral face as a reference, the minute movements of the Action Units, used in FACS, are automatically tracked and mapped onto the avatar using a hybrid method. The hybrid algorithm is composed of Kennedy and Eberhart's Particle Swarm Optimization algorithm (PSO) and Glover's Tabu Search (TS). Distinguishable features portrayed on the avatar ensure a personalized, realistic imitation of the facial expressions. To evaluate the feasibility of using PSO-TS in this approach, a fundamental proof-of-concept test is employed on the system using the OGRE avatar. This method is analyzed in-depth to ensure its proper functionality and evaluate its performance compared to previous work.

No / In this paper we discuss a novel method of mathematically modelling facial action units for accurate representation of human facial expressions in 3- dimensions. Our method utilizes the approach of Facial Action Coding System (FACS). It is based on a boundary-value approach, which utilizes a solution to a fourth order elliptic Partial Differential Equation (PDE) subject to a suitable set of boundary conditions. Here the PDE surface generation method for human facial expressions is utilized in order to generate a wide variety of facial expressions in an efficient and realistic way. For this purpose, we identify a set of boundary curves corresponding to the key features of the face which in turn define a given facial expression in 3-dimensions. The action units (AUs) relating to the FACS are then efficiently represented in terms of Fourier coefficients relating to the boundary curves which enables us to store both the face and the facial expressions in an efficient way.

Die rheumatoide Arthritis (RA) ist eine chronisch-entzündliche Bindegewebserkrankung mit bevorzugtem Befall der Gelenke. Es bestimmen Knorpel- und Knochendestruktionen das Krankheitsbild. Eine Schlüsselrolle in der Pathogenese nehmen proliferierende, synoviale Fibroblasten (RASF) durch Auflösung der extrazellulären Matrix (EZM), durch Interaktion mit immunkompetenten Zellen und durch Produktion pro-inflammatorischer Zytokine ein. Die vorliegende Arbeit zeigt die Migrationseigenschaften von RASF und zwei verschiedenen murinen (LS48) und humanen (TK188) Fibroblastenzelllinien in einem In-vitro-Migrationsassay. Es wird der Einfluss von Antikörpern sowie verschiedener EZM-Komponenten auf die Migration der Zelllinien untersucht. Die nachfolgende Analyse phänotypischer Charakteristika stellt dabei die besondere Rolle der genannten Fibroblastenzelllinien heraus, welche eine Reihe von Gemeinsamkeiten untereinander und mit RASF besitzen. Sie zeigen ebenso erhöhte Migrationsaktivität unter dem Einfluss eines Chemoattraktants und besitzen ähnliche Destruktionsmuster von Kollagenmatrizen. Beide Zellreihen exprimieren mehr RASF-typische Proteasen, Adhäsionsmoleküle und immunologisch agierende Proteine als nicht pathologisch transformierte Fibroblasten. Ebenso weisen sie eine gesteigerte Stoffwechselaktivität und Proliferationstätigkeit auf. Diese in vitro erbrachten Hinweise auf mögliche Knorpeldestruktionen sollten Anlass für weitere In-vivo-Studien zu den genannten Zelllinien geben.

Many normal and cancerous cell lines exhibit a stable composition of cells in distinct states which can, e.g., be defined on the basis of cell surface markers. There is evidence that such an equilibrium is associated with stochastic transitions between distinct states. Quantifying these transitions has the potential to better understand cell lineage compositions. We introduce CellTrans, an R package to quantify stochastic cell state transitions from cell state proportion data from fluorescence-activated cell sorting and flow cytometry experiments. The R package is based on a mathematical model in which cell state alterations occur due to stochastic transitions between distinct cell states whose rates only depend on the current state of a cell. CellTrans is an automated tool for estimating the underlying transition probabilities from appropriately prepared data. We point out potential analytical challenges in the quantification of these cell transitions and explain how CellTrans handles them. The applicability of CellTrans is demonstrated on publicly available data on the evolution of cell state compositions in cancer cell lines. We show that CellTrans can be used to (1) infer the transition probabilities between different cell states, (2) predict cell line compositions at a certain time, (3) predict equilibrium cell state compositions, and (4) estimate the time needed to reach this equilibrium. We provide an implementation of CellTrans in R, freely available via GitHub (https://github.com/tbuder/CellTrans).

The Gram-negative bacterium Photorhabdus luminescens is a pathogen of insects. It is able to secrete a variety of toxins and effectors against its host in order to escape its immune defences. The model insect Manduca sexta is able to mount a variety of humoral and cellular responses against pathogen attack. Ultimately these prove ineffective against P. luminescens. The pre-treatment of M. sexta with Escherichia coli provides protection against the pathogenesis of P. luminescens. Here, I use RNA interference and Fluorescence-assisted cell sorting techniques to investigate interactions between pathogen and host to further elucidate the roles of various host factors in mounting the immune response. I also investigate the nutrient requirements of the bacteria for pathogenesis. I show data that peptidoglycan recognition protein (PGRP) is essential for the up-regulation of antimicrobial peptides, an important immune defence. I also show that P. luminescens has a requirement for two types of iron during pathogenesis of M. sexta. And lastly I show that P. luminescens is able to avoid phagocytosis, another important immune defence.

The primary aim of the work presented in this thesis was to change the inducer specificity of the DntR protein in order to improve the response to DNT. The long-term goal is to use this protein in a biosensor for DNT, a signature compound for detection of the explosive TNT. Another aspect of this work was to understand the mechanisms of inducer binding and how the binding of an inducer molecule changes the DntR structure into a state that triggers transcriptional activation. In the papers included in this thesis the inducer specificity of wt DntR has been investigated under different conditions. The functional effects of specific mutations have also been investigated, in some cases in combination with structure determination using X-ray crystallography. In addition, structural data offering insights into the details of inducer binding and conformational changes upon inducer binding are presented and discussed in terms of mechanisms for transcriptional activation by DntR. Furthermore, a directed evolution strategy was employed in order to find variants of DntR with improved response to DNT. A variant with a large improvement in the DNT response was isolated and characterized. In optimized growth conditions, this DntR variant had a nearly 10-fold increase in fluorescence in response to DNT compared to wt DntR. Specific substitutions found in this DntR variant are suggested to be important for changing the inducer response. / Syftet med denna avhandling har varit att förbättra förmågan hos proteinet DntR att upptäcka DNT. Det långsiktiga målet har varit att använda DntR i en biosensor för att upptäcka sprängämnet TNT, som avger DNT som en ”signaturmolekyl”. En annan aspekt har varit att bättre förstå den detaljerade mekanismen för hur DntR fungerar. DntR är ett protein som binder till en viss DNA sekvens (promotor) och reglerar hur gener intill denna promotorsekvens läses av. När en inducerande molekyl som t.ex. DNT binder till DntR förändras proteinets struktur på ett sådant sätt att DntR kan aktivera transkription av de gener som finns intill promotor-sekvensen. För att mäta hur DntR reagerar på olika inducerande molekyler har DntR uttryckts i bakterien Escherichia coli, som också innehållit promotorn som DntR binder till. Intill promotorn sitter en gen som kodar för proteinet GFP. När en inducerande molekyl binder till DntR, slås avläses gfp-genen, och det fluorescerande proteinet GFP produceras. Ju mer GFP som produceras i cellerna, desto högre fluorescens kan uppmätas när cellerna analyseras.   I de artiklar som presenteras i avhandlingen har vi undersökt hur olika substitutioner i DntR proteinet påverkar specificiten och sensitiviteten och hur dessa egenskaper kan påverkas av olika experimentella faktorer. Effekten av substitutioner har relaterats till strukturdata, där bilder av hur proteinet ser ut på molekylär nivå har tagits fram. Dessutom presenteras även en bild av hur DntR förändras beroende på om inducerande molekyler är bundna eller inte. En sådan strukturbild ökar förståelsen för de mekanismer som gör att bindning av en inducerande molekyl orsakar en förändring av formen hos DntR på så sätt att avläsning av gener kan aktiveras. Vi har också använt en metod där evolutionära processer härmats för att få fram varianter av DntR med förbättrad respons till DNT. En variant med en drastisk ökning av DNT-responsen har isolerats, och dess egenskaper har karaktäriserats. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript

Mucosal associated invariant T (MAIT) cells are a population of innate-like lymphocytes, with an emerging role in tuberculosis (TB). They are characterised by the expression of high levels of CD161 and IL-18Rα, possession of a Vα7.2⁺ T cell receptor (TCR), and restriction by the MHC class I-related protein (MR1). MAIT cells can be activated by MR1 presenting microbe-derived riboflavin metabolites; or, by the cytokines IL-12 and IL-18 in a TCR-independent fashion. How human MAIT cells integrate these signals for their activation in response to Mtb is unclear. Lymphatic TB (LNTB) is a common extra-pulmonary manifestation of TB; however, little is known about the status of MAIT cells in LNTB - or in other granulomatous diseases, such as sarcoidosis. In this study, an in vitro approach was used to probe MAIT cell activation by Mtb, and the roles of IL-12/-18, the TCR, cell-cell contact and the immunological synapse (IS). Following TCR ligation, TNFα expression was rapid and transient, and was enhanced following sustained IL-12/-18 exposure. IFNγ expression occurred following sustained exposure to ng/ml concentrations of IL-12/-18; however, alongside TCR stimulation, pg/ml concentrations were sufficient. Using an artificial bilayer system, CD161 was excluded from the central regions of the MAIT cell IS, whilst the distribution of IL-18Rα remained unaffected. In response to Mtb and BCG, MR1 was necessary for rapid activation and TNFα expression, IL-12/-18 were necessary for robust and sustained IFNy expression, whilst an anti-Mtb effect was indicated in an intracellular infection model. Assessment of patients with TB or sarcoid lymphadenopathy revealed a depletion of MAIT cells in the blood in sarcoidosis, but not LNTB. In both groups, MAIT cells could be detected within a proportion of sampled lymph nodes. Overall, these findings indicate the importance of inflammatory cytokine signals in the induction of high-intensity and sustained MAIT cell effector function, including in response to Mtb. The observation of a numerical deficiency of MAIT cells in sarcoidosis requires further investigation.

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>