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Die vorliegende Arbeit beschäftigt sich mit der genetischen Optimierung der Spalthefe S. pombe für die biotechnologische Produktion von Fremdproteinen. Hierbei werden vor allem zwei Aspekte näher untersucht, zum einen die Stabilität des zu produzierenden Proteins und zum anderen die Bildung von Disulfidbrücken. Von anderen Organismen ist bekannt, dass die N-terminale AS im Verbund mit einem Lysinrest ein Protein destabilisieren kann. Das Modellprotein vVEGF besitzt an Position 2 einen Lysinrest (K2) und damit ein Hauptmerkmal eines derartigen Destabilisierungselementes. Falls das Protein dem Ubiquitin-vermittelten Abbau unterliegt, ist es wahrscheinlich, dass K2 eine essenzielle Rolle für die Stabilität dieses Proteins spielt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass K2 in S. cerevisiae destabilisierend wirkt, während es in S. pombe keinen destabilisierenden Effekt hat. Dieses Ergebnis spricht dafür, dass es Unterschiede im Ubiquitin-vermittelten Abbau von Proteinen in diesen beiden Hefen gibt. Der Schwerpunkt dieser Arbeit lag auf der Analyse und Optimierung der Bildung von Disulfidbrücken in S. pombe. Disulfidbrücken stellen eines der wichtigsten Elemente der korrekten Proteinfaltung dar und werden in Eukaryonten vorwiegend im oxidierenden Milieu des ER in das naszierende Protein eingeführt. Aus diesem Grunde wurden Proteindisulfid-isomerasen (PDIs) und ER-oxidoreduktin (Ero)-ähnliche Proteine, die die Schlüssel-komponenten der Bildung von Disulfidbrücken in Eukaryonten darstellen, näher untersucht. In S. pombe finden sich insgesamt drei PDI-Homologe (SpPdi1p, SpPdi2p und SpPdi3p) sowie zwei Ero-Homologe (SpEro1a p und SpEro1b p). Mit Ausnahme des nicht glycosylierten SpPdi2p, sind alle Proteine Membran-assoziierte glycosylierte Komponenten des ER. SpPdi2p und SpPdi3p sowie SpEro1a p und SpEro1b p liegen in vivo teilweise in oxidiertem Zustand vor. Des Weiteren konnte gezeigt werden, dass SpEro1b p, nicht jedoch SpEro1a p in der Lage ist, die temperatursensitive S. cerevisiae ero1-1-Mutante funktionell zu komplementieren. Interessanterweise ergab die Untersuchung konservierter Cysteine mittels gerichteter Mutagenese einerseits Unterschiede zwischen SpEro1a p und SpEro1b p sowie andererseits zwischen den S. pombe Ero-Proteinen und den Ero-Proteinen anderer Spezies. Im Gegensatz zu Ero1b p wird Ero1a p durch reduzierenden Stress und Hitzestress induziert. Dies deutet darauf hin, dass SpEro1b p für die Bildung von Disulfidbrücken unter normalen Wachstumsbedingungen nötig ist, während SpEro1a p vornehmlich bei der Adaption der Zellen an Stressbedingungen erforderlich ist. Abschließend konnte gezeigt werden, dass die gesteigerte Expression von SpEro1a p und SpEro1b p zu einer deutlich erhöhten Ausbeute des disulfidhaltigen heterologen Proteins Orf19p-HA führt. Dieser Befund impliziert, dass in S. pombe die Oxidation der Disulfidbrücken für die Faltung von Proteinen vermutlich limitierend ist.

I många sammanhang är det önskvärt att importera data från textfiler, excelfiler och liknande till en databas. För detta krävs att data i något skede översätts till en ER-modell (Entity Relationship), en modell som beskriver relevanta delar i ett databasschema. Modellen för hur denna översättning ser ut varierar från fall till fall. I det här examensarbetet har en applikation tagits fram för import av data till en ER-modell ur ett modellperspektiv i linje med Model Driven Architecture (MDA) ™. Vinsten ligger i att använda en metamodell som beskriver hur olika modeller för transformering från tabelldata till en ER-modell får se ut. Modellerna i sin tur beskriver hur transformeringen ska se ut. På så sätt kan flera olika modeller användas utan att ändringar i källkoden behöver göras. Metamodellen som beskriver transformeringen har visualiserats i ett klassdiagram. Klassdiagrammet beskriver schematiskt sambanden mellan tabeller som data ska importeras ifrån och den ER-modell som data ska överföras till. Metamodellen har transformerats till ett XML-schema.  Modellerna som ska användas har skrivits i en XML-fil som följer den transformerade metamodellen. / In many situations it is desirable to import data from text files, excel files and similar to a database. To do so the data needs to be translated at some stage to an ER model (Entity Relationship), i.e. a model describing relevant parts of a database schema. The approach for this translation varies from case to case. During this thesis an application has been developed to import data to an ER model from a modeling perspective, in line with the Model Driven Architecture (MDA) ™. The gain lies in using a metamodel that describes how different models for transformation from grid tables to an ER mode may look like. The models in turn describe how the transformation will look, thus allowing the usage of several different models without any need of changes to the source code. The metamodel describing the transformation of data can be visualized in a class diagram. The class diagram schematically describes the relationships between tables of data to be imported from and the ER model the data will be transferred to. Preferable is to write a model in an XML that conforms to the metamodel. Therefore the class diagram should be transformed into an XML schema that enables validation of the model in the XML file.

Diese Dissertation untersucht einige ungeklärten Aspekte der Sprachproduktion. Das erste Ziel war es zu klären, wie Komposita (z.B. Goldfisch) auf der lexikalisch-syntaktischen Ebene unseres Sprachproduktionssystems repräsentiert sind. Gibt es dort einen einzelnen lexikalischen Eintrag für das gesamte Kompositum (GOLDFISCH) oder mehrere Einträge für jedes seiner Konstituenten (GOLD und FISCH), welche beim Sprechen zusammengesetzt werden? Zur Beantwortung dieser Frage wurde die sogenannte kumulative semantische Interferenz (KSI) verwendet. Dieser semantische Kontexteffekt beschreibt die Beobachtung, dass die Benennlatenzen von Sprechern systematisch länger werden, wenn diese eine Reihe von semantisch verwandten Bildern benennen. Obwohl KSI bereits viel als Instrument in der Sprachproduktionsforschung genutzt wird, sind einige Fragen rund um den Effekt selbst noch offen. Das zweite Ziel dieser Dissertation war es daher einige dieser Fragen mit Hilfe von behavioralen und elektrophysiologischen Maßen zu beantworten, um so unser Verständnis von KSI zu erweitern. Die Ergebnisse deuten darauf hin, dass KSI ihren Ursprung auf der konzeptuellen Ebene des Sprachproduktionssystems hat und dass sie nicht von der morphologischen Komplexität der verwendeten Begriffe moduliert wird, aber davon, wie häufig diese benannt werden. Diese Erkenntnisse ermöglichen es in der Zukunft zielgenauere Vorhersagen zu machen, wenn KSI als Forschungsinstrument verwendet wird. Die Ergebnisse zeigen zudem, dass die Konstituenten von Komposita während deren Produktion aktiviert werden. Dies belegt, dass Komposita in einer komplexen Struktur repräsentiert sind, die aus einem Eintrag für das ganze Kompositum und zusätzlichen Einträgen für die Konstituenten besteht. Somit zeigen diese Ergebnisse, dass die Morphologie bereits die Repräsentationen auf der lexikalisch-syntaktischen Ebene beeinflusst und erweitern somit unser Wissen über den Aufbau unseres Sprachproduktionssystems. / This dissertation addresses unresolved issues concerning speech production processes and the cognitive architecture of our speech production system. The first aim was to answer the question how compounds (e.g., goldfish) are represented on the lexical-syntactic level of our speech production system. Is there a single entry for the whole compound (GOLDFISH) or multiple ones for each of its constituents (GOLD and FISH), which are assembled for each use? To investigate this question, we used the cumulative semantic interference (CSI) effect. This semantic context effect describes the observation that speakers’ naming latencies systematically increase when naming a sequence of semantically related pictures. Although CSI has been extensively used as a tool in language production research, several aspects of it are not fully understood. Thus, the second aim of this dissertation was to close some of these knowledge gaps and gain a more comprehensive understanding of CSI. In three studies, we first investigated the CSI effect, before using it as a tool to study the lexical representation of compounds. Behavioural and electrophysiological data from the first two studies point to a purely conceptual origin of CSI. Furthermore, they revealed that CSI is not influenced by the items’ morphological complexity but affected by item repetition. These findings advance our understanding of CSI and thus allow us to make more informed predictions when using CSI as a research tool. The last study showed that the compounds’ constituents are activated during compound production, which provides evidence for a complex lexical-syntactic representation of compounds, consisting of one entry for the holistic compound and additional entries for each of its constituents. This dissertation thus reveals that the morphological complexity of compounds affects the lexical-syntactic level during speech production and thus advances our understanding of the architecture of our speech production system.

Hepatocellular carcinoma (HCC) is the most common liver malignancy that usually develops in patients suffering from chronic liver diseases. One of the major problems faced in the treatment of HCC is severe chemoresistance. Endoplasmic reticulum (ER) stress and hepatic stellate cells play an important role in tumour survival and growth as well as fibrosis. This study further investigates the crosstalk between ER-stress and hepatic stellate cells in HCC resistant cells as well as their relation to chemoresistance markers expression. Mice with chemically induced HCC were divided in 3 different treatment group; one was only treated with doxorubicin, one only with pharmacological ER-stress inhibitor 4μ8C, and one was treated with a combination of doxorubicin and 4μ8C. Tumour burden, fibrosis and cell proliferation were assessed through histological analysis and ImageJ processing. Chemoresistance markers expression was evaluated through mRNAs determination using real-time qPCR. While the combined treatment consisting of doxorubicin and pharmacological ER-stress inhibitor (4μ8C) has shown to positively reduce tumour progression, ferroptosis and collagen deposition, consequently decreasing fibrosis, drug resistance markers’ expression, on the other hand, seems to be more intricate, thus indicating that further investigations are probably needed. / Hepatocellulärt karcinom (HCC) är den vanligaste maligniteten i levern som vanligtvis utvecklas hos patienter som lider av kroniska leversjukdomar. Ett av de största problemen vid behandling av HCC är svår kemoresistens. Stress i endoplasmatiska retikulum (ER) och hepatiska stellatceller spelar en viktig roll för tumörernas överlevnad och tillväxt samt för fibros. I denna studie undersöks vidare samspelet mellan ER-stress och hepatiska stellatceller i HCC-resistenta celler samt deras relation till uttryck av kemoresistensmarkörer. Möss med kemiskt inducerad HCC delades in i tre olika behandlingsgrupper; en behandlades enbart med doxorubicin, en enbart med den farmakologiska ER-stresshämmaren 4μ8C och en behandlades med en kombination av doxorubicin och 4μ8C. Tumörbörda, fibros och cellproliferation bedömdes genom histologisk analys och ImageJ-bearbetning. Kemoresistensmarkörernas uttryck utvärderades genom bestämning av mRNA med hjälp av qPCR i realtid. Medan kombinationsbehandlingen bestående av doxorubicin och farmakologisk ER-stresshämmare (4μ8C) har visat sig minska tumörprogressionen, ferroptos och kollagenavlagring och därmed minska fibros, verkar uttrycket av läkemedelsresistensmarkörer å andra sidan vara mer invecklat, vilket tyder på att det troligen behövs ytterligare undersökningar.

This paper discusses the metamorphosis in Ingvild Lothe's poetry collection Hvorfor er jeg så trist når jeg er så søt (2016). To investigate the relationship between human and nature Stacy Alaimo's term "trans-corporeality" is being used. In this paper, an analysis is being made of the poetry collection and the book cover regarding these matters. The poetry analysis is split into four sections, following the stages of the butterfly's metamorphosis. When analyzing the book cover, it is mostly viewed as an adaptation of the poems. The findings of this study suggests that there are three different types of metamorphoses in the poems and on the book cover. These are biological, symbolical and psychological. It is also found that the metamorphosis on the book cover is being more positively depicted than in the poetry collection.

The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown. Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis. In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%). The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. The third objective of this work was to obtain a broad picture of the pancreatic beta-cell molecular responses during and after (recovery period) a severe ER stress. For this purpose, we utilized an “in home” spotted microarray, the APOCHIP, containing nearly 600 probes selected for the study of beta-cell apoptosis. Time-dependent gene expression profiles were measured in INS-1E cells exposed to CPA. CPA-induced ER-stress modified expression of 183 genes in at least one of the time points studied. Most of theses genes returned to control levels 3h after CPA removal from the culture medium. We observed full beta-cell recovery and survival, indicating that these cells trigger efficient defenses against ER stress. Beta-cell recovery is associated with a sustained increase in the expression of ER chaperones and a rapid decrease of pro-apoptotic mRNAs following CPA removal. Two groups of genes were particularly affected by CPA, namely those related to the cellular responses to ER stress, which were mostly up-regulated, and those related to differentiated beta-cell functions, which were down-regulated. Among this last group, we observed a 40-90% decrease of the mRNAs for insulin-1 and -2. These findings were confirmed in INS-1E cells exposed to cytokines or thapsigargin (another SERCA blocker), and in primary beta-cells exposed to the same treatments. This decrease in insulin mRNA expression is due to transcript degradation, most probably caused by IRE-1 activation and triggering of its endoribonuclease activity, as recently described in Drosophila cells. In conclusion, our work enabled a better understanding of the pancreatic beta-cell responses to ER stress: 1.)We identified a sensitizing effect of IFN-gamma to ER stress in beta-cells via downregulation of key ER chaperones. 2.)We observed a differential regulation of Chop transcription by different treatments suggesting distinct responses of pancreatic beta-cells to diverse ER stress inducers. 3.)We provided the first global analysis of gene expression modifications in pancreatic beta-cells following ER stress. 4.)We demonstrated a high capacity of beta-cells to cope and recover from a severe ER stress. 5.)We identified a new protective mechanism against ER stress, namely the degradation of insulin mRNA which limits the load posed on the ER by insulin synthesis. This, coupled to a marked increase in ER chaperones and a fast degradation of pro-apoptotic mRNAs, enables beta cells to recover from ER stress after the causes of this stress are removed.

The goal of this project has been to develop therapeutic strategies for protein misfolding diseases caused by excessive degradation of misfolded proteins and loss of protein function. The focus for this work is lysosomal storage disorders (LSDs), a group of more than 50 known inherited metabolic diseases characterized by deficiency in hydrolytic enzymes and consequent buildup of lysosomal macromolecules. Gaucher’s Disease (GD) is used as a representative of the family of LSDs in this study. GD is caused by mutations in the gene encoding lysosomal glucocerebrosidase (GC) and consequent accumulation of the GC substrate, glucocerebroside. The most prevalent mutations among GD patients are single amino acid substitutions that do not directly impair GC activity, but rather destabilize its native folding. GC normally folds in the ER and trafficks through the secretory pathway to the lysosomes. GC variants containing destabilizing mutations misfold and are retrotranslocated to the cytoplasm for ER-associated degradation (ERAD). However, evidence shows that if misfolding-prone, mutated GC variants are forced to fold into their 3D native structure, they retain catalytic activity. This study describes strategies to remodel the network of cellular pathways that maintain protein homeostasis and to create a folding environment favorable to the folding of unstable, degradation-prone lysosomal enzyme variants. We demonstrated that folding and trafficking of mutated GC variants can be achieved by modulating the protein folding network in fibroblasts derived from patients with GD to i) upregulate the expression of ER luminal chaperones, ii) inhibit the ERAD pathway, and iii) enhance the pool of mutated GC in the ER amenable to folding rescue. We also demonstrated that the same cell engineering strategies that proved successful in rescuing the folding and activity of mutated GC enable rescue of mutated enzyme variants in fibroblasts derived from patients with Tay-Sachs disease, a LSD caused by deficiency of lysosomal hexosaminidase A activity. As a result, the current study provides insights for the development of therapeutic strategies for GD based on the modulation of general cellular pathways that maintain protein homeostasis that could in principle be applied to the treatment of multiple LSDs.

When damaged DNA is detected during replication, a checkpoint delays the cell cycle to allow time for repair. Here I show that continually delaying the cell cycle in the G2/M phase of the cell cycle stabilizes the genome of Saccharomyces cerevisiae in both checkpoint proficient and deficient cells; a phenomenon I call slow cycle stabilization (SCS). SCS stabilizes the genome in cells defective for DNA damage response (DDR), spindle checkpoint, and telomere biology, as well as wild type (WT) cells. I verify SCS using genetic and chemical means and further substantiate SCS using three different Saccharomyces cerevisiae chromosome systems.

HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART). HIV PIs are extremely effective at suppressing viral load, but have been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease. Recent studies indicate that activation of endoplasmic reticulum (ER) stress is an important cellular mechanism underlying HIV PI-induced dysregulation of lipid metabolism. However, the exact role of ER stress in HIV PI-associated lipodystrophy and dyslipidemia remains to be identified. Hepatocytes and adipocytes are important players in regulating lipid metabolism and the inflammatory state. Dysfunction of these two cell types is closely linked to various metabolic diseases. In this dissertation research, we aimed to define the role of activation of ER stress in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes and further identifty the potential molecular mechanisms. Both cultured and primary mouse adipocytes and hepatocytes were used to examine the effect of individual HIV PIs on ER stress activation and lipid metabolism. The results indicated that HIV PIs differentially activate ER stress through depletion of ER calcium stores, activating the unfolded protein response (UPR). UPR activation further lead to an alteration of cellular differentiation through downstream transcription factor CHOP. At the same time, HIV PIs also altered adipogenesis via differential regulation of the adipogenic transcription factor PPARγ. HIV PI-induced ER stress was closely linked to dysregulation of autophagy activation through CHOP, and upstream ATF-4, signaling pathways. In hepatocytes, the integrase inhibitor raltegravir abrogated HIV PI-induced lipid accumulation by inhibiting ER stress activation and dysregulation of autophagy pathway. Our studies suggest that both ER stress and autophagy are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes and hepatocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV HAART-treated patients.