In the work presented this thesis, two different conditions that are classified as protein misfolding diseases: Alzheimer's disease and lysozyme amyloidosis and proteins that could have a beneficial effect in these diseases, have been studied using Drosophila melanogaster, commonly known as the fruit fly. The fruit fly has been used for over 100 years to study and better understand fundamental biological processes. Although the fruit fly, unlike humans, is an invertebrate, many of its central biological mechanisms are very similar to ours. The first transgenic flies were designed in the early 1980s, and since then, the fruit fly has been one of the most widely used model organisms in studies on the effects of over-expressed human proteins in a biological system; one can regard the fly as a living, biological test tube. For  most proteins, it is necessary that they fold into a three-dimensional structure to function properly. But sometimes the folding goes wrong; this may be due to mutations that make the protein unstable and subject to misfolding. A misfolded protein molecule can then aggregate with other misfolded proteins. In Alzheimer's disease, which is the most common form of dementia, protein aggregates are present in the brains of patients. These aggregates are composed of the amyloid-β (Aβ) peptide, a small peptide of around 42 amino acids which is cleaved from the larger, membrane-bound, protein AβPP by two different enzymes, BACE1 and γ-secretase. In the first part of this thesis, two different fly models for Alzheimer’s disease were used: the Aβ fly model, which directly expresses the Aβ peptide, and the AβPP-BACE1 fly model, in which all the components necessary to produce the Aβ peptide in the fly are expressed in the fly central nervous system (CNS). The two different fly models were compared and the results show that a significantly smaller amount of the Aβ peptide is needed to achieve the same, or an even greater, toxic effect in the AβPP-BACE1 model compared to the Aβ model. In the second part of the thesis, these two fly models for Alzheimer’s disease were again used, but now to investigate whether lysozyme, a protein involved in our innate immune system, can counteract the toxic effect of Aβ generated in the fly models. And indeed, lysozyme is able to save the flies from Aβ-induced toxicity. Aβ and lysozyme were found to interact with each other in vivo. The second misfolding disease studied in this thesis is lysozyme amyloidosis. It is a rare, dominantly inherited amyloid disease in which mutant variants of lysozyme give rise to aggregates, weighing up to several kilograms, that accumulate around the kidneys and liver, eventually leading to organ failure. In the third part of this thesis, a fly model for lysozyme amyloidosis was used to study the effect of co-expressing the serum amyloid P component (SAP), a protein that is part of all protein aggregates found within this disease class. SAP is able to rescue the toxicity induced by expressing the mutant variant of lysozyme, F57I, in the fly's CNS. To further investigate how SAP was able to do this, double-expressing lysozyme flies, which exhibit stronger disease phenotypes than those of the single-expressing lysozyme flies previously studied, were used in the fourth part of this thesis. SAP was observed to reduce F57I toxicity and promote F57I to form aggregates with more distinct amyloid characteristics. In conclusion, the work included in this thesis demonstrates that: i) Aβ generated from AβPP processing in the fly CNS results in higher proteotoxicity compared with direct expression of Aβ from the transgene, ii) lysozyme can prevent Aβ proteotoxicity in Drosophila and could thus be a potential therapeutic molecule to treat Alzheimer’s disease and iii) in a Drosophila model of lysozyme amyloidosis, SAP can prevent toxicity from the disease-associated lysozyme variant F57I and promote formation of aggregated lysozyme morphotypes with amyloid properties; this is important to take into account when a reduced level of SAP is considered as a treatment strategy for lysozyme amyloidosis.

Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair. During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways. Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension. In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair.

<p>Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.</p>

<p>Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. </p><p>The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton.</p><p>TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). </p><p>The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation.</p><p>Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells.</p><p>Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene <i>c-myc</i>. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis</p>

<p>The parasite <i>Echinococcus granulosus</i> causes hydatid disease, a major zoonosis. A fatty-acid-binding protein, EgFABP1, is important for the parasite, as it must acquire almost all its lipids from its environment or the host. The structure of EgFABP1 has been solved and refined to 1.6 Å resolution. The structure reveals that EgFABP1 has the 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins. </p><p>Human semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6), also known as vascular adhesion protein-1, is a copper-containing monoamine oxidase that occurs both as a membrane-bound protein and in a soluble form in plasma. SSAO has been implicated in glucose transport in adipocytes, the differentiation of adipose cells and the leukocyte extravasation process. Toxic reaction products have been suggested to cause some of the vascular complications associated with diabetes and SSAO is therefore of pharmaceutical interest.</p><p>The structure of a truncated, soluble form of human SSAO has been determined to 2.5 Å resolution. The structure reveals that a leucine residue located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five out of six potential N-glycosylation sites. Carbohydrates attached to Asn 232 flank the active site entrance and might influence substrate specificity. The structure also reveals a vicinal disulfide bridge, which we hypothesise could act as a redox switch involved in the protein’s mechanism of action. The structure of a complex of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 Å resolution. Both structures together will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies. </p>

Transforming growth factor-β (TGF-β) is a member of a large superfamily of cytokines which participate in many different types of cellular processes, such as growth inhibition, cell migration, differentiation, cell adhesion, wound healing and immunosuppression. Alterations of TGF-β superfamily signalling results in several different disorders, including bone disease, vascular disease and cancer. The TGF-β signalling pathways involve several different proteins, such as the Smad proteins, which upon receptor activation are translocated to the nucleus, where they affect transcriptional responses. The actin cytoskeleton is an organised network of filaments with a highly dynamic structure, which is under a continuous reconstruction to control the morphology, survival, growth and motility of eukaryotic cells. The members of the family of small GTP-binding proteins have been shown to be important regulators of the actin cytoskeleton. TGF-β was found to induce short term as well as long term actin reorganisation in prostate cancer cells. The short term response included membrane ruffling, and required signalling by the small GTPases Cdc42 and Rho as well as, the involvement of the mitogen-activated protein kinases p38 (p38 MAPK). The long term response included formation of stress fibers and required a cooperation between Smad and Rho GTPase signalling pathways involving the Rho-associated coiled-coil-containing protein kinase 1 (ROCK1). The TGF-β-induced activation of Cdc42 was, furthermore, shown to require the inhibitory Smad7 and p38 MAP kinase, via a PI3K-dependent pathway. Mixed lineage kinase 3 (MLK3), a mediator downstream of Cdc42, was necessary for the Cdc42-dependent actin filament reorganisation. Apoptosis is an important and carefully regulated process in human development and disease, which allows the multicellular organisms to remove cells that are in excess or potentially dangerous. TGF-β family members can induce apoptosis in many different cell types, in the presence or absence of other growth factors. Smad7 had previously been shown to be necessary for TGF-β-induced apoptosis of epithelial cells. We could show that Smad7 is required for TGF-β-induced activation of the TGF-β activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK pathway, which subsequently leads to apoptosis in prostate cancer cells. Members of the lymphoid enhancer factor-1/T-cell factor (LEF1/TCF) family of transcription factors have, together with β-catenin, been shown to be nuclear effectors in the Wnt-signalling pathway. We investigated a possible cross-talk between the TGF-β and Wnt signalling pathways. We found that TGF-β, in a Smad7-dependent manner induced a nuclear accumulation of β-catenin and enhanced the transcriptional activity of β-catenin and the induction of the downstream target gene c-myc. Since β-catenin and c-Myc has been shown to promote apoptosis, our results suggests the possibility that β-catenin contributes to TGF-β-induced apoptosis

Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.

The parasite Echinococcus granulosus causes hydatid disease, a major zoonosis. A fatty-acid-binding protein, EgFABP1, is important for the parasite, as it must acquire almost all its lipids from its environment or the host. The structure of EgFABP1 has been solved and refined to 1.6 Å resolution. The structure reveals that EgFABP1 has the 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins. Human semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6), also known as vascular adhesion protein-1, is a copper-containing monoamine oxidase that occurs both as a membrane-bound protein and in a soluble form in plasma. SSAO has been implicated in glucose transport in adipocytes, the differentiation of adipose cells and the leukocyte extravasation process. Toxic reaction products have been suggested to cause some of the vascular complications associated with diabetes and SSAO is therefore of pharmaceutical interest. The structure of a truncated, soluble form of human SSAO has been determined to 2.5 Å resolution. The structure reveals that a leucine residue located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five out of six potential N-glycosylation sites. Carbohydrates attached to Asn 232 flank the active site entrance and might influence substrate specificity. The structure also reveals a vicinal disulfide bridge, which we hypothesise could act as a redox switch involved in the protein’s mechanism of action. The structure of a complex of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 Å resolution. Both structures together will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies.

Disturbances of host integrity have the potential to evoke activation of innate immunologic and hemostatic protection mechanisms in blood. Irrespective of whether the activating stimulus is typically immunogenic or thrombotic, it will generally affect both the complement system and platelets to a certain degree. The theme of this thesis is complement and platelet activity, which is intersected in all five included papers. The initial aim was to study the responses and mechanisms of the complement cascade in relation to platelet activation. The secondary aim was to use an applied approach to regulate platelets and complement on model biomaterial and cell surfaces.    Complement activation was found in the fluid phase in response to platelet activation in whole blood. The mechanism was traced to platelet release of stored chondroitin sulfate-A (CS-A) and classical pathway activation via C1q. C3 was detected at the platelet surface, though its binding was independent of complement activation. The inhibitors factor H and C4-binding protein (C4BP) were detected on activated platelets, and their binding was partly dependent on surface-exposed CS-A. Collectively, these results showed that platelet activation induces inflammatory complement activation in the fluid phase. CS-A was shown to be a central molecule in the complement-modulatory functions of platelets by its interaction with C1q, C4BP, and factor H. Platelet activation and surface adherence were successfully attenuated by conjugating an ADP-degrading apyrase on a model biomaterial. Only minor complement regulation was seen, and was therefore targeted specifically on surfaces and cells by co-immobilizing a factor H-binding peptide together with the apyrase. This combined approach led to a synchronized inhibition of both platelet and complement activation at the interface of biomaterials/xenogeneic cells and blood. In conclusion, here presents a novel crosstalk-mechanism for activation of complement when triggering platelets, which highlights the importance of regulating both complement and platelets to lower inflammatory events. In addition, a strategy to enhance the biocompatibility of biomaterials and cells by simultaneously targeting ADP-dependent platelet activation and the alternative complement C3-convertase is proposed.

Alzheimers sjukdom (AD) är den vanligaste formen av demens och kännetecknas av intracellulärt neurofibrillärt trassel (NFT) bestående av proteinet tau och extracellulära plack, uppbyggda av peptiden amyloid beta (Aβ). En växande skara studier har börjat peka mot att AD är en hjärnspecifik typ av diabetes. Insulinresistens följt av hyperinsulinemi och hyperglykemi är kännetecken för diabetes mellitus typ 2 (DMT2) och har visat sig vara en riskfaktor för AD. Insulin, ett hormon som kontrollerar glukoshomeostasen i perifera nervsystemet (PNS) och är viktigt för minne och inlärning, transporteras över blod-hjärnbarriären i en mättnadsbar transportmekanism och dess koncentration i centrala nervsystemet (CNS) minskar vid DMT2 och AD. Insulin-like growth factor 1 (IGF-1), ett neuronskyddande protein som minskar ogynnsam β-sekretasklyvning av amyloid precursor protein (APP) i amyloidkaskadhypotesen, minskar i koncentration i hjärnan när mycket insulin transporteras in i CNS. γ-sekretas ökar sin aktivitet på APP vid höga halter kolesterol som är vanligt vid DMT2, Aβ fungerar då som en negativ inhibitor till HMG-Coa reduktas (HMGR), enzymet som bildar kolesterol och kan därmed reglera kolesterolhalterna. Regleringssystem för Aβ i blod-hjärnbarriären (BBB) som p-GP, LRP-1 och RAGE rubbas vid DMT2. Aβ och insulin delar samma degraderingssystem, insulin degrading enzyme (IDE), som reglerar halterna Aβ och insulin. Dessutom har Aβ oligomerer visat sig kunna bryta ned insulinreceptorer (IR). Vidare har läkemedel mot diabetes visat sig lindra demens hos AD patienter. I den här rapporten gås de molekylärfysiologiska sambanden igenom i detalj. Slutligen finns det fog för ett samband mellan metabolt syndrom, en riskfaktor för DMT2, och AD.