Parkinson’s Disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s and old age is the strongest risk factor for developing PD. PD has traditionally been seen as a motor disorder, but its non-motor symptoms such as dysautonomia, sensory dysfunction, sleeping problems and neuropsychiatric features equally add to the disease burden. There is no cure for PD and this is probably a reflection of our poor understanding of the disease pathogenesis. One way of tackling this is to focus on the small, but significant number of PD patients with a family history compatible with Mendelian autosomal inheritance (10-15%). Hereditary and sporadic PD share important clinical and neuropathological features, and there is reasonable hope that dissecting molecular pathways of PD gene products will have more general implications for the pathophysiology of PD associated neurodegeneration and help device new treatment strategies. Mutations in the FBXO7 gene have recently been shown to cause an autosomal recessive early onset Parkinsonian-pyramidal syndrome and FBXO7 has been designated as PARK 15 (Di Fonzo et al., 2009). FBXO7 is a member of the F-box protein family, which functions as the variable subunit of Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complexes and as such dictate substrate specificity. The canonical outcome of ubiquitylation is proteasomal degradation and my working hypothesis is that FBXO7 may be involved in protein quality control in the brain. A perturbation thereof may be a first step towards FBXO7 dependent disease. At the time of starting with my PhD project, little was known about the molecular function of FBXO7 and how mutations in FBXO7 result in neurodegeneration. In order to learn more and dissect the signalling pathway of FBXO7 I have used tagged stable overexpression cell lines of the FBXO7 wildtype as well as human disease mutant proteins for tag-pulldowns followed by mass-spectrometry to identify interacting partners and possible substrates. With this approach I have been able to confirm the interaction between FBXO7 and its core SCF E3 ligase partners as well as some of the previously reported interacting partners. I have been able to show that not only the FBXO7 wildytpe protein, but also all of the so far reported human disease mutants are able to assemble into an SCF complex. Hence, my fist conclusion is that the human disease mutants do not exert their pathogenicity by SCF complex disruption. Next, a knock-in (KI) mouse model of one of the pathogenic FBXO7 mutations (R378G) was generated and evaluated by molecular and biochemical approaches as well as motor and behaviour phenotyping. In particular, I have used the Fbxo7 mouse model for extensive proteomic screens to identify wildtype (wt) and KI Fbxo7 interactors: endogenous Fbxo7 immunoprecipitations from mouse brain lysates and subsequent fingerprint mass-spectrometry; differential whole proteome: ex vivo differential dimethyl labelling of wt and KI brain samples, and Fbxo7-dependent ubiquitinome analysis: quantitative di-GLY capture proteomics combining in vivo SILAC labelling with antibody-based affinity enrichment of “di-GLY remnant motifs”- containing peptides prior to proteomic profiling of the wild-type in comparison to the homozygous R379G Fbxo7 KI ubiquitinome in MEF lysates. The di-GLY remnant motif is the signature peptide of ubiquitinylated protein sites at peptide level after tryptic digestions. Some of my findings are: • For the first time I show that endogenous Fbxo7 actually assembles into an Skp1-Cullin1-Fbxo7 complex and that the pathogenic R378G does not disrupt SCFFbxo7-KI complex formation in vivo. This is true for the Fbxo7 KI mouse model, but also for patient derived immortalized cell lines carrying the R378G FBXO7 mutation.• Endogenous Fbxo7 interacts with the Sumo E3 ligase complex RanBP2/ RanGAP1*Sumo1/Ubc9 complex. • In the differential enrichment of ubiquitylated protein species in SILAC labelled wild-type and homozygous R379G Fbxo7 KI MEFs, I have clearly identifies 2 highly conserved lysine residues, which are conserved amongst VDAC 1, 2, and 3 in mouse as well as human homologous, to be preferentially ubiquitinylated in a Fbxo7 wild-type background (in collaboration with Dr. Patrick Pedrioli, MRC Programme leader).• There is a significant difference in motor performance between wildtype and homozygous R379G KI Fbxo7 mice at 10 months of age (in collaboration with Dr. Steve Martin, Neuroscience Division, Dundee). • Furthermore, I have successfully set up an in vitro FBXO7 dependent ubiquitinylation assays.

The interferon (lFN) signalling and RNA interference (RNAi) pathways are the major antiviral pathways in animals and plants, respectively. Although the mechanism of RNAi remains to be completely characterised, the genes that encode the proteins involved in this process have been identified in the plant, fungi and animal kingdoms (Fagard et al., 2000, Grishok et aI., 2000, Hall et al., 2003, Kanellopoulou et al., 2005, Kolb et al., 2005); with comparative analyses indicating that RNAi is an evolutionarily conserved mechanism. Several studies have identified RNAi suppressors encoded by animal viruses, suggesting an antiviral role for the RNAi pathway in animals as well as plants (Andersson et al., 2005, Bennasser et al., 2006, Garcia et al., 2006, Li et al., 2004, Lichner et al., 2003, Lingel et al., 2005, Lu & Cullen, 2004, Wang et al., 2006). However, most of these studies were performed in non-mammalian systems and as yet, there is no direct evidence indicating that the RNAi pathway plays a significant antiviral role during the infection of mammalian cells. Interestingly, several viruses have now been shown to express their own microRNA (miRNA) in infected cells (Grey et al., 2005, Pfeffer et al., 2005, Pfeffer et al., 2004, Samols et al., 2005, Sullivan et al., 2005). Further, in the case of hepatitis C virus (HCV), there is evidence that the virus usurps the host cell miRNAs to enhance viral replication (Jopling et al., 2005). The principal aim of this project was to investigate the role of RNAi in mammalian cells during viral infection, particularly infection with the influenza A virus. This thesis is divided into six major chapters followed by a brief general discussion. Chapter 1 contains a general introduction to the RNAi pathway. It describes the history of the discovery of RNAi and summarizes the known and proposed antiviral roles of the RNAi pathway in plants and mammalian cells. Chapter 2 describes the general materials and methods used for this project. There are four main result chapters, each dealing with a specific experimental system. Each chapter is divided into a brief introduction, specific materials and methods used, followed by presentation of the experimental results and a brief discussion. Chapter 3 describes the development of an in vitro Dicer activity assay to study the effect of viral proteins on the activity of the mammalian Dicer protein. It was demonstrated that crude cell lysates derived from influenza A virus infected cells impaired the activity of Dicer and this observation was not due to degradation of the Dicer protein by virally-induced proteases. Chapter 4 describes the use of a GFP reporter assay for screening potential RNAi suppressors. This assay is suitable for studying viral proteins in isolation. The effect of the influenza NS1 protein on the RNAi pathway in HEK293 cells was investigated and it was shown that NS1 could exert modest, but nevertheless significant, suppression of the RNAi pathway. Northern studies, performed to examine the processing of shRNA in the presence of NS1, demonstrated that NSI suppressed the RNAi mechanism through interfering with the maturation ofshRNA into siRNA. Chapter 5 describes the effect of over-expressing components of the RNAi pathway on influenza A virus infection. In these experiments, Exportin 5, which encodes a protein involved in the transport of pre-miRNA/shRNA into the cytoplasm, was over-expressed during influenza A virus infection. Reduced viral infection was observed in cells over-expressing Exportin 5, suggesting that this treatment protects cells from virus infection. Chapter 6 describes the expressed small RNA profile during influenza A virus infection in MDCK cells. Novel canine miRNA homologues were identified through cloning and sequencing. No definitive evidence for virally-derived siRNA/miRNA was found but a general reduction of endogenous miRNA expression was detected.

The exponential growth of information demands the automated movement of data and software via new software models that are able to integrate data and components on their own without scientists’ direct involvement. However, current stand-alone software modeling environments do not support a secure software execution, nor do client server applications allow user customization of the software running on the servers. To address this problem, a biological pathway modeling environment was built as a stand-alone Rich Internet Application (RIA). The modeling environment was tested by constructing a simulation of the glycolysis pathways in the human erythrocytes, and the results were compared against one of the latest and richest erythrocyte metabolism models developed by Kuchel and Mulquiney. The working simulation was able to settle into a quasi-stable state, with substrate concentrations close to what Kuchel and Mulquiney presented. It was also found that while the browser environment does allow for dynamic applications to be developed, speed and performance do become major issues. In later versions, it is hoped that the performance of the simulator can be increased and that it will become possible to link models together and add collaboration tools.

Genes can be associated in numerous ways, e.g. by co-expression in micro-arrays, co-regulation in operons and regulons or co-localization on the genome. Association of genes often indicates that they contribute to a common biological function, such as a pathway. The aim of this thesis is to predict metabolic pathways from associated enzyme-coding genes. The prediction approach developed in this work consists of two steps: First, the reactions are obtained that are carried out by the enzymes coded by the genes. Second, the gaps between these seed reactions are filled with intermediate compounds and reactions. In order to select these intermediates, metabolic data is needed. This work made use of metabolic data collected from the two major metabolic databases, KEGG and MetaCyc. The metabolic data is represented as a network (or graph) consisting of reaction nodes and compound nodes. Interme- diate compounds and reactions are then predicted by connecting the seed reactions obtained from the query genes in this metabolic network using a graph algorithm. In large metabolic networks, there are numerous ways to connect the seed reactions. The main problem of the graph-based prediction approach is to differentiate biochemically valid connections from others. Metabolic networks contain hub compounds, which are involved in a large number of reactions, such as ATP, NADPH, H2O or CO2. When a graph algorithm traverses the metabolic network via these hub compounds, the resulting metabolic pathway is often biochemically invalid. In the first step of the thesis, an already existing approach to predict pathways from two seeds was improved. In the previous approach, the metabolic network was weighted to penalize hub compounds and an extensive evaluation was performed, which showed that the weighted network yielded higher prediction accuracies than either a raw or filtered network (where hub compounds are removed). In the improved approach, hub compounds are avoided using reaction-specific side/main compound an- notations from KEGG RPAIR. As an evaluation showed, this approach in combination with weights increases prediction accuracy with respect to the weighted, filtered and raw network. In the second step of the thesis, path finding between two seeds was extended to pathway prediction given multiple seeds. Several multiple-seed pathay prediction approaches were evaluated, namely three Steiner tree solving heuristics and a random-walk based algorithm called kWalks. The evaluation showed that a combination of kWalks with a Steiner tree heuristic applied to a weighted graph yielded the highest prediction accuracy. Finally, the best perfoming algorithm was applied to a microarray data set, which measured gene expression in S. cerevisiae cells growing on 21 different compounds as sole nitrogen source. For 20 nitrogen sources, gene groups were obtained that were significantly over-expressed or suppressed with respect to urea as reference nitrogen source. For each of these 40 gene groups, a metabolic pathway was predicted that represents the part of metabolism up- or down-regulated in the presence of the investigated nitrogen source. The graph-based prediction of pathways is not restricted to metabolic networks. It may be applied to any biological network and to any data set yielding groups of associated genes, enzymes or compounds. Thus, multiple-end pathway prediction can serve to interpret various high-throughput data sets.

Syfte: Att beskriva sjuksköterskors upplevelser av att vårda patienter i livets slutskede enligt en vårdplan utformad som en journal med riktlinjer för vård i livets slutskede, den så kallade Liverpool Care Pathway (LCP). Metod: Studien är utförd som en litteraturöversikt. Artiklarna söktes via databaserna CINAHL, PubMed, MedLine, SAGE Journals och Scopus. Tolv artiklar valdes ut för kvalitétsgranskning enligt en förutbestämd granskningsmall. Elva artiklar godkändes och utgjorde grunden för resultatet. Resultat: En tydlig effekt av införandet av LCP var att sjuksköterskorna upplevde ett ökat självförtroende och en ökad kunskap i vårdandet. Detta bidrog till förbättringar gällande symtomkontroll samt ifrågasättande av fortsatt rutinmässig behandling. LCP anågs vara en bra utgångspunkt för samtal och undervisning i möten med patienter och anhöriga. LCP upplevdes ge ramar och gemensamma utgångspunkter i teamarbetet runt patienten vilket bidrog till en bättre planering och tydligare mål i vårdandet. Trots alla positiva upplevelser kände en del sjuksköterskor att de kunde uppstå svårigheter i vårdandet enligt LCP. Brister i vårdmiljön ansågs av sjuksköterskorna göra att målen med vården inte kunde tillgodoses. Tidsbrist och underbemanning ledde ofta till att patienter i livets slutskede fick en lägre prioritet. Vidare ansågs kriterierna vara för hårda enligt vissa sjuksköterskor gällande införande av LCP.

This thesis describes the characterization of benzene-degrading denitrifying cultures. Four objectives were pursued. The first objective was to identify conditions that promote or inhibit benzene decomposition and thus, to improve the biodegradation capacity of the cultures. FeS, resazurin, and nitrite had a detrimental impact on benzene degradation, whereas addition of supernatant from an active culture improved the benzene degradation activity by reducing the lag times. The second objective was to determine the microbial community composition in enrichment cultures and to identify the bacterial species that mediate benzene mineralization. Five dominant bacterial Operational Taxonomic Units (OTUs) were identified. The most abundant phylotype was related to the gram-positive Peptococcaceae family. Other bacteria present were closely affiliated with Dechloromonas, Azoarcus, Chlorobi and Anammox species. To correlate the growth of these specific microbes with benzene degradation, the abundance of specific 16S rRNA genes was monitored during mineralization process using quantitative polymerase chain reaction (qPCR). Based on the result of qPCR experiments and information about the metabolisms of the above bacteria, a syntrophic mode of benzene degradation was hypothesized to occur under denitrifying conditions. In this process, Peptococcaceae initiate attack on benzene, and ferment benzene to hydrogen and low molecular weight products such as acetate. These products are then consumed by nitrate-respiring Azoarcus and Dechloromonas or phototrophic Chlorobi. Anammox bacteria recycle and detoxify nitrite, and stabilize the culture. The third objective was to isolate and characterize pure cultures with the ability to mineralize benzene anaerobically. Dechloromonas- and Dechlorosoma-like microorganisms were isolated from several benzene-degrading microcosms. Theses bacteria, however, were not able to metabolize benzene anaerobically. The fourth objective was to investigate the key metabolic steps in the anaerobic benzene degradation pathway and to identify enzymes that are involved in this process. Differential transcription during growth of the culture on benzene versus growth on a metabolite of benzene degradation, i.e. benzoate was examined. Carboxylase-related genes were specifically transcribed in the presence of benzene. Furthermore, mRNA sequences corresponding to the genes that encode different enzymes of the benzoyl-CoA degradation pathway were present in the culture. These findings suggest that mineralization of benzene starts by its activation to benzoate through a carboxylation reaction catalyzed by benzene carboxylase. Benzoate is further metabolized through benzoyl-CoA pathway.

The secretory pathway of plant cells consists of several organelles that are connected by vesicle and tubular transport. Every compartment has a distinct function and the specificity of vesicle fusion is essential to maintain the organelles identity. N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in the secretory pathway driving specific vesicle fusions. A vesicle SNARE (v-SNARE) on a vesicle specifically interacts with two or three target SNAREs (t-SNAREs) on the target compartment. This event leads to vesicle membrane fusion with the membrane of the target compartment and the release of cargo molecules into the organelle lumen.<p> The aim of this work was the characterization of two <i>Arabidopsis thaliana</i> SNAREs. The first one is a v-SNARE, Bet11 that is the Arabidopsis ortholog of the yeast and mammal ER-Golgi v-SNARE, Bet1. In these organisms, Bet1 is involved in trafficking between the ER and Golgi apparatus. The second protein studied is a putative SNARE called Bet12 that shares high sequence identity with Bet11. In particular, I was interested in studying the sorting of these two proteins and their role in the secretory pathway of plant cells. By confocal laser microscopy, I demonstrated that these two proteins have different intracellular localization: Bet11 was mainly localized on the ER, Golgi stacks and punctate structures that I have identified as endosomes. Bet12 was localized only on the Golgi stacks. The identification of signal(s) involved in targeting of Bet11 and Bet12 were studied. To reach this aim I generated different mutant chimeras of Bet11 and Bet12. The co-expression of these chimeras with specific protein markers suggested that the distribution of these proteins was the result of a combined influence of multiple domains.<p> A serine in the Bet11 sequence was identified as a putative phosphorylation site and appeared important for proper Bet11 intracellular distribution.<p> The different intracellular distributions of Bet11 and Bet12 suggest different biological roles for the two proteins. To functionally characterize these two proteins homozygous knock-down mutants of Bet11 were screened. These plants had no evident phenotype, suggesting a possible genetic redundancy in this SNARE family.

The global gene expression profiles of industrial strains of <i>Saccharomyces cerevisiae</i> responding to nitrogen deficiency and very high sugar concentrations stresses were determined by oligonucleotide microarray analysis of ~ 6200 yeast open reading frames. Genomics analysis showed that 400 genes in S. cerevisiae was differentially expressed by more than 1.5-fold compared with controls at late-logarithmic phase of fermentation, as the yeast adapted to changing nutritional, environmental and physiological conditions. The genes of many pathways are regulated in a highly coordinated manner. The repressed expression of GDH1 and up-regulation of ARO10 within the contrast of Q270/Q10 indicated high energy demanding of yeast cells under high sugar stress. Activities of G3P shuttle indicated that under very high gravity environment, sufficient assimilatory nitrogen enhances yeasts ability of redox balancing, and therefore higher stress-tolerance and higher fermentation efficiency of yeast. Under contrast W270/Q270, the up-regulation of DUR1,2 responsible for urea degradation induces the glutamate biosynthesis and the consumption of -ketoglutarate. This may indicate that higher nitrogen level would enable higher activities in the TCA cycle, and therefore generate more energy for biosynthesis and yeast cell proliferation under very high gravity fermentation conditions. Nitrogen metabolism was also stimulated by high nitrogen level when yeast was grown in very high gravity environment.

The fundamental principles governing the natural phenomena of life is one of the critical issues receiving due importance in recent years. Most complex real-world systems are found to have a similar networking model that manages their behavioral pattern. Recent scientific discoveries have furnished evidence that most real world networks follow a scale-free architecture. A number of research efforts are in progress to facilitate the learning of valuable information by recognizing the underlying reality in the vast amount of genomic data that is becoming available. A key feature of scale-free architecture is the vitality of the highly connected nodes (hubs). This project focuses on the multi-cellular organism <i>Drosophila melanogaster</i>, an established model system for human biology. The major objective is to analyze the protein-protein interaction and the metabolic network of the organism to consider the architectural patterns and the consequence of removal of hubs on the topological parameters of the two interaction networks. <p> Analysis shows that both interaction networks pursue a scale-free model establishing the fact that real networks from varied situations conform to the small world pattern. Similarly, the topology of the two networks suffers drastic variations on the removal of the hubs. It is found that the topological parameters of average path length and diameter show a two-fold and three-fold increase on the deletion of hubs for the protein-protein interaction and metabolic interaction network, respectively. The arbitrary exclusion of the nodes does not show any remarkable disparity in the topological parameters of the two networks. This aberrant behavior for the two cases underscores the significance of the most linked nodes to the natural topology of the networks.

This thesis describes the characterization of benzene-degrading denitrifying cultures. Four objectives were pursued. The first objective was to identify conditions that promote or inhibit benzene decomposition and thus, to improve the biodegradation capacity of the cultures. FeS, resazurin, and nitrite had a detrimental impact on benzene degradation, whereas addition of supernatant from an active culture improved the benzene degradation activity by reducing the lag times. The second objective was to determine the microbial community composition in enrichment cultures and to identify the bacterial species that mediate benzene mineralization. Five dominant bacterial Operational Taxonomic Units (OTUs) were identified. The most abundant phylotype was related to the gram-positive Peptococcaceae family. Other bacteria present were closely affiliated with Dechloromonas, Azoarcus, Chlorobi and Anammox species. To correlate the growth of these specific microbes with benzene degradation, the abundance of specific 16S rRNA genes was monitored during mineralization process using quantitative polymerase chain reaction (qPCR). Based on the result of qPCR experiments and information about the metabolisms of the above bacteria, a syntrophic mode of benzene degradation was hypothesized to occur under denitrifying conditions. In this process, Peptococcaceae initiate attack on benzene, and ferment benzene to hydrogen and low molecular weight products such as acetate. These products are then consumed by nitrate-respiring Azoarcus and Dechloromonas or phototrophic Chlorobi. Anammox bacteria recycle and detoxify nitrite, and stabilize the culture. The third objective was to isolate and characterize pure cultures with the ability to mineralize benzene anaerobically. Dechloromonas- and Dechlorosoma-like microorganisms were isolated from several benzene-degrading microcosms. Theses bacteria, however, were not able to metabolize benzene anaerobically. The fourth objective was to investigate the key metabolic steps in the anaerobic benzene degradation pathway and to identify enzymes that are involved in this process. Differential transcription during growth of the culture on benzene versus growth on a metabolite of benzene degradation, i.e. benzoate was examined. Carboxylase-related genes were specifically transcribed in the presence of benzene. Furthermore, mRNA sequences corresponding to the genes that encode different enzymes of the benzoyl-CoA degradation pathway were present in the culture. These findings suggest that mineralization of benzene starts by its activation to benzoate through a carboxylation reaction catalyzed by benzene carboxylase. Benzoate is further metabolized through benzoyl-CoA pathway.