Prolidase has potential applications in cheese debittering, organophosphate detoxification and as an enzyme replacement therapy in prolidase-deficient patients. Recombinant Lactococcus lactis prolidases and their catalytic properties have previously been characterized in Dr. Tanaka's research group. Unlike other prolidases, L. lactis prolidase shows allosteric behaviour, metal-dependent substrate specificity and substrate inhibition. The current project focuses on elucidating the three-dimensional structure of L. lactis prolidase using X-ray crystallography. Hexagonal plate-like crystals of wild-type L. lactis prolidase were grown by the hanging drop vapour diffusion method, allowing the crystals to grow to about 50 µm in their longest dimension. The crystallization cocktail in which they grew contained 0.08 M sodium cacodylate (pH 6.5), 0.16 M calcium acetate, 14 % PEG 8000 and 18 % glycerol. Crystal diffraction data was collected at a wavelength of 0.9795 Å on beamline 08ID-1 of the Canadian Macromolecular Crystallography Facility at the Canadian Light Source and was processed using X-ray Detector Software. The crystals belonged to space group C2 and estimated to contain three molecules in an asymmetric unit. The electron density map of this structure was solved by the molecular replacement method and the structure model was refined against 2.25 Å resolution data. Molecule A forms a dimer with molecule B, while molecule C forms a dimer with molecule C', which is located in the neighbouring crystal asymmetric unit. The electron density of molecule A was well-defined and complete. Therefore, all the 362 amino acid residues of L. lactis prolidase were fitted. The other two molecules were incomplete and less defined. Only 360 and 352 residues could be fitted in molecules B and C, respectively. Molecule C, the worst of the three, compromised the overall quality of the refined structure. However, the functional interpretation of the structure was not compromised since the well-defined molecules form a dimer with each other and the biologically-functional form of L. lactis prolidase is a homodimer. The final Rwork and Rfree are 22.39 and 27.77, respectively. Comparison with other known prolidases revealed that Asp 36 and His 38 are unique to L. lactis prolidase. These residues have been shown to be involved in the allosteric behaviour and substrate inhibition of this enzyme, respectively. Therefore, this crystal structure further supports their suggested contribution in L. lactis prolidase's unique catalytic properties.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5 ' and 3 ' UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5 ' end (2-21 nt) of the 5 ' UTR. Nucleolin binds the hairpin loop structure in the 5 ' UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3 ' UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)(n), <= 26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

This dissertation concerns the study and prediction of loops in protein structures. Proteins perform crucial functions in living organisms. Despite their importance, we are currently unable to predict their three dimensional structure accurately. Loops are segments that connect regular secondary structures of proteins. They tend to be located on the surface of proteins and often interact with other biological agents. As loops are generally subject to more frequent mutations than the rest of the protein, their sequences and structural conformations can vary significantly even within the same protein family. Although homology modelling is the most accurate computational method for protein structure prediction, difficulties still arise in predicting protein loops. Protein loop structure prediction is therefore a bottleneck in solving the protein structure prediction problem. Reflecting on the success of homology modelling, I implement an improved version of a database search method, FREAD. I show how sequence similarity as quantified by environment specific substitution scores can be used to significantly improve loop prediction. FREAD performs appreciably better for an identifiable subset of loops (two thirds of shorter loops and half of the longer loops tested) than ab initio methods; FREAD's predictive ability is length independent. In general, it produces results within 2Å root mean square deviation (RMSD) from the native conformations, compared to an average of over 10Å for loop length 20 for any of the other tested ab initio methods. I then examine FREAD’s predictive ability on a specific type of loops called complementarity determining regions (CDRs) in antibodies. CDRs consist of six hypervariable loops and form the majority of the antigen binding site. I examine CDR loop structure prediction as a general case of loop structure prediction problem. FREAD achieves accuracy similar to specific CDR predictors. However, it fails to accurately predict CDR-H3, which is known to be the most challenging CDR. Various FREAD versions including FREAD with contact information (ConFREAD) are examined. The FREAD variants improve predictions for CDR-H3 on homology models and docked structures. Lastly, I focus on the local properties of protein loops and demonstrate that the protein loop structure prediction problem is a local protein folding problem. The end-to-end distance of loops (loop span) follows a distinctive frequency distribution, regardless of secondary structure elements connected or the number of residues in the loop. I show that the loop span distribution follows a Maxwell-Boltzmann distribution. Based on my research, I propose future directions in protein loop structure prediction including estimating experimentally undetermined local structures using FREAD, multiple loop structure prediction using contact information and a novel ab initio method which makes use of loop stretch.

La flexibilité est une caractéristique intrinsèque des protéines qui doivent, dès le mo- ment de leur synthèse, passer d’un état de chaîne linéaire à un état de structure tridimen- sionnelle repliée et enzymatiquement active. Certaines protéines restent flexibles une fois repliées et subissent des changements de conformation de grande amplitude lors de leur cycle enzymatique. D’autres contiennent des segments si flexibles que leur structure ne peut être résolue par des méthodes expérimentales. Dans cette thèse, nous présentons notre application de méthodes in silico d’analyse de la flexibilité des protéines : • À l’aide des méthodes de dynamique moléculaire dirigée et d’échantillonnage pa- rapluie, nous avons caractérisé les trajectoires de liaison de l’inhibiteur Z-pro- prolinal à la protéine Prolyl oligopeptidase et identifié la trajectoire la plus pro- bable. Nos simulations ont aussi identifié un mode probable de recrutement des ligands utilisant une boucle flexible de 19 acides aminés à l’interface des deux domaines de la protéine. • En utilisant les méthodes de dynamique moléculaire traditionnelle et dirigée, nous avons examiné la stabilité de la protéine SAV1866 dans sa forme fermée insérée dans une membrane lipidique et étudié un des modes d’ouverture possibles par la séparation de ses domaines liant le nucléotide. • Nous avons adapté auproblème de la prédiction de la structure des longues boucles flexibles la méthode d’activation et de relaxation ART-nouveau précédemment uti- lisée dans l’étude du repliement et de l’agrégation de protéines. Appliqué au replie- ment de boucles de 8 à 20 acides aminés, la méthode démontre une dépendance quadratique du temps d’exécution sur la longueur des boucles, rendant possible l’étude de boucles encore plus longues. / Flexibility is an intrinsic characteristic of proteins who from the moment of synthesis into a linear chain of amino acids, have to adopt an enzymatically active tridimensionnel structure. Some proteins stay flexible once folded and display large amplitude confor- mational changes during their enzymatic cycles. Others contain parts that are so flexible that their structure can’t be resolved using experimental methods. In this thesis, we present our application of in silico methods to the study of protein flexibility. • Using steered molecular dynamics and umbrella sampling, we characterized the binding trajectories of the Z-pro-prolinal inhibiter to the Prolyl oligopeptidase pro- tein and we identified the most probable trajectory. Our simulations also found a possible ligand recrutement mechanism that involves a 19 amino acids flexible loop at the interface of the two domains of the protein. • Using traditional and steered molecular dynamics, we examined the stability of the SAV1866 protein in its closed conformation in a lipid membrane and we studied one of its proposed opening modes by separating its nucleotide binding domains. • We also adapted the activation-relaxation technique ART-nouveau which was pre- viously used to study protein folding and aggregation to the problem of structure prediction of large flexible loops. When tested on loops of 8 to 20 amino acids, the method demonstrate a quadratic execution time dependance on the loop length, which makes it possible to use the method on even larger loops.

La flexibilité est une caractéristique intrinsèque des protéines qui doivent, dès le mo- ment de leur synthèse, passer d’un état de chaîne linéaire à un état de structure tridimen- sionnelle repliée et enzymatiquement active. Certaines protéines restent flexibles une fois repliées et subissent des changements de conformation de grande amplitude lors de leur cycle enzymatique. D’autres contiennent des segments si flexibles que leur structure ne peut être résolue par des méthodes expérimentales. Dans cette thèse, nous présentons notre application de méthodes in silico d’analyse de la flexibilité des protéines : • À l’aide des méthodes de dynamique moléculaire dirigée et d’échantillonnage pa- rapluie, nous avons caractérisé les trajectoires de liaison de l’inhibiteur Z-pro- prolinal à la protéine Prolyl oligopeptidase et identifié la trajectoire la plus pro- bable. Nos simulations ont aussi identifié un mode probable de recrutement des ligands utilisant une boucle flexible de 19 acides aminés à l’interface des deux domaines de la protéine. • En utilisant les méthodes de dynamique moléculaire traditionnelle et dirigée, nous avons examiné la stabilité de la protéine SAV1866 dans sa forme fermée insérée dans une membrane lipidique et étudié un des modes d’ouverture possibles par la séparation de ses domaines liant le nucléotide. • Nous avons adapté auproblème de la prédiction de la structure des longues boucles flexibles la méthode d’activation et de relaxation ART-nouveau précédemment uti- lisée dans l’étude du repliement et de l’agrégation de protéines. Appliqué au replie- ment de boucles de 8 à 20 acides aminés, la méthode démontre une dépendance quadratique du temps d’exécution sur la longueur des boucles, rendant possible l’étude de boucles encore plus longues. / Flexibility is an intrinsic characteristic of proteins who from the moment of synthesis into a linear chain of amino acids, have to adopt an enzymatically active tridimensionnel structure. Some proteins stay flexible once folded and display large amplitude confor- mational changes during their enzymatic cycles. Others contain parts that are so flexible that their structure can’t be resolved using experimental methods. In this thesis, we present our application of in silico methods to the study of protein flexibility. • Using steered molecular dynamics and umbrella sampling, we characterized the binding trajectories of the Z-pro-prolinal inhibiter to the Prolyl oligopeptidase pro- tein and we identified the most probable trajectory. Our simulations also found a possible ligand recrutement mechanism that involves a 19 amino acids flexible loop at the interface of the two domains of the protein. • Using traditional and steered molecular dynamics, we examined the stability of the SAV1866 protein in its closed conformation in a lipid membrane and we studied one of its proposed opening modes by separating its nucleotide binding domains. • We also adapted the activation-relaxation technique ART-nouveau which was pre- viously used to study protein folding and aggregation to the problem of structure prediction of large flexible loops. When tested on loops of 8 to 20 amino acids, the method demonstrate a quadratic execution time dependance on the loop length, which makes it possible to use the method on even larger loops.

Background Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species. Methodology/Principal Findings We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster. Conclusions/Significance The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

The power distribution system is the final link to transfer the electrical energy to the individual customers. It is distributed in a complex technical grid but is associated with the majority of all outages occurring. Improving its reliability is an efficient way to reduce the effects of outages. A common way of improving the reliability is by designing loop structures containing two connected feeders separated by a section point. The location of the section point will decide how the system structure is connected and its level of reliability. By finding the optimal location, an improved reliability may be accomplished. This Master’s thesis has developed a method of finding optimized section points locations in a primary distribution system in order to improve its reliability. A case study has been conducted in a part of Mälarenergi Elnät’s distribution system with the objective of developing an algorithm in MATLAB able to generate the optimal section points in the area. An analytical technique together with a method called Failure Modes and Effect Analysis (FMEA) as preparatory step, was used to simulate the impact of outages in various components based on historical data and literature reviews. Quantifying the impact was made by calculating the System Average Interruption Duration Index (SAIDI) and the Expected Cost (ECOST) which represented the reliability from a customer- and a socio-economic perspective. Using an optimization routine based on a Greedy algorithm an improvement of the reliability was made possible. The result of the case study showed a possible improvement of 28% on SAIDI and 41% on ECOST if optimizing the location of section points. It also indicated that loop structures containing mostly industry-, trade- and service-sectors may improve ECOST considerably by having a relocated section point. The analysis concluded that based on the considerable improvement the case study showed, a distribution system could be highly benefitted by optimizing the location of section points. The created algorithm may provide a helpful tool well representative for such a process in a cost-effective way. Applying it into a full size system was considered being possible but it would first require some additional improvements of reliability inputs and to resolve some fundamental issues like rated current in lines and geographical distances to substations.

Background Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species. Methodology/Principal Findings We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster. Conclusions/Significance The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.

In today’s society the electric grid has become one of the most important infrastructures and industries as well as other infrastructures, and individuals rely on its functionality. The unavailability of electricity caused by outages is increasingly rare but when it occurs it can have serious consequences. An electric grid’s reliability is now a requirement, not only from customers but also from the regulatory authority.The electric grid in Stockholm is owned and operated by Ellevio AB. Over the past decade the reliability, measured in SAIDI (System Average Interruption Duration Index), in the area has deterioated, mainly due to failures of the medium voltage grid (11- kV, MV). The network structure on the MV- grid consists primarily of two parallel medium voltage cables each of which acts redundantly to its pair cable. The technique, called dual cable structure, works most optimally with substations with automatic switching, which automaticly switch over to the redundant cable when failure occurs. However substations without this automatic switching are widely used resulting in a longer interruption time. Another network structure is the ring or loop structure, which still has redundancy, but when failure occurs a load- break switch must close so that the grid can be fed from another direction. The substation in the loop structure can also be remotely controlled, making the load-break swith operable from the control center, resulting in a shorter interruption time.This thesis evaluates and compares different network structures consisting of dual cable with automatic switching and loop structure with remotely controlled substations, based on economy and reliability, on Stora Essingen. The two structures have been designed in the network information system program Trimble NIS and divided into two different environments; one developed and one undeveloped environment. The results show that the technical solutions in network structures of MV- grid do not necessarily mean major differences in economy nor reliability, rather the choice between few but long interruptions and many but short interruptions is central. Results show however that a combination of the dual cable structure with remotely controlled substations in the undeveloped environment is economically motivated while the reliability can be maintained at the same tame.