<p>Abstract</p><p>Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.</p>

DNA samples from crime scenes or mass disasters are often limited and degraded which limits the possibility of successful traditional STR analysis. Moreover, there is a need to decrease the turnaround time in criminal investigations. These circumstances require a wider set of assays and technologies to be investigated for potential use in forensic DNA analysis, which has been explored in this thesis work. DNA analysis can also provide a useful tool in forensic pathology investigations. In a search for mutations involved in The Sudden Infant death Syndrome (SIDS), the entire mitochondrial genome was sequenced in six SIDS infants and shorter mtDNA regions were analysed in paraffin-embedded tissues from an additional 14 SIDS cases. In this sample material no mutations associated with SIDS were found that could explain the death of these infants. To reduce time, cost and effort related to sequencing of the mtDNA HVI/HVII regions in caseworks, a HVI/HVII mtDNA linear array assay was used as a pre-screening for exclusions of suspects or evidence samples. Using this assay, 56% of the samples involved in casework analysis could be excluded before sequencing was undertaken. The possibility to use the new array technology was explored in a SNP assay targeting both mtDNA and nuclear SNPs. The system relies on minisequencing in solution prior to hybridisation to tag arrays. Using this system, we demonstrate a rapid, highly multiplexable and flexible array-format for SNP analysis. The properties of the Pyrosequencing technology being a fast and user-friendly assay was utilised in a study to investigate the possibility to use this method for limited and degraded samples. Ten STR loci, overlapping with standardised kits, were genotyped in 114 Swedish individuals. We found additional variation and higher resolution of repeats at some of these loci that are not detected using standard fragment analysis.

Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used. This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible. The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies. / QC 20101006

Abstract Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.

The aim of this study was to find out how features that have been found to be typical of women’s language, such as hedges, tag questions and a high level of talkativeness etc., are represented in a popular TV series. Five cross-sex conversations from one episode of the sitcom The New Adventures of Old Christine were analyzed, and the results show that many of the features of interest, as for instance tag questions, minimal responses and indirect style, are unexpectedly used more frequently by men in this small investigation. In fact, the only feature that was used more frequently by the female main character was hedges. Several factors affect the results of the study, as for instance the fact that the conversations are fictional. The special characteristics of the speakers also affect the results, as well as the tone and the topic of the chosen conversations. Many of the features of interest were used to a very small extent, which is probably a result of the fact that the language in a sitcom is to be entertaining and rather quick, which leaves little or no room for the features studied.

Interactive packaging is an emerging research area in recent years. It brings people convenient and smart lives, reduces consumption of traditional packaging materials and direct or indirect labor costs as well. Being integrated in interactive packaging, Radio Frequency Identification (RFID) technology becomes one of the most proactive development enablers. In this paper, an interactive and intelligent packaging solution integrating passive RFID system and Controlled Delamination Material (CDM) is given at first. Package opening action is electrically controlled by the RFID system. CDM is primarily used in aerospace applications in the past and the conductor/adhesive joint can be easily opened by applying a little electric power on to the material. Some related works will be shown about the electrochemical characteristics of CDM in order to facilitate the system design. A demonstration system was developed and the test results have proved feasibility of the solution and shown the potential of low cost for mass production. Based on this solution, an interactive medication package for pervasive healthcare is further developed, using EPCglobal Gen2 RFID technology. It will make the medication being accessible for patient only at the prescribed dose and time, and medication taking information will be delivered as well. Such medication package will not only give unprecedented high patient compliance, but also improve the communication between patients and healthcare staffs. / QC 20111202

Motivation: As the human genome project progresses and some microbial and eukaryotic genomes are recognized, numerous biotechnological processes have attracted increasing number of biologists, bioengineers and computer scientists recently. Biotechnological processes profoundly involve production and analysis of highthroughput experimental data. Numerous sequence libraries of DNA and protein structures of a large number of micro-organisms and a variety of other databases related to biology and chemistry are available. For example, microarray technology, a novel biotechnology, promises to monitor the whole genome at once, so that researchers can study the whole genome on the global level and have a better picture of the expressions among millions of genes simultaneously. Today, it is widely used in many fields- disease diagnosis, gene classification, gene regulatory network, and drug discovery. For example, designing organism specific microarray and analysis of experimental data require combining heterogeneous computational tools that usually differ in the data format; such as, GeneMark for ORF extraction, Promide for DNA probe selection, Chip for probe placement on microarray chip, BLAST to compare sequences, MEGA for phylogenetic analysis, and ClustalX for multiple alignments. Solution: Surprisingly enough, despite huge research efforts invested in DNA array applications, very few works are devoted to computer-aided optimization of DNA array design and manufacturing. Current design practices are dominated by ad-hoc heuristics incorporated in proprietary tools with unknown suboptimality. This will soon become a bottleneck for the new generation of high-density arrays, such as the ones currently being designed at Perlegen [109]. The goal of the already accomplished research was to develop highly scalable tools, with predictable runtime and quality, for cost-effective, computer-aided design and manufacturing of DNA probe arrays. We illustrate the utility of our approach by taking a concrete example of combining the design tools of microarray technology for Harpes B virus DNA data.

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.

This thesis provides an in-depth analyze of expressed sequence tags (EST) that represent pieces of eukaryotic mRNA by using unsupervised hidden Markov model (HMM). ESTs are short nucleotide sequences that are used primarily for rapid identificationof new genes with potential coding regions (CDS). ESTs are made by sequencing on double-stranded cDNA and the synthesizedESTs are stored in digital form, usually in FASTA format. Since sequencing is often randomized and that parts of mRNA contain non-coding regions, some ESTs will not represent CDS.It is desired to remove these unwanted ESTs if the purpose is to identifygenes associated with CDS. Application of stochastic HMM allow identification of region contents in a EST. Softwares like ESTScanuse HMM in which a training of the HMM is done by supervised learning with annotated data. However, because there are not always annotated data at hand this thesis focus on the ability to train an HMM with unsupervised learning on data containing ESTs, both with and without CDS. But the data used for training is not annotated, i.e. the regions that an EST consists of are unknown. In this thesis a new HMM is introduced where the parameters of the HMM are in focus so that they are reasonablyconsistent with biologically important regionsof an mRNA such as the Kozak sequence, poly(A)-signals and poly(A)-tails to guide the training and decoding correctly with ESTs to proper statesin the HMM. Transition probabilities in the HMMhas been adapted so that it represents the mean length and distribution of the different regions in mRNA. Testing of the HMM's specificity and sensitivityhave been performed via BLAST by blasting each EST and compare the BLAST results with the HMM prediction results.A regression analysis test shows that the length of ESTs used when training the HMM is significantly important, the longer the better. The final resultsshows that it is possible to train an HMM with unsupervised machine learning but to be comparable to supervised machine learning as ESTScan, further expansion of the HMM is necessary such as frame-shift correction of ESTs byimproving the HMM's ability to choose correctly positioned start codons or nucleotides. Usually the false positive results are because of incorrectly positioned start codons leadingto too short CDS lengths. Since no frame-shift correction is implemented, short predicted CDS lengths are not acceptable and is hence not counted as coding regionsduring prediction. However, when there is a lack of supervised models then unsupervised HMM is a potential replacement with stable performance and able to be adapted forany eukaryotic organism.

I Vänern, Sverige, fångades årligen ca 75 ton lax och öring av yrkesfisket, sportfisket och fritidsfisket under 1990-talet och början av 2000-talet. Fångsterna av lax och öring verkar ha sjunkit under de senaste åren men det råder stor osäkerhet över fångstuppskattningar. För en ökad förståelse över smolts mortalitet släpps varje år ett visst antal Carlin-märkta laxar och öringar ut i Vänern och Klarälven. Syftet med studien var att sammanställa och analysera databasen för Carlin-märkt lax och öring i Vänern under åren 1965 till 2005. Målet var att åskådliggöra långsiktiga tendenser över återfångster av Carlin-märkt lax och öring i både Vänern och Klarälven samt bedöma om återfångster varierar beroende på utsättningsplats (Vänern eller Klarälven). Microsoft Excel användes för att sammanställa och analysera återfångad och inrapporterad lax och öring från 1965 till 2005. För att bedöma trender för återfångster av de fyra stammarna, Gullspångs- och Klarälvs-, lax och öring, användes linjär regression. Totalt 299 165 Carlin-märkta fiskar fördelade över 388 utsättningsgrupper har släppts ut i Vänern med tillflöden under 40 års tid. Sammanlagt återfångades 14 504 fiskar, vilket motsvarar knappt 5 % av antalet Carlin-märkta och utsläppta fiskar. Återfångsterna har varierat genom åren (&lt;1 % - &gt;20 %), högst var återfångsterna under 1970-och 1980-talet, sedan 1990-talet har de minskat betydligt. Dessa tendenser är liknande för alla fyra stammar. De flesta återfångsterna sker i Vänern. Fisk utsläppt i Vänern återfångas som regel i något högre grad i Vänern än fisk utsläppt i Klarälven. Slutligen framkom att ingen lax eller öring utsläppt i Vänern återfångades i Klarälven. / In Lake Vänern, Sweden, commercial, sport, and subsistence fisheries in the 1990s and the beginning of 2000 caught about 75 tons of salmon and trout. Catches of salmon and trout appeared to have declined in recent years, although there is much uncertainty in catch estimates. In order to better understand smolt-adult mortality, a number of Carlin-tagged salmon and trout are released in Vänern and Klarälven each year. The aim of the study was to assemble and analyze the Carlin-tag database for salmon and trout in Vänern during the years 1965 to 2005. My objectives were to identify long-term trends in tag returns rates in both Vänern and Klarälven, and to assess whether return rates varied by release location (Vänern or Klarälven). Microsoft Excel was used to compile and analyze reported recoveries of Carlin-tagged salmon and trout from 1965 to 2005. Linear regression was used to assess trends in return rates of four stocks, Gullspångsälven and Klarälven salmon and trout. Total 299 165 tagged fish in 388 release groups have been released in Vänern over the 40-year period. Total recaptures were 14 504, which equates to just under 5 % of the number of Carlin-tagged and released fish. Recapture rates have varied throughout the years (&lt;1 % - &gt;20 %), the highest return rates were in the 1970s – 1980s, but they have decreased significantly since the 1990s. These trends are similar for all four stocks. Most of the recaptures occur in the lake, return rates of fish released in the lake are most often caught in the lake than are fish released in Klarälven. Finally, revealed that no salmon or trout that were released in the lake were recaptured in the Klarälven.