The fern genus Pteridium comprises a number of closely related species distributed throughout the world. Collectively they are called bracken ferns and have historically been treated as a single species, Pteridium aquilinum. Bracken is notorious as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily cultured and has become one of the most intensively studied ferns. Bracken has been used as a model system for the study of the fern life cycle, fern gametophyte development, the pheromonal mechanism of sex determination, toxicology, invasion ecology, and climate change. This dissertation places bracken within a global evolutionary perspective and establishes bracken as an emerging system for evolutionary genomics in ferns. Bracken samples from around the world were examined for chloroplast DNA variation to infer historical phylogenetic and biogeographic evolutionary events. New high-throughput DNA sequencing technologies and bioinformatic approaches were used to determine the complete chloroplast genome sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to identify gene sequences that are expressed in the gametophyte stage of the fern life cycle. These data represent an important genomic resource in ferns and were examined within a functional and evolutionary perspective. Several novel approaches and analyses were developed in the course of this research. Results presented in this dissertation provide novel insights into fern biology and land plant evolution.

<p><strong>Background: </strong>Lung cancer is the leading cause of cancer-related death and one of the most common cancer types worldwide. Epidermal growth factor receptor (EGFR) has been shown to be an important therapeutic target in non-small cell lung cancer. Kirsten rat sarcoma viral oncogene homologue (KRAS) is a downstream signalling molecule in the EGFR pathway. Lung cancer patients with <em>EGFR </em>mutations respond to tyrosine EGFR inhibitor therapy, in contrast, patients with <em>KRAS </em>mutations do not benefit of such treatment.</p><p><strong>Methods: </strong>This study investigates the frequency of <em>EGFR </em>and <em>KRAS </em>mutations in non-small cell lung cancer patients. Fifty-one lung cancer patients with primary non-small cell lung cancer diagnosed between 1995 and 2005 in the Uppsala-Örebro region were analysed by Sanger sequencing and Pyrosequencing to determine the mutation status of these genes.</p><p><strong>Results: </strong>Five <em>EGFR </em>mutations were found in four patients (8%), two deletions in exon 19, one point mutation in exon 20 and two point mutations in exon 21. <em>KRAS </em>mutations were found in 12 patients (24%), ten codon 12 mutations and two codon 61 mutations.</p><p><strong>Conclusions: </strong>This study confirms previous observations regarding the frequency of <em>EGFR </em>and <em>KRAS </em>mutations in non-small cell lung cancer. Mutations in <em>EGFR </em>and <em>KRAS </em>were mutually exclusive, indicating that both mutations present relevant tumorigenic genomic aberrations.</p>

Pyrosequencing is a DNA sequencing method based on thedetection of released pyrophosphate (PPi) during DNA synthesis.In a cascade of enzymatic reactions, visible light isgenerated, which is proportional to the number of nucleotidesincorporated into the DNA template. When dNTP(s) areincorporated into the DNA template, inorganic PPi is released.The released PPi is converted to ATP by ATP sulfurylase, whichprovides the energy to luciferase to oxidize luciferin andgenerate light. The excess of dNTP(s) and the ATP produced areremoved by the nucleotide degrading enzyme apyrase. The commercially available enzymes, isolated from nativesources, show batch-tobatch variations in activity and quality,which decrease the efficiency of the Pyrosequencing reaction.Therefore, the aim of the research presented in this thesis wasto develop methods to recombinantly produce the enzymes used inthe Pyrosequencing method. Production of the nucleotidedegrading enzyme apyrase by Pichia pastoris expression system,both in small-scale and in an optimized large-scale bioreactor,is described. ATP sulfurylase, the second enzyme in thePyrosequencing reaction, was produced inEscherichia coli. The protein was purified and utilizedin the Pyrosequencing method. Problems associated with enzymecontamination (NDP kinase) and batch-to-batch variations wereeliminated by the use of the recombinant ATP sulfurylase. As a first step towards sequencing on chip-format,SSB-(single-strand DNA binding protein)-luciferase and KlenowDNA polymerase-luciferase fusion proteins were generated inorder to immobilize the luciferase onto the DNA template. The application field for the Pyrosequencing technology wasexpanded by introduction of a new method for clone checking anda new method for template preparation prior the Pyrosequencingreaction. Keywords:apyrase, Pyrosequencing technology, Zbasictag fusion, luciferase, ATP sulfurylase, dsDNAsequencing, clone checking, Klenow-luciferase, SSB-luciferase,Pichia pastoris, Echerichia coli.

The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin. Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique. <b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras

Carrion decomposition is an essential ecosystem function as it is an important component of nutrient cycling. Carrion decomposition has primarily been attributed to insect consumption, with little attention given to microbial communities or their potential interactions with insects. The first objective was to use passive insect-trapping methods to assess primary colonizer communities on swine carcasses between two treatments: 1) carrion with access to insects and 2) carrion excluded from insect access for five days using exclusion cages. Despite similarities between succession patterns within each treatment, carcasses initially exposed to insects had significantly fewer insect taxa. Therefore, collections of adult insect communities associated with carrion are promising as an indication of whether or not there has been a delay in insect colonization of a resource. There has yet to be a study documenting bacterial communities during carrion decomposition. The second objective was to describe bacterial community succession and composition during decomposition in the presence and absence of naturally occurring insects. Total genomic DNA was used to identify bacterial community composition via a modified bacterial tagged encoded FLX amplicon pyrosequencing. I obtained 378,904 sequences and documented distinct bacterial community successional trajectories associated with insect access and exclusion carcasses. By the fifth day of decomposition, Proteus was the dominant (72%) bacterial genus on exclusion carcasses while Psychrobacillus (58%) and Ignatzschineria (18%) were dominant bacterial genera on insect carcasses. These data are the first to document bacterial community composition and succession on carrion. My final objective was to assess microbial community function in response to carrion insect colonization using metabolic profiling. I characterized microbial community metabolic function in the presence and absence of the primary necrophagous insects. I documented significant microbial community metabolic profile changes during active decomposition of carcasses. Mean carcass microbial community metabolic function with insect access continuously decreased over decomposition during both field seasons. Thus demonstrating microbial metabolic activity may have discriminatory power to differentiate early and late stages of decomposition. Overall, my data contributes to an understudied area of microbial research important to organic matter decomposition, forensic entomology, and microbial-insect ecological interactions.

All contemporary taurine cattle originated some 10,000 years ago when their wild ancestor, the aurochs, was domesticated in the Near East. Although the aurochs was widespread also in Europe, there is no evidence for a local domestication. The aurochs has been extinct since 1627 and therefore little is known about its biology. Following domestication, cattle were selected for traits of interest to humans. All modern cattle breeds were developed in the 19th century and the only sources of information about prehistoric breeding practices, and breeds, come from a few ancient Roman Empire and medieval European written accounts. The aim for this thesis was to investigate the effects early selection may have had on the cattle genome and to investigate genetic variation in European aurochs. Using second-generation sequencing and coalescent simulation analyses of aurochs Y chromosomal DNA, I estimated effective population size to between 20,000-80,000 aurochs bulls, indicating that a large population was present when domestic cattle entered Europe. A Y chromosomal SNP revealed that the two male lineages present in modern cattle were also present in European aurochs, and that the frequency of these lineages in domestic cattle fluctuated over time. This indicates that cattle were mobile and that bottlenecks, possibly due to selective breeding, occurred. I used nuclear SNPs to trace genetic variation in North European cattle through time and show that when genetics is combined with archaeology and osteology, even small but notable changes in the use of cattle can be detected. There has been a significant decrease in genetic variation over time, with the most dramatic changes associated with the formation of breeds during the 19th century.

DNA as forensic evidence is valuable in criminal investigations. Implementation of new, sensitive and fast technologies is an important part of forensic genetic research. This thesis aims to evaluate new sensitive methods to apply in forensic DNA analysis including analysis of old skeletal remains. In Paper I and II, two novel systems for analysis of STRs, based on the Pyrosequencing technology, are presented. In Paper I, Y chromosomal STRs are analysed. Markers on the male specific Y chromosome are especially useful in analysis of DNA mixtures. In Paper II, ten autosomal STRs are genotyped. The systems are based on sequencing of STR loci instead of size determination of STR fragments as in routine analysis. This provides a higher resolution since sequence variants within the repeats can be detected. Determination of alleles is based on a termination recognition base. This is the base in the template strand that is excluded from the dispensation order in the sequencing of the complementary strand and therefore terminates the reaction. Furthermore, skeletal remains are often difficult to analyse, due to damaging effects from the surrounding environment on the DNA and the high risk of exogenous contamination. Analysis of mitochondrial DNA is useful on degraded samples and in Paper III, mtDNA analysis of 700 years old skeletal remains is performed to investigate a maternal relationship. The quantity and quality of DNA are essential in forensic genetics. In Paper IV the efficiency of DNA isolation is investigated. Soaking skeletal remains in bleach is efficient for decontamination but result in a lower DNA yield, especially on pulverised skull samples. In conclusion, this thesis presents novel sequencing systems for accurate and fast analysis of STR loci that can be useful in evaluation of new loci and database assembly as well as the utility of mtDNA in forensic genetics.

Cancer is an important public health concern in the western world, responsible for around 25% of all deaths. Although improvements have been made in the diagnosis of cancer, treatment of disseminated disease is inefficient, highlighting the need for new and improved methods of diagnosis and therapy. Tumours arise when the balance between proliferation and differentiation is perturbed and result from genetic and epigenetic alterations. Due to the heterogeneity of cancer, analysis of the disease is difficult and a wide range of methods is required. In this thesis, a number of techniques are demonstrated for the analysis of genetic, epigenetic and transcriptional alterations involved in cancer, with the purpose of identifying a number of molecular signatures. Pyrosequencing proved to be a valuable tool for the analysis of both point mutations and CpG methylation. Using this method, we showed that oncogenes BRAF and NRAS, members of the Ras-Raf-MAPK pathway, were mutated in 82% of melanoma tumours and were mutually exclusive. Furthermore, tumours with BRAF mutations were more often associated with infiltrating lymphocytes, suggesting a possible target for immunotherapy. In addition, methylation of the promoter region of the DNA repair gene MGMT was studied to find a possible correlation to clinical response to chemotherapy. Results showed a higher frequency of promoter methylation in non-responders as compared to responders, providing a possible predictive role and a potential basis for individually tailored chemotherapy. Microarray technology was used for transcriptional analysis of epithelial cells, with the purpose of characterization of molecular pathways of anti-tumourigenic agents and to identify possible target genes. Normal keratinocytes and colon cancer cells were treated with the antioxidant N-acetyl L-cysteine (NAC) in a time series and gene expression profiling revealed that inhibition of proliferation and stimulation of differentiation was induced upon treatment. ID-1, a secreted protein, was proposed as a possible early mediator of NAC action. In a similar study, colon cancer cells were treated with the naturally occurring bile acid ursodeoxycholic acid (UDCA) in a time series and analysed by microarray and FACS analysis. Results suggest a chemopreventive role of UDCA by G1 arrest and inhibition of cell proliferation, possibly through the secreted protein GDF15. These investigations give further evidence as to the diversity of cancer and its underlying mechanisms. Through the application of several molecular methods, we have found a number of potential targets for cancer therapy. Follow up studies are already in progress and may hopefully lead to novel methods of treatment. / QC 20110121

<p>Digital microfluidics as implemented in electrowetting-on-dielectric (EWD) technology has been widely used as a platform for miniaturizing the biomedical or biochemical laboratory on a chip in recent years. DNA pyrosequencing, one of the DNA sequencing-by-synthesis methods, has been successfully integrated on EWD devices. However, this platform requires microliters of reagents and 200~300V of applied voltages, which contributes to higher costs and limits the feasibility of a portable system. This dissertation proposes a low voltage EWD device using multi-layer insulators that can manipulate picoliter droplets on chip. A 300pl droplet was dispensed and actuated at voltages as low as 11.4Vrms and 7.2Vrms respectively on a 95um electrode a EWD device with a 20um SU8 gasket. The stacked insulators in the actuator consisted of 135nm tantalum pentoxide (Ta2O5) and 180nm parylene C films deposited and coated with 70 nm of CYTOP. The physical scaling of electrodes was further demonstrated for 33um and 21um electrode devices, resulting in droplets of 12pl and 5pl respectively in conjunction with 3um gaskets. Manipulation of magnetic beads during dispensing, droplet splitting and merging, and droplet transport were also demonstrated on the scaled EWD devices. The chemiluminescent light produced by the on-chip reaction of 100pl ATP-luciferin and luciferase could be detected with an external cooled CCD camera, but detecting this reaction with smaller-scale droplet reactions was limited by the external detector's sensitivity. Based on fundamental theories and experiments, the actuation voltage and dimensional scaling of EWD devices have been demonstrated, but the use of picoliter droplets in biochemical applications will required improved sensing methods.</p> / Dissertation

<p>The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin.</p><p>Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis.</p><p>After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique.</p><p><b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras</p>