It is well known that genetic variation and ability to adapt is crucial for the survival of anypopulation. Whether it be about a natural population’s ability to respond to changes in itsenvironment, or a population of livestock’s ability to produce more milk, genetic variation is akey element. Effectively small populations have an increased risk of extinction caused byreduced ability to adapt or respond to selection. Small populations are also more affected bygenetic drift, which can cause deleterious mutations to fixate, reducing the populations’ fitnesspossibly to the point where it is unable to survive. Models describing changes in allelefrequencies in a population under selection can be used to study a population’s response toselection. A limitation to such models is they often assume infinite population size and neglectthe effects of genetic drift, making them unable to implement when working with effectivelysmall populations.Here, an individual-based model of a quantitative trait affected by selection, mutation andgenetic drift is used to study the adaptive potential of effectively small populations. In a series ofsimulations, changes in the trait are explored under directional selection and stabilizingselection with adaptation to one, and several repeated shifts in optimum. Results of simulationinclude that populations under strong directional selection, such as breeding, potentially risklosing all adaptive potential. Results also suggest that effects of strong directional selectionmight be irreversible, even if the strong selective pressure is removed.

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Sepsis, a life-threatening condition with alarmingly high mortality rates, demands the development of improved diagnostic methods to better understand and manage the disease. Enterococcus spp., significant contributors to sepsis and known for their multidrug resistance, urgently require thorough and detailed investigation to devise effective treatment strategies and healthcare interventions. This study explores the genomic characterization of Enterococcus spp. isolates from patients with suspected community-onset sepsis in Sweden, using whole-genome sequencing. Data was processed through an in-house developed bioinformatics pipeline, including quality control with FastQC, sequence trimming with Trimmomatic, and assembly with Unicycler and QUAST, generating reliable FASTA files for the downstream analysis. Subsequently, different gene annotation tools were applied for the genotypic species identification, prediction of antibiotic resistance genes, plasmid replicons, and determination of sequence type. Moreover, the results from the genotypic characterization were compared to those obtained using routine microbiological methods based on cultures followed by MALDI-TOF MS for species identification and disk diffusion for antimicrobial resistance testing. The results revealed a high prevalence of resistance genes, particularly for macrolide, lincosamide, and streptogramin antibiotics. A notable finding was the high discordance (83.2%) between phenotypic and genotypic methods in detecting resistance, highlighting the complexity of correlating phenotypic antibiotic resistance with genotypic predictions. Additionally, a statistically significant higher prevalence of antibiotic resistance genes was observed in E. faecium compared to E. faecalis (p=0.001). Furthermore, a high diversity of sequence types among Enterococcus spp. isolates was detected by multi-locus sequencing typing, with ST6 (14%) as the most prevalent for E. faecalis and ST192 (37%) for E. faecium. In conclusion, this comprehensive genomic approach enhances the understanding of antibiotic resistance spread and informs strategies for improved clinical and public health interventions in sepsis management. The study also underscores the importance of integrating genomic data with traditional diagnostic methods to develop effective strategies for managing antibiotic resistant infections. / <p>Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.</p><p>There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.</p>

Ascetosporea is a class of poorly studied unicellular eukaryotes that function as parasites of marine invertebrates. These parasites cause mass mortality events in aquaculture species such as oysters and mussels. The economic importance of these aquaculture species should lead to more attention on the genomics of Ascetosporea and their place on the evolutionary tree of life. With the onset of global warming and rising sea levels and temperatures, many emerging pathogens have been seen and until these are sequenced and analysed, it is difficult to make any conclusions about their relationships and evolution. As there aren’t many genomes and transcriptomes available for Ascetosporea, their position in the larger eukaryotic tree of life remains hypothetical. To attempt to remedy this lack of information, the Burki lab has recently generated sequencing data through sample collection and sequencing for these organisms (genomes and transcriptomes). A curated dataset of the various eukaryotic species was previously created and newly sampled and sequenced Ascetosporean genomes of Paramarteilia sp., Marteilia pararefringens, Paramikrocytos canceri, etc. from multiple sampling locations like Ireland, Norway, Sweden, and the UK were included. These could increase the genomic and transcriptomic data available for Ascetosporea and help to resolve the relationships within Ascetosporea. A few reasons why this group has not yet been placed on the tree of life are that the samples are from host tissue, which makes it difficult to sequence these parasites. These Ascetosporeans have also been seen to be very fast-evolving. After building phylogenetic relationships with single gene trees to allow for the identification of possible contaminants and paralogs, it was seen that there was a lot of contamination in Ascetosporea, due to the sampling being from host tissue material (hosts are open to the environment). After cleaning and filtering the possible contaminated genes, the trees were remade and a possible link between a fungal group called Microsporidia and Ascetosporea was observed in a few genes. This was hypothesized to be lateral gene transfer between the two groups resulting from their similar lifestyles and infection of invertebrates. There were complications like contamination and short blast hits that arose during analysis, and these could be caused by problems by fragmentation in the genome. This fragmentation could have negative effects on genome annotation predictions and consequently phylogenetic and phylogenomic analysis. Due to this and the challenging nature of collecting samples, the read coverage for the genomes is low but it can be used to perform phylogenetic and phylogenomic studies using currently available data and methods. Another expected result was that the sequenced data had contaminants, and a thorough and comprehensive search would have to be conducted on a dataset-wide level to remove any contaminants.

<p>Of the pattern recognition receptors involved in innate immunity, the class A Scavenger receptors are involved in the recognition and clearance of bacteria, yeast, and senescent molecules. Previous research has implicated the intracellular region of these receptors as essential for the clearance of these substances via endocytosis. In this work, I used a bioinformatic approach to define the evolutionary history of the class A Scavenger receptor family while elucidating areas of conservation within the cytoplasmic domains of these proteins. With this information, in addition to further predictions of post translational modifications and potential docking motifs for interacting proteins, I conducted molecular biology experiments to study the in vitro functionality of the macrophage receptor with collagenous domain (MARCO), a member of the class A Scavenger receptors.</p> <p>Evolutionary analyses of the 5 class A Scavenger receptors identified a shared ancestry between these proteins and allowed me to postulate that 4 distinct gene du- plication events in addition to subsequent domain fusions, internal repeats, and dele- tions are responsible for the diverse protein structures and functions of this family. Despite some variation in domain structure, I found highly conserved regions across all 5 members, including a negatively charged region in the cytoplasmic domain. Further analyses of MARCO across organisms identified other conserved regions, in- cluding 2 residues predicted to be ubiquitinated, sumoylated, or phosphorylated by in silico predictive methods. However, molecular biology experiments demonstrated that these post translational modifications to not occur in the steady state. Addi- tional in vitro experiments, including isolations of MARCO and an artifical construct containing only the intracellular regions of the protein, were unable to identify any candidate adaptor binding proteins. Further research is needed to determine whether modifications in this region occur in the presence of bound ligands and/or known co-receptors.</p> / Master of Science (MSc)

<p>We introduce new software (ISAAC: Improved Structural Annotation of attC) to annotate cassette arrays in bacterial integrons by finding attI and attC sites, and to provide detailed annotation of the attC sites for analysis. We demonstrate an initial application of ISAAC by annotating the cassette complements of all the integrons we identified in the RefSeq bacterial genome database, and providing an analysis of the patterns of nucleotide frequencies at the structurally important positions in the attCs we’ve found.</p> / Master of Science (MSc)

Satellite DNA (satDNA) is among the fastest evolving elements in the genome and is highly abundant in some eukaryotic genomes. Its highly repetitive nature means it is challenging to assemble, and thus underrepresented in most assemblies and often understudied as a result. Birds are an ideal model organism for the study of satDNA and its evolution, since the large amount of available sequenced genomes of this clade allows for dense sampling across various evolutionary timescales, and the low number of satDNA families within their satellitomes facilitates their study and comparison between species. Here, we characterize satDNA and its evolution across Passeriformes, an avian clade containing two-thirds of all bird species spanning ~50 million years of evolution. With this goal we use both short-read data and long-read assemblies of species representative of over 30 passerine families in this clade to shed light on the evolution of its satellitome. We focus on examining the phylogenetic relationships between satellites common to most species as well as characterizing satellite array structure and location in genome assemblies. We also analyse satellite abundance in each genome, focusing on differences in the satellite content between male and female individuals to look for satellites present in the female-specific W sex chromosome and the germline-restricted chromosome. Seven satDNA families shared by a quarter of the species were found, that were likely present in an ancestral species shared by most, if not all the species of Passeriformes. We observed that satDNA evolution is complex and does not follow species phylogeny and that satellite arrays generally have a simple head-to-tail conformation, with evidence in four of the sampled species of satDNA arrays with higher-order repeats. We also found two satDNA families with fairly consistent monomer length and conserved regions that we hypothesise to might be functional.

Salmonella is one of the most important pathogens of mankind and animals alike, causing several billion pounds worth of damage worldwide each year. We have sequenced, annotated and published 4 genomes of Salmonella of well-defined virulence in farm animals. This provides valuable measures of intraserovar diversity and opportunities to formally link genotypes to phenotypes in target animals. Specifically, we have examined pathway detrition and mutagenesis and linked this to host specificity of the serovars. With the advent of next generation sequencing there has been a boom in genomic sequence submission, and an onslaught of -omics data has ensued. Integrating these different data types is complex and there is little available to visualise this data in the context of its genome. We present GeneBook, a web-based tool that synchronously integrates disparate datasets, displaying a fully annotated genome, enriched with publicly available data and the user's private experiments. It is accessed through a user-friendly interface that allows scientists to interrogate genomic features across multiple, heterogeneous, experiments.

The airway epithelium demonstates the ability to quickly repair following physical injury. The morphologic features of this dynamic repair process have been well characterised at the anatomic and cellular level using a number of animal model systems and these studies have provided a solid foundation upon which our understanding of normal repair is build. With the advent of molecular and bioinformatic tools and resources the opportunity exists to extend the value of these models in defining the molecular pathways and interactions that underlay the normal repair process. This thesis represents a realisation of this opportunity. A large animal model was developed in which selected areas of airway epithelium were subjected to bronchial brush biopsy as part of routine bronchoscopic examination prcedures in anaethetised sheep. The process resulted in a physical perturbation of the normal pseudostratified structure of the sheep airwway epithelium at specific locations. By careful experimental design it was possible, within the same animals. to identify and sample from sites undergoing repair at different intervals subsequent in injury. To supplement the histological evaluation of the repair process and align findings with extablished small animal models of airway epithelial repair proliferative cell labelling strategies were implemented in order to study the location and extent of cellular proliferation occurring duringthe repair process. Molecular approaches towards defining the transcriptional response to physical injury comprised application of microarray technology using a commercially sources array platform. Such approach demanted preliminary effort directed towards optimising RNA integrity and yield from airway samples. Following preliminary studies directed towards optimising the model conditions patterns of airway epithelial repair following bronchial brush biopsy were studies in eight sheep at degined time points (6 hours, 1,3, & 7 days) post-injury. Bronchial brush biopsy resulted in the acute removal of the epithelial cell layer and components of the underlying structures. repair processes were rapidly implemented through initial epithelial dedifferentiation, proliferatino and migration at the wound margins and subsequent time-depentend changes in the proportion of subepithelial structures, including smooth muscle and blood vessels, as the epithelial surface moved towards repair. Transcriptional analysis revewaled that over 13,000 probes showed evidence of differential expession at some point during the repair process (p<0.05), whilst of these, 1491 probes had in excess of a two-fold change in expression. array results were validated against conventional semi-quantitative RT-PCR for selected genes. Differentially expressed genes with previously characterised roles in epithelial migration, prolifereation and differentiation were identified during the repair process. The relative emphasis of gene products with particular functional roles varied during the course of repair. Indeed gene ontology (GO) terms identified included those associated with the inflammatory response, cellular migration, extracellular matrix activities, differentiation, proliferation, cellular development, cell cycle activities, cellular adhesion, apoptosis and mitosis. In addition the Kyoto Encyclopedia of Genes and Gneomes (KEGG) databases were queried and such process indicated the involvement of cell communication, 053 and complement and coagulation cascade pathways throughout the repair process, initial (6h) Toll-like receptor and cytokine-cytoine receptor interaction pathways, and the progressive involement of cell cycle, focal adhesion and extracellulaar matrix (ECM)-receptor, and cytokine interaction pathways as the epithelium repaired. The model of airway epithelial injury developed in this thesis generated features broadly consistent with those previosly described in relation to various small animal model systems. Importantly, and in addition, this thesis defines the molecular features associated with repair in this model system and provides a useful resource with which to assess the comparative fetures of the airway transcriptional response to physical injury, It is through such comparison, using analogous methodology, that the fundamental pathways and interactions that underlay normal repair and regeneration can be identified and therafter extended towards inderstanding the basis for variation associated with natural and experimental disease.

<p>The partitioning of translation to the outer membrane of the endoplasmic reticulum is a problem that has been the subject of inquiry since the discovery of the ribosome. The large degree to which ribosomes were found to be tethered to the membrane led to intense investigation of a series of related questions regarding the identity of those mRNAs that are translated on the endoplasmic reticulum, and the functions of that localization in cell stress. In this dissertation, I approach each of these questions in turn and work to reconcile my observations with those models that have been previously proposed. A theme of this work is the application of modern methods, particularly deep sequencing technology, to address problems that had largely been considered solved. The most prominently featured method is ribosome profiling, which is paired with classical biochemical and cell biological techniques. I arrive at several conclusions: 1) a significant fraction of all mRNAs is well represented on the endoplasmic reticulum membrane, 2) the properties of translation diverge substantially between membrane-associated and free ribosomes, and 3) the compartmentalization of translation can serve as an important variable in cell stress.</p> / Dissertation