| 1. |
- Thegerström, Johanna, 1972-
(författare)
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Mycobacterium avium infections in children
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mycobacterium avium belongs to a group of over 130 species of non-tuberculous mycobacteria (NTM) or environmental mycobacteria. The subspecies Mycobacterium avium avium was originally described as the causative agent of bird tuberculosis, but was later found to cause disease also in humans. Small children display a special form of infection that is seldom detected in other age groups. It manifests as a chronic lymphadenitis usually in the head and neck region. The incidence rate is approximately 1-5/100,000 children/year. However, exposure to this bacterium is high as judged by sensitin skin test studies. Even if a lot of persons are infected with M. avium, a majority of them do not develop disease and the bacterium is therefore considered to be of low virulence, causing disease mainly in immunocompromised persons. Children with M. avium lymphadenitis, however, usually do not have any known deficiencies in the immune system.This thesis elucidates why small children are prone to develop disease by M. avium. Investigation of a possible zoonotic spread of this bacterium to children involved analysis and comparison of different strains isolated from birds and other animals and from children, using the restriction fragment length polymorphism (RFLP) method on insertion sequence IS1245, resulting in the finding that the children were infected exclusively with the new proposed subspecies M. avium hominissuis. Animals in general and birds in particular were infected with the subspecies M. avium avium (using the more narrow definition). Moreover, when investigating the immunological response of human peripheral blood mononuclear cells (PBMCs) to stimulation with M. avium hominissuis and M. avium avium, respectively, it was found that the former subspecies induced lower IFN-γ and IL-17 than the latter, but higher levels of Il-10, which might contribute to explain the higher pathogenicity of M. avium hominissuis in humans.Through studies of the geographical distribution of cases of M. avium infection in children in Sweden and the seasonal variation of the disease, a fluctuation of the incidence over the year was detected, with higher numbers of cases in the autumn months and lower numbers in the late spring. There was a higher incidence rate in children living close to water than in those living in the inland or in the urban areas. Therefore, outdoor natural water is the most probable source of infection in children with M. avium lymphadenitis. Through a descriptive clinical retrospective study, complete surgical removal of the affected lymph node was found to lead to better results than treatment by incision and drainage of abscess or expectation only.Finally there might be several explanations as to why an individual develops disease after infection with M. avium, such as, exposure, bacterial virulence factors or possible specific deficiencies of the immune system of the host or a combination of these factors. Which are the more important factors regarding children with M. avium lymphadenitis is still an open question.
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| 2. |
- Behra, Phani Rama Krishna
(författare)
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Comparative genomics of the genus Mycobacterium : Genome evolution, phylogeny and diversity
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The genus Mycobacterium includes more than 190 species, and many cause severe diseases such as tuberculosis and leprosy. According to the "World Health Organization", in year 2019 alone, 10 million people developed TB, and 1.4 million died. TB had been in decline in developed countries, but made its reappearance as an opportunistic pathogen targeting immuno-compromised AIDS victims. Also, non-tuberculosis mycobacteria (NTM) infections have emerged as a major infectious agent in recent times. NTM occupy diverse ecological niches and can be isolated from soil, tap water, and groundwater. This thesis has investigated the Mycobacterium species from a genomic perspective, focusing on the biology of virulence factors, mobile genetic elements, tRNAs, and non-coding RNAs and their evolutionary distribution and possible relationship with phenotypic diversity. As part of this study, we have sequenced 153 mycobacterial genomes, including type strains, environmental samples, isolates from hospital patients, infected fish, and outbreak samples in an animal facility at Uppsala University. We have provided a phylogenetic tree based on 387 (and 56) core genes covering most species (244 genomes) constituting the Mycobacterium genus. The core gene phylogeny resulted in 33 clades. Subsequently, we have covered different clade groups, such as, M. marinum, M. mucogenicum, M. chelonae and M. chlorophenolicum and investigated the NTM clade-specific genome diversity and evolution. Our examination of non-coding genes showed that the total number of tRNA genes per species varies between 42 and 90. Among the species with more than 50 tRNAs, additional tRNA genes are likely acquired through horizontal gene transfer (HGT), as supported by the presence of closely linked HNH endonuclease gene and GOLLD RNA. We have explored the presence of selenocysteine utility and the gene for selenoprotein "formate dehydrogenase" among 244 mycobacterial genomes. For the M. chlorophenolicum clade, we have explored genes with a role in the bioremediation process. Comparative genomics of M. marinum and M. chelonae clade groups suggest new clusters or subspecies. Mutational hotspots are relatively higher in M. marinum compared to that in M. tuberculosis and M. salmoniphilum. Relatively higher number of hotspots in M. marinum is likely related to its ability to occupy different ecological niches. Finally, the thesis uncovered IS elements, phage sequences, plasmids, tRNA, and ncRNA contributing to mycobacterial evolution.
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| 3. |
- Larsson, Pontus, 1977-
(författare)
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Computational Approaches to the Identification and Characterization of Non-Coding RNA Genes
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Non-coding RNAs (ncRNAs) have emerged as highly diverse and powerful key players in the cell, the range of capabilities spanning from catalyzing essential processes in all living organisms, e.g. protein synthesis, to being highly specific regulators of gene expression. To fully understand the functional significance of ncRNAs, it is of critical importance to identify and characterize the repertoire of ncRNAs in the cell. Practically every genome-wide screen to identify ncRNAs has revealed large numbers of expressed ncRNAs and often identified species-specific ncRNA families of unknown function. Recent years' advancement in high-throughput sequencing techniques necessitates efficient and reliable methods for computational identification and annotation of genes. A major aim in the work underlying this thesis has been to develop and use computational tools for the identification and characterization of ncRNA genes.We used computational approaches in combination with experimental methods to study the ncRNA repertoire of the model organism Dictyostelium discoideum. We report ncRNA genes belonging to well-characterized gene families as well as previously unknown and potentially species-specific ncRNA families. The complicated task of de novo ncRNA gene prediction was successfully addressed by developing a method for nucleotide composition-based gene prediction using maximal-scoring partial sums and considering overlapping dinucleotides.We also report a substantial heterogeneity among human spliceosomal snRNAs. Northern blot analysis and cDNA cloning, as well as bioinformatical analysis of publicly available microarray data, revealed a large number of expressed snRNAs. In particular, U1 snRNA variants with several nucleotide substitutions that could potentially have dramatic effects on splice site recognition were identified.In conclusion, we have by using computational approaches combined with experimental analysis identified a rich and diverse ncRNA repertoire in the eukaryotes D. discoideum and Homo sapiens. The surprising diversity among the snRNAs in H. sapiens suggests a functional involvement in recognition of non-canonical introns and regulation of messenger RNA splicing.
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| 4. |
- Pettersson, B. M. Fredrik, 1974-
(författare)
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tRNA Gene Structures in Bacteria
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In bacteria, tRNA molecules are produced as precursors with additional nucleotides both upstream and downstream of the tRNA coding sequence. To generate a mature tRNA, the endoribonuclease RNase P removes the upstream sequence, while a number of enzymes can remove the downstream sequence. In this thesis, the influence of such upstream and downstream sequences on the expression of mature functional tRNA was studied. It was found that in Escherichia coli, the presence of an upstream sequence positively influences tRNA expression. Furthermore, it was shown that the identity of the nucleotide immediatedly 5' of the canonical RNase P cleavage site in a tRNA precursor influenced RNase P cleavage site selection in vivo in E. coli, but not in Pseudomonas aeruginosa. Additionally, a stem-loop in the precursor just downstream of the tRNA increased this "miscleavage". This stem-loop resembled a rho-independent transcription terminator and overlapped the trpT gene in P. aeruginosa, but it only marginally influenced the expression of the downstream secE gene. The trpT and secE genes were found to be cotranscribed. The trpT-secE gene order was also conserved in the majority of bacteria investigated. The expression of tRNA genes during development in Streptomyces coelicolor was also studied. Here, the expression of most tRNA genes tested increased with time during development. Similarly, the expression of the RNase P RNA increased. The relative increase was quantified and correlated with different criteria related to gene structure and gene organisation, but no significant differences could be found. Moreover, a tRNALeuCAA UUA codon suppressor as well as the cognate bldA tRNA could restore differentiation to a developmentally blocked bldA deletion strain. For both tRNAs, the efficiency of restoration depended on the 5'- and 3'-flanks. In conclusion, both 5'- and 3'-flanking sequences influence tRNA expression, and bacteria respond differently to changes in their tRNA gene structures.
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| 5. |
- Wu, Shiying, 1978-
(författare)
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Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RNase P is a ubiquitous ribonuclease responsible for removing the 5’ leader of tRNA precursor. Bacterial RNase P contains one RNA (RPR) and one protein (RPP) subunit. However, the number of protein variants depends on the origin. The RNA subunit is the catalytic subunit that in vitro cleaves its substrate with and without the protein subunit. Therefore RNase P is a ribozyme. However, the protein subunit is indispensable in vivo. The objective of this thesis was to understand the mechanism of and substrate interaction in RPR-mediated cleavage, in particular the contributions of the two domains of RPR and the roles of the base at the -1 residue in the substrate. As model systems I have used bacterial (Eco) and archaeal (Pfu) RPRs. The TSL (T-stem-loop) region of a tRNA precursor and the TBS (TSL-binding site) in the RPR S-domain interact upon RPR-substrate complex conformation. A productive TSL/TBS-interaction affects events at the cleavage site by influencing the positioning of chemical groups and/ or Mg2+ such that efficient and correct cleavage occurs consistent with an induced fit mechanism. With respect to events at the cleavage site, my data show that the identity of the residue immediately upstream the 5’ of the cleavage site (at -1) plays a significant role for efficient and accurate cleavage although its presence is not essential. My data also show that the RPR C-domain can cleave without the S-domain. However, the presence of the S-domain increases the efficiency of cleavage but lowers the accuracy. The structure of the S-domain of Pfu RPR differs from that of Eco RPR and my data suggest that the Pfu S-domain does not affect the accuracy in the same way as for Eco RPR. It also appears that the proteins that bind to the Pfu S-domain play a role in formation of a productive TSL/TBS-interaction. It is therefore possible that the proteins of Pfu RNase P have evolved to take over the role of the S-domain with respect to the interaction with the TSL-region of the substrate.
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| 6. |
- Kikovska, Ema, 1977-
(författare)
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Versatile and Antique World of RNA : The Simplicity of RNA Mediated Catalysis
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RNA is the only biological molecule that can function both as a repository of information and as a catalyst. This, together with the ability to self-replicate, led to recognition of RNA as ‘prelude to life’.My work highlights some of the important features of RNA as a catalyst, exemplified by RNase P. It addresses questions of evolutionary preservations of residues and structure, involvement of metal ions and finally structure evolution towards minimal catalytically competent RNA motifs.RNase P is the only enzyme involved in 5’ end processing of all pre-tRNAs. Until recently, it was believed that the RNA moiety of RNase P is responsible for mediating catalysis only in Bacteria. However, my recent study conclusively demonstrated that eukaryotic RNase P RNA is catalytically competent in vitro in absence of proteins. These findings evidenced evolutionary preservation of RNA-mediated catalysis in RNase P.RNase P RNA is a metalloeznyme. In my studies I analyzed the contributions of individual chemical groups at the cleavage site to catalysis. My findings suggested that the 2’OH of N-1 and the exocyclic amine of G+1 are involved in positioning of functionally important metal ions. Additionally, data appointed the function of Pb2+ as both structural metal ion and important in generating the nucleophile. My studies further indicate a conformational change upon RNase P RNA -substrate complex formation in keeping with an induced fit mechanism. Studying the effects of reducing the ribozyme size upon dissection of bacterial RNase P RNAs, we defined the smallest catalytically competent domain i.e. P15-loop. Derivatives of this autonomous metal ion binding domain, (the smallest being 31nt-s), are able to cleave both whole-length pre-tRNAs as well as hairpin substrates, though with severely reduced rates relative to their parent ribozymes. The study has inferred that partite ES interactions at the cleavage site prove sufficient for catalysis.
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| 7. |
- Lu, Jian, 1974-
(författare)
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The Kluyveromyces lactis killer toxin is a transfer RNA endonuclease
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Killer strains of the yeast Kluyveromyces lactis secrete a heterotrimeric protein toxin (zymocin) to inhibit the growth of sensitive yeasts. The cytotoxicity of zymocin resides in the γ subunit (γ-toxin), however the mechanism of cytotoxicity caused by γ-toxin was previously unknown. This thesis aimed to unravel the mode of γ-toxin action and characterize the interaction between γ-toxin and its substrates.Previous studies suggested a link between the action of γ-toxin and a distinct set of transfer RNAs. In paper I, we show that γ-toxin is a tRNA anticodon endonuclease which cleaves tRNA carrying modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at position 34 (wobble position). The cleavage occurs 3’ to the wobble uridine and yields 2’, 3’-cyclic phosphate and 5´-hydroxyl termini.In paper II, we identified the determinants in tRNA important for efficient γ-toxin cleavage. The modifications present on the wobble uridines have different effects on tRNA cleavage by γ-toxin. The Saccharomyces cerevisiae wobble modification mcm5 group has a strong positive effect, whereas the Escherichia coli wobble modification 5-methylaminomethyl group has a strong inhibitory effect on tRNA cleavage. The s2 group present in both S. cerevisiae and E. coli tRNAs has a weaker positive effect on the cleavage. The anticodon stem loop (ASL) of tRNA represents the minimal structural requirement for γ-toxin action. Nucleotides U34U35C36A37C38 in the ASL are required for optimal cleavage by γ-toxin, whereas a purine at position 32 or a G at position 33 dramatically reduces the reactivity of ASL.Screening for S. cerevisiae mutants resistant to zymocin led to the identification of novel proteins important for mcm5s2U formation (paper III). Sit4p (a protein phosphatase), Sap185p and Sap190p (two of the Sit4 associated proteins), and Kti14p (a protein kinase) are required for the formation of mcm5 side chain. Ncs2p, Ncs6p, Urm1p, and Uba4p, the latter two function in a protein modification (urmylation) pathway, are required for the formation of s2 group. The gene product of YOR251C is also involved in the formation of s2 group. The involvement of multiple proteins suggests that the biogenesis of mcm5s2U is very complex.
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| 8. |
- Ramesh, Malavika
(författare)
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Morphological changes in mycobacteria
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Morphological variations at different stages and conditions of growth has been observed frequently in various mycobacteria. Despite years of research, the molecular basis of such variations is still not clear and requires further elucidation. Owing to the pathogenic significance of mycobacteria, currently, mycobacterial research emphasizes on understanding responses to oxygen deprivation, starvation, various antibiotic treatments, latency and dormancy.In this thesis, change in cell shape from rod to coccoid, occurrence of refractive spore-like phase grey, phase bright bodies and branching are some of the morphological variations discussed. Further, this thesis also discusses microaerobic condition (oxygen deprivation) and the localisation pattern of the stressosome complex.
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