Coming dissertations at Uppsala university
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Sequencing and comparative analyses of diplomonad genomes : Beyond the Gut: The Lake of Forgotten Dreams
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-537396
This thesis presents a comprehensive analysis of diplomonad genomes, focusing on both parasitic and free-living species, to explore the evolutionary adaptations within this group of anaerobic, flagellated eukaryotes. I first generated and analyzed the reference genome of Hexamita inflata, the first free-living diplomonad to be sequenced. The genome of H. inflata is significantly larger than those of parasitic diplomonads, with 142 Mbp encoding 79,341 proteins. The expansion of protein-encoding capacity and a high proportion of interspersed repeats contribute to the large genome size, providing a foundation for future studies on the evolution of parasitism and free-living lifestyles in diplomonads.
In a subsequent study, I improved the genome assembly of Spironucleus salmonicida, a diplomonad responsible for systemic infections in salmon, using PacBio long-read sequencing and optical mapping. The new assembly consolidates the genome into nine near-complete chromosomes, providing a more comprehensive view of the gene families, gene organization, and chromosomal structure. This high-quality reference genome will facilitate comparative genomic studies at the chromosomal level and serve as a valuable resource for researchers studying diplomonads and other protists.
Further, I generated the draft genome of the commensal diplomonad Spironucleus barkhanus, revealing a genome size (26.9 Mbp) larger than the morphologically similar S. salmonicida due to extensive duplications, expansions in protein-coding capacity, and a higher content of interspersed repeats. Comparative analysis between these two diplomonads highlighted key genomic differences, which are likely related to their distinct lifestyles. The S. barkhanus genome will contribute to understanding the pathogenicity of S. salmonicida and aid in the development of diagnostic tools to differentiate between these species in salmonid fish.
Finally, I performed a comparative genomic analysis using the GenoDiplo and CompareDiplo bioinformatics pipelines across various diplomonads with different lifestyles, revealing significant expansions in seven key protein superfamilies. The data suggest that environmental factors drive the evolution of these protein and multi-gene families, resulting in organisms that are well-adapted to their specific habitats. This work enhances our understanding of the diversity and evolutionary history of eukaryotes, particularly the adaptations to anaerobic lifestyles and the evolution of key eukaryotic cellular mechanisms.
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Growing up with pediatric hip disease– a companion for life : Exploring the multifaceted experiences throughout life, with special attention to Legg-Calvé Perthes disease
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-536903
Pediatric hip diseases such as developmental dysplasia of the hip, Legg-Calvé-Perthes disease, and slipped capital femoral epiphysis can lead to lasting hip deformities and early-onset osteoarthritis. This thesis explores the long-term impacts of these conditions, combining patient experiences with large-scale data.
Study I used data from the Swedish Pediatric Orthopedic Quality Register to examine the incidence, demographics, and risk factors for containment surgery in patients with unilateral Legg-Calvé-Perthes disease. 309 patients registered between 2015 and 2024. The study found a national yearly incidence of 4.2 per 100,000 children aged 2–12 years, somewhat lower than previously reported in Sweden. The disease was more common in boys (77%), with an average diagnosis age of 6 years. Notably, 30% of patients were overweight or obese at diagnosis, increasing to around 40% at the 2-year follow-up. The study also found that older age, a positive Trendelenburg sign, and limited hip abduction at diagnosis increased the likelihood of surgical intervention.
Study II focused on the long-term impact of Legg-Calvé-Perthes disease as experienced by patients through qualitative interviews. Themes that emerged included self-reinvention, concerns about future health, surgical interventions, and challenges with physical activities leading to sedentary lifestyles. The study also highlighted patients' sense of disconnection during the transition from pediatric to adult care and emphasized the need for continuous, age-appropriate information and structured healthcare transitions.
Study III explored whether individuals with a history of Legg-Calvé-Perthes disease or slipped capital femoral epiphysis are at increased risk of receiving prescriptions for pain or antidepressant medications in adulthood. The study, which included 1,292 Legg-Calvé-Perthes disease patients and 1,613 slipped capital femoral epiphysis patients, found a higher risk of analgesic prescriptions in both groups compared to matched controls. Additionally, slipped capital femoral epiphysis patients had a slightly higher risk of antidepressant prescriptions. The findings underscore the need for long-term pain management and health support.
Study IV investigated mortality risks after total hip arthroplasty in patients with a history of pediatric hip disease. Among 4,043 patients identified from the Swedish Hip Arthroplasty Register, the study found no increased 90-day mortality risk compared to the general population. These patients’ overall mortality risk was lower, suggesting that total hip arthroplasty does not increase mortality risks despite their pre-existing conditions.
In conclusion, this thesis provides a comprehensive view of the long-term impacts of pediatric hip diseases, highlighting the need for improved, continuous, and age-appropriate care strategies to enhance patient outcomes.
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Pathway Engineering for Butanol Production in Cyanobacteria
Link: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-537808
Cyanobacteria, photosynthetic microorganisms, are emerging as promising platforms for sustainable biofuel production, leveraging their capabilities to utilize solar energy, carbon dioxide, and water in a direct process. Among various biofuels, isobutanol (IB) and 3-methyl-1-butanol (3M1B) are superior cadidates as gasoline replacements. The unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) has been successfully engineered for IB and 3M1B production via heterologous expression of α-ketoisovalerate decarboxylase (KivdS286T). However, the production levels remain low. This thesis focuses on further enhancing photosynthetic IB and 3M1B bioproduction via the 2-keto acid pathway in Synechocystis through multifaceted metabolic engineering strategies.
For the first time, a functional 2-keto acid pathway, consisting of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), KivdS286T, and alcohol dehydrogenase (Adh), was successfully introduced in Synechocystis. By modulating kivdS286T copy number, a stepwise increase of IB and 3M1B production was observed. Building on these successes, additional genetic engineering strategies were employed. First, by overexpressing selected genes involved in central carbon metabolism, substantial production improvements were achieved. Second, a dCas12a-mediated CRISPR interference (CRISPRi-dCas12a) system was developed and integrated into an IB/3M1B producer strain to allow for elimination of potential competing pathways. Repression of ten out of fifteen target genes resulted in improved IB and 3M1B production. A strain with dual repression of ppc and gltA demonstrated 2.6-fold and 14.8-fold increases in IB and 3M1B production per cell, respectively. In addition to genetic engineering, protein engineering is another powerful tool in metabolic engineering. Manipulating kivdS286T copy number effectively increased overall cellular KivdS286T expression level, while KivdS286T directed evolution offered an opportunity to improve catalytic activity. After screening 1,600 variants, one KivdS286T variant (1B12), featuring dual T186S and K419E substitutions, exhibited a significant increase of 55% in IB and 50% in 3M1B production per cell. This is the first demonstration of using directed evolution to enhance bioproduction in Synechocystis. Long-term cultivation of two selected strains in plug-sealed tissue culture flasks resulted in maximal cumulative IB and 3M1B titers of 1.2 g L-1 and 0.4 g L-1, respectively, representing new records of photosynthetic IB and 3M1B production levels in cyanobacteria.
In summary, these advancements highlight the feasibility of applying various metabolic engineering strategies to develop Synechocystis as a biofuel producer. Furthermore, the engineered biofuel-producing strains establish a strong foundation for further research into industrial-scale biofuel production process, aiming to replace fossil fuels with sustainable, carbon-neutral alternatives.