| 1. |
- Bergman, Jessica M.
(författare)
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Genetics and Growth Regulation in Salmonella enterica
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications.Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response.To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E.Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA.Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria.
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| 2. |
- Dyrhage, Karl
(författare)
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Multi-omics investigation into bacterial evolution
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The focus of this thesis is the investigation of the evolution and cellular processes of Tuwongella immobilis and Apilactobacillus kunkeei, two bacterial species with different levels of genomic and cellular complexity, using a multi-omics approach.In the first study we examined the proteome of T. immobilis with LC-MS/MS after fractionation by differential solubilisation, yielding fractions corresponding to the cytoplasm, inner membrane, and outer membrane. The experiment was repeated with Escherichia coli and the results were compared. T. immobilis had five times as many predicted cytoplasmic proteins in the most hydrophobic fraction as E. coli. Among these are innovations in the Planctomycetota lineage and protein families that have undergone recent paralogisation followed by domain shuffling, including many enzymes related to information processing.The remaining three studies dealt with honeybee symbiont A. kunkeei. In the first of these, we sequenced and compared the chromosomal and extrachromosomal content of 102 novel A. kunkeei strains. We found that A. kunkeei has an open pangenome and an active set of transposable elements. Within the population we discovered three plasmids between 19.5 and 32.9 kb, one of which codes for enzymes involved in the synthesis of the antimicrobial compound kunkecin A which inhibits growth of the bee pathogen Melisococcus plutonius.In the next study we collected transcriptomic, proteomic, and metabolomic data from two growth phases from A. kunkeei strain A1401 and mapped the results to a metabolic pathway model. Enzymes involved in fermentation of fructose were highly expressed during the exponential growth phase. Enzymes involved in UMP biosynthesis were upregulated during stationary phase, as were protein involved in stress response and detoxification.The last study concerned the secretome of A. kunkeei. We characterised two types of extracellular particles from A. kunkeei strains A1401 and A0901. One type of particle was found to be proteinaceous, while the other type constituted membrane vesicles containing RNA. Comparison of transcriptomic data from the membrane vesicles and whole cells showed that the packing of the RNA was largely untargeted, but with a bias towards highly expressed mRNAs. We suggest that the cell uses membrane vesicles as a mechanism to get rid of superfluous mRNAs after rapid-response overexpression.Together these studies provide insights into the processes driving evolution in T. immobilis and A. kunkeei, and generate several testable hypotheses for future studies.
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| 3. |
- Ancillotti, Mirko, 1981-
(författare)
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Antibiotic Resistance: A Multimethod Investigation of Individual Responsibility and Behaviour
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rapid development of antibiotic resistance is directly related to how antibiotics are used in society. The international effort to decrease and optimise the use of antibiotics should be sustained by the development of policies that are sensitive to social and cultural contexts.The overarching aim of the thesis was to explore and discuss the Swedish public’s beliefs, values and preferences influencing engagement in judicious antibiotic behaviour.Study I explored through focus group discussions lay people’s perceptions and beliefs about antibiotics and antibiotic resistance. The Health Belief Model was used to identify factors that could promote or hinder engagement in judicious antibiotic behaviour. Participants found antibiotic resistance to be a serious problem but were not equally worried about being affected by it. There was a tension between individual and collective reasons for engaging in judicious behaviour.Study II explored lay people’s views on the moral challenges posed by antibiotic resistance through focus group discussions. Participants identified in the decreasing availability of effective antibiotics a problem of justice, which involves individual as well as collective moral responsibility. Different levels of policy demandingness were discussed in light of these results.Study III investigated, through an online Discrete Choice Experiment, public preferences regarding antibiotic treatment and the relative weight of antibiotic resistance in decision-making. Public behaviour may be influenced by concerns over the rise of antibiotic resistance. Therefore, stressing individual responsibility for antibiotic resistance in clinical and societal communication may affect personal decision-making.Study IV clarified the notions of collective and individual moral responsibility for antibiotic resistance and suggested a virtue-based account thereof. While everyone is morally responsible for minimising his/her own contribution to antibiotic resistance, individuals do or do not engage in judicious antibiotic behaviour with different degrees of voluntariness.The findings suggest that people could change their behaviour due to concerns over their own contribution to antibiotic resistance. Effective health communication should be developed from an appraisal of people’s attitudes, beliefs and social norms that influence antibiotic resistance related behaviours. Policy demandingness should take into account socioeconomic factors characterising local realities.
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| 4. |
- Andersson, Dan, 1967-
(författare)
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Exploring Perceptions of Route Environments in Relation to Walking
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Every walk takes place in a route environment, which can play an important role in deterring or facilitating walking. The focus of this thesis is on the perceptions of environmental variables, and how they relate to appraisals of route environments as hindering – stimulating for walking and unsafe – safe for reasons of traffic, in two metropolitan environments. Another focus is to expand the state of knowledge concerning the criterion-related validity of the Active Commuting Route Environment Scale (ACRES).Methods: Commuting pedestrians in Greater Stockholm, Sweden, were recruited via advertisements. From the inner urban area there were 294 participants (77% women), aged 49.5 years, and from the suburban areas there were 233 participants (82% women), aged 50.0 years. 77 individuals walked in both areas. The participants evaluated their commuting route environments using the ACRES. Correlation, multiple regression, and mediation analyses were used to explore the relationships between the variables. Comparisons of environmental ratings between groups and settings were performed with t-tests. Studies 1 and 2 focused on the inner urban area and studies 3 and 4 on the suburban areas. Studies 1 and 3 focused exclusively on the relations between the four motorized traffic variables (vehicle speed, vehicle flow, noise, and exhaust fumes), and their relations to the outcome variables (hinders – stimulates walking and unsafe – safe traffic). Studies 2 and 4 utilised proxies from studies 1 and 3 and combined them with other environmental variables to further the understanding of route environmental variables in relation to walking.Results: In both areas, aesthetics and greenery were positively related to stimulating walking, whereas noise, a proxy for motorized traffic, was negatively related. Aesthetics was also positively related to unsafe – safe traffic in the inner urban area, whereas greenery had the corresponding role in suburbia. Another important finding was that greenery also influenced aesthetics positively in both areas. Thus, greenery had both a direct and an indirect positive effect. On the other hand, noise influenced aesthetics negatively in the inner urban area, whereas vehicle flow had the corresponding role in suburbia. A number of variables conjointly influenced the outcome unsafe – safe traffic negatively in both areas (speeds of motor vehicles, noise, conflicts, congestion: pedestrians, red lights, and course of the route). The route environment profiles differed distinctly between the two areas.Conclusions: Several route environmental variables appear to be particularly influential in relation to pedestrian commuting, e.g., aesthetics, greenery, and noise. An important finding is that both positive and negative interactions, between certain predictor variables, were disclosed. The contrasting route environment profiles in the different settings strengthen the criterion-related validity of the ACRES. The findings expands the state of knowledge concerning the relations between the environment and walking. If implemented, these findings can influence public health positively.
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| 5. |
- Geli Rolfhamre, Patricia, 1979-
(författare)
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From Penicillin Binding Proteins to Community Interventions : Mathematical and Statistical Models Related to Antibiotic Resistance
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance has become a major public health concern and mathematical models are important analytical tools for the understanding, evaluation and prediction of the resistance problem and related control strategies.The risk of emerging antibiotic resistance and selection has rarely been a concern in the design of antibiotic drug dosing regimens. In the first paper, a selection of antibiotic resistant subpopulations for different antibiotic dosing regimens was studied in vitro. The demonstrated complex relationship was influenced by both the rise of new mutants and a postantibiotic effect (PAE) (continued inhibition of bacterial growth after removal of the antibiotic drug). By constructing a mathematical model that incorporated biologically relevant parameters, we were able to assess the risks of resistance development under different dosing strategies.In the second paper, the model for PAEs is further developed to determine the implications for different dosing regimens. The result challenges the conventional notion that long PAEs promote extended drug dosing intervals and it allows new hypotheses to be tested experimentally based on the findings from the theoretical framework.Since PAE experiments often are time-consuming and laborious, very few studies have been reporting variation for this phenomenon. In the third paper, an extension to capture the stochastic behavior of bacterial population growth under drug exposure is made. The stochastic nature of the model is also an important complement to the existing deterministic models on drug dose drug effect relationships.The last paper describes a controlled clinical intervention study aiming at determining whether the frequency of trimethoprim resistance in E. coli can be decreased by a sudden and drastic reduction in trimethoprim use. In addition to evaluating the intervention effect, the model, given estimated parameters, is also used for predicting other interesting outcomes.
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| 6. |
- Malik, Sohaib Zafar
(författare)
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Interaction of cyclotides and bacteria : A study of the cyclotide action and the bacterial reaction
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). Thesepotent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults. Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics. Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.
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| 7. |
- Mellenius, Harriet, 1983-
(författare)
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Exploring and predicting DNA template dependent variation in transcription
- 2012
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Reliable transmission of information from DNA to proteins is a pre-requisite for all life, where substitution errors in the polypeptide chain may arise from transcription, aminoacylation of tRNAs or translation. The fidelity control mechanisms in transcription have nevertheless received little attention, based on the assumption that the transcriptional error is masked by the translational error. This thesis shows how accuracy theory can be applied to transcription to elucidate the principles of transcriptional accuracy. The DNA template dependent transcriptional accuracy variation is studied through modelling based on transition state theory, using thermodynamic properties of the nucleic acids in the transcription bubble. The models show that the error frequency variation in transcription causes it to surpass the translational error in some sequence contexts, making transcription a significant source of amino acids substitution errors.
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| 8. |
- Simm, Niklas, 1994-
(författare)
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Sharing Resources for Greener Logistics
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Freight transportation, a significant contributor to the environmental impact of logistics, is projected to grow, thereby increasing its environmental footprint. With many actors contributing to the greening of logistics, their roles, and responsibilities for the greening of logistics differs. One mean for progressing the greening of logistics, is to share resources between actors needed for implementing different green logistics practices. Resource sharing among these actors will be a key strategy for advancing the greening of logistics.The purpose of this thesis is to increase the understanding how resources can be shared between actors to facilitate the implementation of green logistics practices. It focuses on the different green logistics practices that can be implemented to green logistics through a resource perspective, where resources and actors that controls resources and implements green logistics practices are the subject of analysis. Through the application of resource dependency theory, this thesis sets out to understand why actors need to share resources with other actors, and how they can manage their dependency onto resources controlled by other actors.The findings of suggest that there are different resources needed for green logistics: knowledge, technical resources, financial resources, time, employees, and IT-systems. The actors that control these resources or that implements green logistics practices are: logistics service providers, shippers, customers, technology providers, and governmental authorities. Through the lens of resource dependence, it is suggested how the dependencies can be managed through resources sharing between different actors. These ways of sharing resources are characterized by three parameters: duration (short-term vs long-term), directness (direct vs indirect), and scope (resource sharing with one or multiple actors). Through a combination of these parameters, different ways of sharing resources between actors are proposed.The contributions of this thesis are primarily aimed at enriching the green logistics literature and enlightening logistics practitioners. Theoretically, it extends our understanding of interaction for greener logistics by focusing on resource sharing between different actors. By applying resource dependency theory, a deeper understanding of why and how actors can share resources to enable the greening of logistics is given. Additionally, this thesis proposes a categorizations of green logistics practices, actors, and resource. This can be used to further understand green logistics, whilst simultaneously be a tool for practitioners to understand their role in the greening of logistics.
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| 9. |
- Sun, Song, 1982-
(författare)
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Dynamics and Mechanisms of Adaptive Evolution in Bacteria
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics.In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.
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| 10. |
- Berglund, Björn, 1983-
(författare)
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Deliberations on the impact of antibiotic contamination on dissemination of antibiotic resistance genes in aquatic environments
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The great success of antibiotics in treating bacterial infectious diseases has been hampered by the increasing prevalence of antibiotic resistant bacteria. Not only does antibiotic resistance threaten to increase the difficulty in treating bacterial infectious diseases, but it could also make medical procedures such as routine surgery and organ transplantations very dangerous to perform. Traditionally, antibiotic resistance has been regarded as a strictly clinical problem and studies of the problem have mostly been restricted to a clinical milieu. Recently, non-clinical environments, and in particular aquatic environments, have been recognised as important factors in development and dissemination of antibiotic resistance. Elevated concentrations of antibiotics in an environment are likely to drive a selection pressure which favours resistant bacteria, and are also believed to promote horizontal gene transfer among the indigenous bacteria. Antibiotic resistance genes are often located on mobile genetic elements such as plasmids and integrons, which have the ability to disseminate among taxonomically unrelated species. The environmental bacteria can thus serve as both reservoirs for resistance and hot spots for the development of new antibiotic resistance determinants.There is still a lack of data pertaining to how high antibiotic concentrations are necessary to drive a selection pressure in aquatic environments. The aim of this thesis is to determine the effect of high and low concentrations of antibiotics on environmental bacterial communities from different aquatic environments. In the studies performed, antibiotics were measured using liquid chromatography-mass spectrometry. Bacterial diversity and evenness were assessed using molecular fingerprints obtained with 16S rRNA gene-based denaturing gradient gel electrophoresis, and antibiotic resistance genes and class 1 integrons were quantified using real-time PCR.Water and sediment samples were collected from different rivers and canals in Pakistan. The environments differed in anthropogenic exposure from undisturbed to heavily contaminated. A general trend could be observed of high concentrations of antibiotics correlating to elevated concentrations of antibiotic resistance genes and integrons. Extremely high concentrations of antibiotic resistance genes and integrons were found in the sediments downstream of an industrial drug formulation site, which likely correlated to the high load of antibiotics found in the water. Antibiotic and antibiotic resistance gene concentrations were also shown to increase downstream of Ravi river, which flows through Lahore, a city of more than 10 million inhabitants. Rivers not impacted by anthropogenic contamination were found to contain antibiotics and resistance gene concentrations of similar levels as in Europe and the U.S. Similar measurements were performed in the Swedish river Stångån. The concentrations of antibiotic resistance genes and class 1 integrons were shown to increase in the river after it had passed, and received urban wastewater effluent from the city of Linköping.A series of constructed wetlands were exposed to a mixture of different antibiotics at environmentally relevant concentrations over a few weeks. The antibiotic exposure did not observably affect the bacterial diversity or integron concentrations. Antibiotic resistance genes were found at low background concentrations, but the antibiotic exposure did not observably affect the concentrations. The constructed wetlands were also found to reduce most antibiotics at levels comparable to conventional wastewater treatment schemes, suggesting that constructed wetlands may be useful supplementary alternatives to conventional wastewater treatment.To investigate the effect of antibiotics on an uncontaminated aquatic environment in a more controlled setting, microcosms were constructed from lake water and sediments and subsequently exposed to varying concentrations of antibiotics (ranging from wastewater-like concentrations to 1,000 times higher). The water and sediments were gathered from the lake Nydalasjön, near Umeå, which is not exposed to urban waste. While antibiotic resistance genes and class 1 integrons were found in the lake sediments, no increase in the concentrations of these genes could be observed due to the antibiotic additions.In conclusion, although antibiotic resistance genes and integrons are part of the environmental gene pool, low concentrations of antibiotics do not seem to immediately impact their prevalence. However, aquatic environments exposed to anthropogenic waste do exhibit elevated levels of antibiotic resistance genes and integrons. Aquatic environments heavily polluted with antibiotics also clearly display correspondingly high concentrations of antibiotic resistance genes and integrons. These results clearly indicate the necessity to keep down pollution levels as well as the need to establish the range of antibiotic concentrations which do promote resistance. This must be done in order to enable risk assessments and to establish acceptable levels of antibiotic pollution. It should also be stressed that more research is required to elucidate what effect low levels of antibiotic exposure has on environmental bacterial communities.
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