På huden lever en sammansättning av olika mikroorganismer såsom bakterier, svampar och virus. Dessa mikroorganismer kallas hudens mikrobiom. Sammansättningen av en individs mikrobiom kan ge mycket information om en individens hälsa. För att undersöka sammansättningen av bakterier på hudytan med exempelvis qPCR, behöver bakterier samlas in och DNA extraheras. Bakteriekoncentrationen på hudens torrare områden som exempelvis armar har normalt en relativt låg bakteriekoncentration vid 102-104 bakterier per cm2. Huden koloniseras till stor del av grampositiva bakterier. Grampositiva bakterier är i regel svårare att lysera än andra bakterier och kräver därför hårdare lysering. En bra extraktionsmetod ska erhålla mycket DNA utan att påverka dess kvalité. I detta arbete utvärderades initialt fem olika extraktionsmetoder på bakteriesuspension med Staphylococcus aureus (S. aureus), både direkt på bakteriesuspension men också från svabb. Utvärdering gjordes på PureLink Microbiome DNA Purification Kit, QlAamp PowerFecal Pro, QlAamp DNA Mini Kit och KOH-EDTA. Metoden med QlAamp DNA Mini Kit testades med två olika protokoll och räknades som två separata metoder. Metoderna som gav bäst resultat vid initial utvärdering var PureLink Microbiome och KOH-EDTA. Därefter utvärderades dessa två metoderna på prov insamlat med svabb från huden på 10 frivilliga deltagare.

Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis, with an incidence in a quarter of the world population. Despite the scientific and technological advances, an effective diagnostic method has not yet been found that allows an early diagnosis and, also, to detect the strain present in the patient. The combination of nanotechnology with molecular diagnostics has shown promising advances offering new possibilities, such as the development of nanoflares.  Nanoflares represent a new class of molecular probes, composed of gold nanoparticles functionalized with a recognition sequence that can be amplified by rolling circle amplification (RCA) technique, producing a fluorescence signal.  This thesis focuses in the synthesis of gold nanoparticles, with different coatings and sizes, as well as their subsequent application in the preparation and optimization of nanoflares for the genotyping of synthetic M. tuberculosis targets using RCA technique. The different preparations of nanoflares have an impact in the assay sensitivity, showing two times increase in sensitivity for citrate-coated nanoparticles with respect to those coated with PEG. Furthermore, it was observed that the sensitivity is directly related to the synthesized particle size.  Sensitivity is also affected by the application of a purification post-treatment of the synthesis product. This post-treatment reduces the sensitivity of nanoflares by up to 37% but, by contrast, extends its useful life.  The results obtained are shown as a proof of concept for a future cost-effective, rapid and robust in situ diagnostic method that identifies the strain of tuberculosis present in the patient.

Climate change and its effects on our society is a steadily growing problem. In 2010, the industry sector accounted for more than 30% of the global greenhouse gas emissions. The chemical industry is one of the industrial subsectors responsible for the highest emissions of greenhouse gas. To reach the climate goals it is therefore urgent to find more sustainable options for production of chemicals in general. Synthetic biology and microbial cell factories are growing fields that have received much attention for inferring promising sustainable alternative production routes for various compounds. When it comes to microbial cell factories, cyanobacteria infer many advantages over heterotrophs. Cyanobacteria can for instance convert atmospheric CO2 into valuable compounds through photosynthesis using the light reaction and the Calvin-Benson cycle. In the present work, the freshwater cyanobacterium Synechocystis sp. PCC 6803 is metabolically engineered to produce 1,2-propanediol; an important chemical feedstock for which there is a great interest in finding a sustainable production route as an alternative to the current petrochemical one. Seven different constructs are designed for introduction and expression of a three-step heterologous metabolic pathway for 1,2-propanediol production. Two strains of Synechocystis are successfully engineered, with the heterologous pathway chromosomally integrated at the Neutral Site I through homologous recombination with an integrative plasmid targeting this genomic site. One of the three heterologous genes (mgsA) of the pathway was successfully translated as shown in a Western immunoblot. In a SDS-PAGE a band of 40 kDa was detected, corresponding to the size of both the sADH and YqhD enzymes.

No description available.

C. jejuni, a pathogenic gram-negative bacterium infecting the human gastrointestinal tract has lately been shown to cause bacteraemia to a wider extent than previously known. In some genotypes, this is thought to be related to GDP-Mannose 4,6 dehydratase encoded by the gene wcbK in the capsule polysaccharide operon and its potential phase variated regulated nature mediated by a homopolymeric guanine tract. This potential regulatory tract has been reported to be controlling the survival in serum by switching expression of wcbK “ON” or “OFF”. This master thesis report evaluates C. jejuni’s ability to survive human serum and low pH, as proxies for the conditions that bacteria meet in human blood or the stomach, respectively. By next generation sequencing, I evaluated the correlation between survival in human serum and the wcbK gene’s “ON” or “OFF” state. Furthermore, the temporal stability of the serum resistant phenotype was assessed over multiple generations. I found that a serum resistant fraction of the C. jejuni population could be enriched by selection in normal human serum. The serum resistant part of the population did not decrease during repeated subculture for 10 generations in bacterial culture medium. However, there was no correlation between the extent of serum resistance in the population and the “ON” or “OFF” state of the wcbK gene.

The understanding of basic cellular processes has been greatly facilitated through investigation of the behaviour of mutant forms. In a similar way the mechanisms of genetic recombination may be clarified by a study of strains which are known to show inherited differences in recombination behaviour at meiosis. The haploid fungus Neurospora crassa is particularly well suited to such an investigation since recombination frequency heterogeneity has been extensively reported in that organism, and the differences are believed to be, to a large extent, under genetic control. Strains showing recombination frequency heterogeneity over a marked genetic region have been extensively analysed in the present work and the mode of action of the factors controlling recombination frequency has been investigated by combining differing strains in heterokaryons. / Thesis / Doctor of Philosophy (PhD)

Immunoassays have become an essential tool in several fields of bioanalytics with tremendous advances in sensitivity and formats seen through the last three decades. Many diseases are today diagnosed solely based on biomarker concentrations evaluated through immunoassays. More biomarkers will be unravelled for diseases that have low serum concentrations once highly sensitive analyzes can be performed routinely, highlighting the importance of improved sensitivity. The aim of this project was to increase the sensitivity of detection on the Gyrolab immunoassay platform. This was done by optimizing the conditions for fluorescence and by reduction of light scattering in the system. Four red-emitting fluorophores were investigated and assays were performed using additives with the purpose to reduce solvent-assisted quenching by water, and to avoid scattering of light in the system’s column. Deuterated solvents and encapsulation strategies were employed to reduce deactivation processes caused by water, and refractive index matching was used to limit the refraction of light. By using heavy water in assay sensitivity was increased by 17-25% for two biomarkers and with the use of different fluorophores showing consistency for the method. With the use of a refractive index matching liquid, it was possible to increase the depth at which the maximum fluorescence intensity occurred three-fold, using confocal microscopy. However, implementing found advantages in assay proved to be difficult due to mediums being hydrophobic and viscous, highlighting the complexity of the microfluidic system.

Legionella pneumophila is a human pathogen commonly found in natural and artificial aquatic environments and can cause a condition called legionellosis. Monitoring for legionellae is therefore important for protecting public health and identifying its environmental sources is a way to prevent illness. This has resulted in development of several control strategies to identify these sources. One of these strategies is to construct a valid method to detect Legionella pneumophila and monitoring these methods is a way to ensure the method remain effective at tracing infection.  The current version of standardized method is called ISO 11731:2017 and supersedes its former version called ISO 11731:1998. The former version uses a combination of heat and acid solution treatment to reduce interfering microorganisms in water with high background, whereas the current version separates the treatment by subdividing the sample in three parts. One part is subjected to heat treatment, one with acid solution treatment and one remains untreated. Therefore, the aim of this study is to analyse how this difference in method strategy will affect detection of Legionella pneumophila between the current and its former version of ISO 11731. To do this, this study divided the experiment into two parts: experiment A was aimed at evaluating the validity of the method and experiment B was designed to study repeatability in terms of dispersion and performance data range. For experiment A: 14 samples were tested using both ISO 11731:2017 and 11731:1998 to see how the results differentiated. Six are natural samples and was appointed based on their previous results that showed positive for Legionella. Four samples were spiked with different serotypes of Legionella and the remaining four were spiked with both Legionella and Legionella-inhibited bacteria. For experiment B, three certified reference material with different concentration of Legionella pneumophila serotype 1 was tested in repeatability conditions with each sample producing ten replicates.  In conclusion, based on results assessed in this study ISO 11731:1998 was more suitable to analyse water with higher concentration of interfering microorganisms. By a combination of heat and acid solution treatment: it maximizes the reduction of interfering microorganisms which facilitates Legionella to cultivate on agar. ISO 11731:2017 was more efficient in recovering different serotypes of Legionella. Although, there were a significant increase in dispersion and performance data range results in ISO 11731:2017. This indicates that since there is an additional dilution step added in acid solution treatment: it increases the risk of human error and therefore a greater vulnerability to the method.

Affinity proteins constitute an important category of cancer therapeutics. Owing to properties such as high target affinity and selectivity, therapeutic proteins offer more targeted therapy than small molecule drugs. The target molecules are typically proteins that are overexpressed on the surface of tumour cells, such as membrane-bound receptors. However, these surface proteins are usually expressed in normal tissues as well, resulting in on-target off-tumour toxicity. Proteins with a higher tissue selectivity are thus needed. Here, this has been addressed by developing prodrug proteins dependent on cancer-specific proteases for activation. The prodrugs were composed of a target-binding affibody (active domain) connected to a masking affibody (masking domain) by a peptide linker including a protease substrate. The target of the prodrugs developed in this project was the HER2 receptor, which is overexpressed in several cancer types. Three prodrug candidates were developed, produced and characterised based on their ability to be activated by their respective protease. The hypothesis that the prodrugs could be activated and thus bind to HER2 in cancer cells was tested using biosensor assays, as well as preliminary cancer cell assays. One of the three candidates showed strong potential to be used as a targeted therapy for cancer treatment in the future. / Affinitetsproteiner utgör en viktig kategori av cancerläkemedel. Jämfört med småmolekylära läkemedel är affinitetsproteiner mer riktade, då de har högre affinitet och selektivitet än små molekyler. Oftast utgörs det molekylära målet av ett protein som överuttrycks på ytan av cancerceller, så som membranbundna receptorer. Dessvärre uttrycks de flesta cancerspecifika proteiner i mindre mängd även i normal vävnad. Detta leder till oönskade effekter som kan ge upphov till biverkningar. I syfte att utveckla mer vävnadsspecifika läkemedel har här affibody-baserade “prodrugs”, beroende av cancerspecifika proteaser för aktivering, tagits fram. Prodrug-proteinerna i detta projekt är riktade mot HER2-receptorn, som är överuttryckt i flera typer av cancer. Tre kandidater togs fram och utvärderades med avseende på deras förmåga att aktiveras av sina respektive proteaser. För att testa hypotesen att kandidaterna kunde binda till HER2 på cancerceller efter proteasaktivering användes biosensoranalys samt experiment med cancerceller. En av kandidaterna visade stark potential att kunna användas som ett riktat läkemedel mot cancer i framtiden.

Trigger Factor (TF) is a prokaryotic chaperone protein that exerts its major chaperone activity while associated with translating ribosomes, assisting de novo folding of the emerging nascent chain. Although much is known about the kinetics behind TF-ribosome binding, most results are based on in vitro experiments which fail to mimic the cellular environment. Single-particle approaches have gained increasing power for studying binding kinetics of biomolecules in living cells. One such method is single-particle tracking by super-resolution fluorescence microscopy, where the position of a fluorescently labelled particle is recorded over time, giving information about the movement of the particle inside the cell. Changes in diffusion behaviour is then used as an indicator of changes in biological activities. In this work, a diffusion model that qualitatively and quantitatively describes TF’s binding to ribosomes is presented. The model was obtained by single-particle tracking of TF labelled with HaloTag. Particle movements were analysed with a Hidden Markov Model-based algorithm that fit the trajectories to a defined set of different diffusion states, where fast diffusion could be related to free TF and slow diffusion to a ribosome-bound state. Moreover, the model could distinguish between two types of ribosome interactions: TF’s stable binding to ribosomes and a faster sampling behaviour. The average time spent stably bound to ribosomes is 670 ms and these interactions account for 53% of TF’s activity. TF is one of many processing proteins that interact with the emerging peptide chain during translation. By using the same approach on more of these factors, the interplay between them and the growing nascent chain can be characterized, giving an increased understanding of the highly complex translation machinery.