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.

The prokaryotic adaptive immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a defense mechanism that helps to protect the prokaryotic cell from invading mobile genetic elements. This project was performed at Uppsala University and served to answer whether the expression of Cascade, which is part of the CRISPR defense system, will have a negative effect on the cell that expresses it and to also determine whether the CRISPR defense system is effective enough to stop the spread of a conjugative plasmid. A microfluidic system was used in order to perform the experiments and images were taken with the help of fluorescent microscopy. Three different donor strains from E.coli were used. These strains had their own version of the RP4 conjugative plasmid which had the ability to infect recipient E.coli cells with said plasmid. The recipient cells had the ability to express the CRISPR system in order to defend themselves from the plasmid and CRISPR was also inducible with the help of IPTG. The different versions of the RP4 conjugative plasmid had different amounts of spacer targets that Cascade, the recognition complex in the CRISPR system, could recognize. When the recipient cells were induced and had a known target sequence of the plasmid they were able to defend themselves and keep the number of transconjugant cells low. When the recipient cells did not know the target the amount of transconjugant cells were higher. It was also noted that when the cells were induced inside the microfluidic PDMS chip they had a slower generation time. It was also noted that recipient cells had begun to die towards the end of the microfluidic experiments when the cells were induced. This raised the question as to whether the CRISPR defense system was targeting itself as well as the RP4 conjugative plasmid.

No description available.

L-valine is a branched chain amino acid often used in food, pharmaceutical, cosmetic, and animal feed industries. The most used production method for L-valine and other branched chain amino acids is bacterial fermentation through Escherichia coli or Corynebacterium glutamicum, both of which are heterotrophic bacteria in need of added sugars and energy demanding bioreactors. Synechocystis sp. PCC 6803 is a model cyanobacteria that only uses carbon dioxide and sunlight as energy source and naturally can biosynthesize L-valine, which makes it a suitable platform for sustainable production. The regulation of the L-valine biosynthesis pathway is not fully understood why more research is needed to be able to optimize the production of L-valine. In other organisms, there is feedback inhibition by L-valine that limit the biosynthesis which might be the case for Synechocystis as well. During this project, adaptive laboratory evolution was used to increase the valine tolerance of Synechocystis sp. PCC 6803, by evolving the cells to grow in increasing concentrations of L-valine over multiple generations. This resulted in a final strain that had a tenfold increase in tolerance compared to non-evolved wild type Synechocystis. Whole genome sequencing was used to determine if and what mutations had led to the increased tolerance. Another aim of the project was to evolve a strain that overproduced L-valine. This was done by adaptive laboratory evolution with norvaline as a selection pressure. Norvaline is an amino acid analogue that has a very similar structure to valine, why it can be mis incorporated during aminoacyl-tRNA synthetase. We hypothesized that the Synechocystis cells would overproduce L-valine to outcompete the increased norvaline, thereby increasing the norvaline tolerance. Through adaptive laboratory evolution the norvaline tolerance was increased, but the mechanism behind the tolerance could not be determined during this project. The production of all branched chain amino acids by the evolved strains first needs to be measured to determine if they are in fact producing more L-valine. Then, transcriptomics and/or whole genome sequencing can be used to investigate what genes are regulated or mutated to obtain the increased L-valine production.

In the future, food security will face significant challenges due to climate change and a growing world population. One approach to make agriculture more sustainable is to preserve biodiversity by utilizing crop wild relatives as a source of genetic material. These crop wild relatives are closely related to today’s cultivated crops and can be an important asset to combat food insecurity. Gaining more knowledge about a species’ genetic diversity through microsatellite analysis is an important step for future conservation and potential utilization in crop improvement. However, before these studies can take place the microsatellite markers have to be optimized for PCR. In this study, eleven microsatellite markers were optimized for bilberry individuals. Optimized annealing temperatures were found for all markers and most of them had amplification in three or more of the individuals tested from Sweden, Finland and Iceland. Ten out of the eleven tested markers were regarded suitable for future genetic diversity analyses. The eleventh, VCB-C00694, was considered unsuitable due to formation of primer dimers and not amplifying in several individuals. By assessing the genetic diversity of bilberry, Vaccinium myrtillus, it opens up the possibility to enrich their domesticated relative the American blueberry, Vaccinium corymbosum, by introducing new genetic variety. In relation to this, the increased knowledge about genetic diversity among bilberries in the Nordic can lead to better understanding of their need for conservation.

Single-cell proteomics is a useful tool for measuring differences in cell populations for clinical trials. In this report we have conducted a literary review where we looked at 23 different single-cell proteomics methods and their advantages and disadvantages. We have looked at both mass spectrometry-based and affinity-based methods to find upcoming methods in the field of single-cell proteomics. Our findings show that there are multiple promising techniques that can be applied in different contexts. Moreover, we recommended combining different protocols, for instance Capillary zone electrophoresis (CZE) with a microfluidic platform or Optidrop with one of the barcoding methods for better results. When conducting this review it became clear that most methods could be improved by implementing software programs such as PEPerMINT and Infinity flow. Therefore, we encourage that such data acquisition and analysis methods are implemented to yield more accurate characterization and quantification of the single-cell proteome.

Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.

Bacteria of the genus Streptococcus are common asymptomatic colonisers of humans and animals. As opportunistic pathogens they can however, depending on their host’s immune status and other circumstances, cause mild to very severe infections. Streptococci are highly intertwined with specific host species, but can also cause zoonosis or anthroponosis in more uncommon hosts. Prolonged and reoccurring infections require immune evasion strategies to circumvent detection and eradication by the host’s immune defence. A substantial part of the immune defence against bacterial pathogens is mediated by immunoglobulins. This thesis is based on work to identify and characterise immunoglobulin degrading enzymes secreted by different Streptococcus species as a means to sabotage and evade antibody-mediated immune responses. Stoichiometric and kinetic analysis of the IgG degrading enzyme IdeS from the important human pathogen S. pyogenes revealed that IdeS cleaves IgG, opposed to previous publications, as a monomer following classical Michaelis-Menten kinetics. The IdeS homologue of S. suis, IdeSsuis, did however not cleave IgG, but was highly specific fo rporcine IgM. S. suis was found to possess yet another protease, IgdE, capable of cleaving porcine IgG. Both of these proteases were shown to promote increased bacterial survival in porcine blood during certain conditions. IgdE is the founding member of a novel cysteine protease family (C113). Novel streptococcal members of this protease family were shown to specifically degrade certain IgG subtypes of the respective Streptococcus species’ main host. The observed substrate specificity of IgdE family proteases reflects the host tropism of these Streptococcus species, thereby giving insights into host-pathogen co-evolution. The abundance of immunoglobulin degrading enzymes among Streptococcus species indicates the importance of evasion from the antibody mediated immune responses for streptococci. These novel identified immunoglobulin degrading enzymes of the IdeS and IgdE protease families are potential valid vaccine targets and could also be of biotechnological use.