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.

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.

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.

Alzheimers sjukdom (AD) är den vanligaste formen av demens och kännetecknas av intracellulärt neurofibrillärt trassel (NFT) bestående av proteinet tau och extracellulära plack, uppbyggda av peptiden amyloid beta (Aβ). En växande skara studier har börjat peka mot att AD är en hjärnspecifik typ av diabetes. Insulinresistens följt av hyperinsulinemi och hyperglykemi är kännetecken för diabetes mellitus typ 2 (DMT2) och har visat sig vara en riskfaktor för AD. Insulin, ett hormon som kontrollerar glukoshomeostasen i perifera nervsystemet (PNS) och är viktigt för minne och inlärning, transporteras över blod-hjärnbarriären i en mättnadsbar transportmekanism och dess koncentration i centrala nervsystemet (CNS) minskar vid DMT2 och AD. Insulin-like growth factor 1 (IGF-1), ett neuronskyddande protein som minskar ogynnsam β-sekretasklyvning av amyloid precursor protein (APP) i amyloidkaskadhypotesen, minskar i koncentration i hjärnan när mycket insulin transporteras in i CNS. γ-sekretas ökar sin aktivitet på APP vid höga halter kolesterol som är vanligt vid DMT2, Aβ fungerar då som en negativ inhibitor till HMG-Coa reduktas (HMGR), enzymet som bildar kolesterol och kan därmed reglera kolesterolhalterna. Regleringssystem för Aβ i blod-hjärnbarriären (BBB) som p-GP, LRP-1 och RAGE rubbas vid DMT2. Aβ och insulin delar samma degraderingssystem, insulin degrading enzyme (IDE), som reglerar halterna Aβ och insulin. Dessutom har Aβ oligomerer visat sig kunna bryta ned insulinreceptorer (IR). Vidare har läkemedel mot diabetes visat sig lindra demens hos AD patienter. I den här rapporten gås de molekylärfysiologiska sambanden igenom i detalj. Slutligen finns det fog för ett samband mellan metabolt syndrom, en riskfaktor för DMT2, och AD.

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

Calmodulin is a highly conserved calcium ion binding protein expressed in all eukaryotic species. The 149 amino acid residues in the primary structure are organized in seven α helices with the highly flexible central α helix connecting the two non-cooperative domains of calmodulin. Each domain contains two EF-hand motifs to which calcium ions bind in a cooperative manner, hence the binding of four calcium ions saturate one calmodulin molecule. In the cardiovascular area calmodulin is involved in the activation of cardiac muscle contraction, and mutations that arise in the genetic sequence of the protein often have severe consequences. One such consequential mutation that can arise brings about the replacement of the highly conserved aspartic acid with glycine at position 130 in the amino acid sequence. In this research, the thermal and chemical stability within the C domain of the D130G variant of human calmodulin was investigated using a new method only requiring circular dichroism spectroscopic measurements. Affinity studies within the C domain of the D130G variant of human calmodulin were performed using fluorescence spectroscopy, and the ligands chosen for this purpose were trifluoperazine and p- HTMI. All analytical experiments were performed with the C domain of wild type human calmodulin as a reference. From the new method, it was concluded that the C domain of the D130G variant of human calmodulin has a slightly decreased stability in terms of Tm and Cm values compared to the C domain of wild type human calmodulin. The affinity analyses indicated that neither trifluoperazine nor p-HTMI discriminates between the C domain of the D130G variant of human calmodulin and the C domain of wild type human calmodulin in terms of dissociation constants. The pivotal outcome from this research is that the new method is applicable for determination of the stability parameters Tm and Cm of heat stable proteins.

This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from Candida albicans as a tool for discovery of anti-candida drugs. C. albicans causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since C. albicans needs Saps during virulent growth, these enzymes are good targets for drug development. In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions. Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.

Wing polyphenism is a type of phenotypic plasticity present in several insect species whereby a genotype have the ability to develop alternative wing morphs when exposed to different environmental cues. One organism demonstrating a clear case of wing polyphenism is the water strider species, Gerris buenoi, which develop long- or short wings depending on exposure to different photoperiods (the time the organism is exposed to light during a 24 h period). The molecular mechanism behind wing polyphenism in insects in general, and in water striders in particular, is largely unknown. From a study on wing polyphenism in the Brown planthopper (Nilaparvata lugens), some candidate genes have been identified and include two insulin receptor genes and the Forkhead transcription factor (FOXO). Since these genes have been demonstrated to affect wing polyphenism in Brown planthopper (BPH) and since G. buenoi contains an additional insulin receptor homolog, the potential role of these genes in regulating wing polyphenism in G. buenoi have in this project been investigated. The functional genetic technique RNA interference (RNAi) was used to evaluate the function of the genes. This method knock down gene expression in the genes mentioned above, one at a time, to investigate if they have a function in wing polyphenism in G. buenoi. DsRNA with specific homology to each target gene was successfully produced. However, when attempting to inject the dsRNA through micro injection all injected liquid leaked out from the body cavity, and the RNAi was therefore not successful. Further optimisation of the injection protocol has to be done to be able to perform RNAi properly in the future. Thereafter, RT-qPCR was used to evaluate whether the insulin receptor genes and FOXO are differentially expressed between the two photoperiods giving rise to the different wing morphs. The differential gene expression experiment showed differences between the mRNA levels of all target genes between G. buenoi being reared in the two different photoperiods. More specific upregulation of the genes FOXO and insulin receptor 2 in short winged G. buenoi were demonstrated. Further, insulin receptor 1-like, was also demonstrated to be upregulated in the short winged morph. Results presented in this project are in line with the previously identified regulation pattern in BPH, still the results need further evaluation. Since gene expression differences were present for all candidate genes between G. buenoi reared in the different photoperiods, theses genes could still be seen as potential candidate genes in wing polyphenism in water striders.