The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles. The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems. We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

Glaucoma is one of the leading causes of vision loss in the world and it is characterized by the dysfunction of retinal ganglion cells (RGCs). An early pathomechanism of glaucoma is the degeneration of the axons of RGCs and finding new treatments that could prevent axon degeneration is of great interest. Increasing the concentration of coenzyme nicotinamide adenine dinucleotide (NAD) has been shown to be axon protective in a number of neurodegenerative systems. Increasing NAD could potentially be achieved by increasing the catalytic properties of the terminal enzyme involved in the cytosolic production of neuronal NAD, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2). NMNAT2 is thus an ideal therapeutic target. Polyphenol A (PA), which is a polyphenol that will not be disclosed, has been demonstrated to be NAD-boosting through positive modulation of NMNAT2. The aim was to develop a cell-based assay for screening PA and 12 novel analogues of PA for their NAD-boosting effects in brain cortex, retinal and liver cells isolated from C57BL/6J mice. The protocol involved using a bioluminescence assay for which optimization of variables such as cell concentration, substrate (nicotinamide) concentration, PA concentration and incubation time was performed. The method development resulted in a one-day protocol for testing PA and its analogues in cortical cells. PA and several of its analogues exhibited NAD-boosting effects. This protocol along with the results from the screening can be further used for the development of novel drugs that could prevent glaucoma and other axon and neurodegenerations.

Sjukdom på grödor orsakad av bakterier kan bidra till ekonomiska förluster för bönder samt brist på mat, därför är det viktigt att utveckla nya hållbara sätt att motverka och behandla grödor mot bakterier. Idag är det mest vanliga tillvägagångssättet antibiotika men detta är inte hållbart p.g.a uppkomst av antibiotikaresistens. Antibiotika är inte heller tillgängligt för alla bönder och grödor då kostnaden blir för hög. Världsbefolkningen växer och om 80 år beräknas det bo mellan 9.9 och 12.7 biljoner (95% konfidens) människor på jorden. Växande befolkning samt ökande klimatförändringar, som torka och höjda temperaturer kräver nya bekämpningsmetoder mot bakterier för att tillgodose behoven i framtiden. Det saknas information om hur växter hanterar och reglerar bakteriella hot, därför är målet med denna studie att bidra med kunskap kring den transkriptionella regleringen av växters immunsystem mot bakterier. För att göra detta har promotorsekvenser hos gener som är förknippade med immunförsvaret i växter undersökts efter konserverade regulatoriska element. En känd receptor, FLS2 har en stor roll i växters försvar mot bakterier och känner igen en peptid från bakteriers flagell. Denna studie har undersökt FLS2 och den sammankopplade receptorn SERK1. Hos FLS2 kunde ingen konserverad modul hittas i uppströmsekvensen, däremot observerades ett 8 bp långt motiv, CAACTTG, i alla undersökta arter. I SERK1 hittades en lång konserverad modul bestående av flera motiv. Både FLS2-motifet och två motiv i SERK1-modulen binds av transkriptionsfaktorn MYC2. För att testa hypotesen att MYC2 bidrar till den transkriptionella regleringen av FLS2 och SERK1 har en experimentell plan utformats, där Nicotiana benthamiana transfekteras av Agrobacterium tumefaciens innehållandes promotorsekvenserna samt generna till transkriptionsfaktorn MYC2. En ökad förståelse kring de olika delarna och mekanismerna som medverkar inom växters immunförsvar kan bidra till den fortsatta forskningen mot hållbara lösningar till att säkra mat i framtiden. / Several factors contribute to the demand of new, sustainable solutions to bring food security to the world population. The United Nations predicts, with a confidence of 95%, that the world population will be between 9.9 and 12.7 billion by year 2100. At the same time plant agriculture as seen today is threatened by climate changes e.g., rising temperatures and more extreme weather conditions. In addition, plant bacterial pathogens reduce yields, and cause losses of over $1 billion dollars worldwide every year to the food production chain. The currently most used and effective treatment against bacterial infections on crops is antibiotics, but this is not a viable alternative for most growers due to increasing antibiotic resistance and the high development, production, and distribution cost. During the upcoming years development of new approaches against bacterial infections on crops is of high importance but currently there are information gaps in the field of plant defense regulation systems. This study was aimed to provide knowledge about the transcriptional regulation of genes that are included in plant immune system towards bacteria. To investigate this, conserved regulatory elements of the upstream sequences of two defense-related plant receptor kinases, FLS2 and SERK1, was searched for in different species. FLS2 is a surface receptor that recognizes a peptide derived from the bacterial flagellin protein, and is part of the pathogen-triggered immunity response of most of higher plants. In FLS2 no conserved module was found but a single motif, CAACTTG, is conserved in all chosen species. In SERK1 a strikingly long and conserved module was found. Both the FLS2 motif and two motifs in the SERK1 module are recognition motifs with MYC2, a transcription factor involved in different plant mechanisms and the regulation of phytohormones like abscisic acid and auxin. To address whether MYC2 is involved in the transcriptional regulation of FLS2, an experimental approach is described, involving transactivation by MYC2 of FLS2 reporter constructs, studies using agroinfiltration in Nicotiana benthamiana. An increased knowledge about the different components and mechanisms of plant defense regulation will help the research towards new bactericides, transgenic plants, and other ways to secure food for upcoming generations.

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.

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.