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.

Genome editing is an exciting field that allows for the precise modification of an organism's DNA. One of the most advanced tools in this area is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which creates a DSB (Double-strand break) at a specific location in the genome. This break can then be repaired by the cell using one of two pathways – NHEJ (nonhomologous end joining) or HDR (homology-directed repair) HDR leads to more precise repair and is used to create KI (Knock-In) modifications by introducing a homologous piece of DNA with the desired changes. However, HDR is a rare event that competes with the error prone NHEJ pathway, limiting its efficiency. HDR mainly occurs in the G2 and S phases of the cell cycle, making it a challenge to control and target. To improve KI efficiency, researchers have used strategies such as inhibiting NHEJ or activating HDR. This study focuses on identifying direct and indirect activators of HDR through a library assay screening. We established a robust method for screening compounds in HEK293 cells that relies on a plasmid-based delivery Cas9, gRNA (guide RNA), and synthetic ssDNA (single strand DNA). Out of 3,000 compounds screened, 1% showed a higher signal than the positive control, and approximately 10% presented a higher signal than untreated cells. The top 5 compounds were further validated in dose response. Our system opens new avenues for improving the efficiency of KI modifications.

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.

The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown. Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In paper I, genetic alterations in Notch1 and the Notch1 regulating gene CDC4 were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating Notch1 mutations in 39% of the lymphomas, suggesting that Notch1 is an important target gene for mutations in chemically induced lymphomas. While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes per se are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In paper II, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including Notch2, were transcriptionally downregulated. We also confirmed binding of Lef1 to Notch2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the Notch2 promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target cyclin D1. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines. In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases. / Studies on molecular genetic alterations in colorectal cancer

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.

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.