Douglas-fir (Pseudotsuga menziesii) is an economically important softwood that is clonally propagated for reforestation purposes by somatic embryogenesis. The molecular basis of embryogenesis in conifers is largely unknown and this prevents progress in somatic embryogenesis protocols. In angiosperms, the LEAFY COTYLEDON1 (LEC1) gene, encoding the HAP3 subunit of the eukaryotic CCAAT box-binding factor, is important in embryo formation, and necessary for somatic embryogenesis. A candidate gene strategy was employed to isolate the Douglas-fir LEC1 homologue, PmLEC1, via the polymerase chain reaction (PCR) with degenerate primers based on the Arabidopsis conserved domain, and the full-length cDNA sequence was obtained by rapid amplification of cDNA ends-PCR (RACE-PCR). The putative protein sequence shared high sequence identity with Arabidopsis LEC1. Northern analysis and quantitative real-time PCR indicate that this is an embryo-specific gene, expressed with the highest abundance during early embryogenesis. Antibodies were raised against a synthetic 18-amino acid PmLEC1 peptide, and in contrast to mRNA expression, Western blotting shows that PmLEC1 protein expression persists until the seedling stage. To gain insight into modulation of PmLEC1 expression and its inducibility in mature tissues, stress and hormone treatments were performed on mature seed and the promoter sequence was isolated by genome walking. Sorbitol, mannitol and 2,4-epibrassinolide were found to significantly up-regulate PmLEC1 expression. The PmLEC1 promoter contains a 5’ UTR intron with numerous enhancer elements, and factors that bind to these elements mediate responses to auxin, UV light and developmental cues, osmotic stress, biotic stress, and tissue culture. Some of the regulatory elements are binding sites for seed-specific transcription factors that are well known from angiosperms, providing new evidence that AGL15, ABI3 and VP1 proteins have a direct role on LEC1 expression. In investigating the embryogenic capacity of PmLEC1, ectopic expression of PmLEC1 in the embryo lethal Arabidopsis lec1-1 null mutant complemented the mutation and permitted the production of viable, desiccation tolerant seeds. In addition, transgenic seedlings produced embryo-like structures from vegetative organs and expressed seed-specific genes. In wild type plants, ectopic expression of PmLEC1 resulted in a bushy phenotype but expression of seed-specific genes was not observed. Taken together, these results show that PmLEC1 is an embryo-specific gene with an essential role throughout embryogenesis, and PmLEC1 expression may be induced in mature seeds by stress and hormone treatments. Because mature seeds show only trace amounts of PmLEC1 transcripts and Douglas-fir somatic embryogenesis can only be induced from immature embryos, this information provides useful insight into initiation of embryogenesis from vegetative tissues. The identification of binding sites for transcription factors known from angiosperms in the promoter region of PmLEC1 has revealed the identity of several genes which are expected to play pivotal roles in conifer embryogenesis.

Multiresistenta gramnegativa bakterier (MRGN) som Escherichia coli och Pseudomonas aeruginosa utgör ett globalt hälsoproblem och på grund av resistensutveckling hos bakterierna behövs nya behandlingsalternativ. Extended Spectrum β-Lactamase (ESBL) är vanligt förekommande hos MRGN och det finns olika typer av ESBL (exempelvis ESBLA och ESBLCARBA). Gemensamt är att det finns få behandlingsalternativ för ESBL-producerande MRGN. Syftet med studien var att undersöka effekten av potentiellt nya behandlingsalternativ mot MRGN i form av cykloserin och ceftazidim tillsammans med β-laktamasinhibitorn avibaktam. För att undersöka minimum inhibitory concentration (MIC) för cykloserin (CYK) mot E. coli (n=26) användes en egenutvecklad buljongspädningsmetod. Isolat av P. aeruginosa med och utan karbapenemasproduktion (n=25) undersöktes avseende MIC för ceftazidim/avibaktam (CZA) med två kommersiella buljongspädningsmetoder.  För CYK låg medianen för E. coli-stammarnas MIC-värden vid 32 mg/L (16 – 64 mg/L) vilket ligger kring det epidemiologiska cut-off värdet för Mycobacterium tuberculosis (32 mg/L), för vilken CYK idag används som behandling.  För CZA låg medianen för P. aeruginosa-stammarnas MIC-värden vid 8 mg/L (<1 - ≥8 mg/L), och 40% (2/5) av de karbapenemasproducerande isolaten var känsliga enligt kliniska brytpunkter (S≤8 mg/L). Sammanfattningsvis visar studien att CYK har MIC-värden mot non-ESBL- och ESBL-producerande E. coli i nivå med andra patogener där preparatet används. Studien visar också att CZA kan ha effekt mot isolat med nedsatt känslighet mot meropenem eller ESBLCARBA-producerande isolat av P. aeruginosa, men isolaten måste resistensbestämmas innan behandling sätts in. / Multiresistant gramnegative bacteria (MRGN) like Escherichia coli and Pseudomonas aeruginosa, constitute a global health issue. Due to the resistance development among bacteria, new options for treatment are needed. Extended Spectrum β-Lactamase (ESBL) is common among MRGN, and there are different types of ESBLs (as ESBLA and ESBLCARBA). The increasing lack of treatment alternatives is mutual for the different ESBLs. The purpose of this study was to examine cycloserine and ceftazidime with the β-lactamase-inhibitor avibactam, two potentially new options for treatments effective against MRGN. To examine the minimum inhibitory concentration (MIC) for cycloserine (CYK) against E. coli (n=26) a in-house broth microdilution-method was used. Using two commercial broth microdilution-methods, isolates of P. aeruginosa with and without carbapenemaseproduction (n=23) were examined regarding MIC for ceftazidime/avibactam (CZA).  Regarding CYK, the median MIC-value of the E. coli-strains was 32 mg/L (16 - 64 mg/L), which is around the epidemiological cut-off-value (32 mg/L) for Mycobacterium tuberculosisfor which CYK is being used as treatment. The median of the MIC-values for CZA was 8 mg/L (<1 - ≥8 mg/L) for all P. aeruginosa-strains and 40% (2/5) of the carbapenemaseproducing isolates were sensitive according to the clinical breakpoint (S≤8 mg/L). In summary, this study shows that CYK has MIC-values against non-ESBL- and ESBL-producing E. coli at the same level as other pathogens where CYK is being used. Further, CZA may have effect against the isolates with reduced susceptibility against meropenem and ESBLCARBA-producing P. aeruginosa, although the isolates need to be susceptibility-determined before treatment.

No description available.

The elucidation of regulatory mechanisms in higher organisms represents a front line problem in biochemical genetics. In Man the only material available for experimental studies of regulatory mechanisms is cells cultured in vitro. Enzymes which are differentiated into isozymes may have a complexgenetic background involving the action of more than one gene locus. The study of isozyme systems in cultured cells has developed into a valuable tool of increasing importance for the understanding of the genetic regulatorymechanisms in normal cells as well as in cancer cells. The purposes of this investigation were: 1. to elucidate the isozyme differentiation of amino acid naphthylamidasein cultured human cancer cells and normal cells. 2. to study the regulatory effects of steroid hormones especially hydrocortisoneon the levels of the different isozymes. / digitalisering@umu.se

The F1 part of F0F1-ATP synthase in the proteobacterium Rhodobacter blasticus contains five different proteins, but when the DNA was sequenced a sixth gene was found in the operon. The protein that corresponds to the sixth gene has been named Majastridin. When an amino acid BLAST search is performed with the Majastridin sequence, protein sequences have been found that are similar to Majastridin in other bacterial strains, and one of them is Streptococcus pneumonia. The hypothetical protein from Streptococcus pneumonia contains 242 amino acids and has a molecular weight around 30 kDa.   In this work the Majastridin-like protein from Streptococcus pneumonia was expressed in E. coli cells and purified with nickel affinity chromatography and size exclusion chromatography. The result was verified with SDS-PAGE and western blot. The purified protein was then crystallized with the hanging drop method, where crystals were formed and optimization was made. The protein was also used to produce antibodies.

digitalisering@umu.se

Some steroids in the brain and peripheral nervous system have been shown to have neuroprotective effects but the knowledge is limited. The present study examines the effects of steroids including oxysterols, vitamin D and vitamin D analogs on cell viability/growth and steroidogenesis in the nervous system. Both 24- and 27-hydroxycholesterol reduced staurosporine-induced toxicity in human neuroblastoma SH-SY5Y cells. In addition, 27-hydroxycholesterol decreased the staurosporine-mediated induction of caspases, known to be important in apoptotic events. From the findings it may be concluded that effects of oxysterols on cellular viability are dependent on the concentration and on the type of oxysterol. 24-Hydroxycholesterol was also found to attenuate oxidative stress both in SH-SY5Y cells and astrocytes. The results indicate that during some conditions, oxysterols may have neuroprotective effects. The vitamin D analogs tacalcitol and calcipotriol strongly reduced proliferation, cell viability and migration of human glioblastoma T98G cells, similarly as 1,25(OH)2D3 , the physiological form of vitamin D. Glioblastoma is the most lethal type of primary tumors in the CNS. These findings suggest that vitamin D analogs are potential candidates in treatment of brain tumors, most likely in combination with other therapies. Astrocytes were found to be a major site for expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) whereas expression of CYP17A1 was found in both astrocytes and neurons. 3β-HSD and CYP17A1 are important steroidogenic enzymes. Vitamin D inhibited both CYP17A1- and 3β-HSD -mediated activity and mRNA levels, with a stronger effect on mRNA expression than on enzyme activity. This indicates that 1,25(OH)2D3 could affect the production of sex hormones in the brain. In summary, results from this thesis contribute to the knowledge on the effects of oxysterols on cell viability and oxidative stress in cells from the CNS. Also the results provide data on the effects of vitamin D in the brain and suggest that vitamin D analogs may be promising candidates for treatment of certain brain tumors.

Campylobacter are probably the most important bacterial pathogen related to food-borne illnesses; specifically, gastroenteritis and diarrheal diseases. These bacteria can be isolated from various environments, but always originate from the intestine of warm blooded animals. Particularly, Campylobacter are found in the intestinal tract of poultry, and due to contamination of poultry meat and also further contamination of other food they can cause human infections. Sometimes this results in larger outbreaks, such as during 2016-2017 in Sweden where thousands of persons got infected by a single strain of Campylobacter jejuni sequence type 918 (ST-918). The same strain was also identified amongst a large number of poultry farms and suspicions were directed towards dirty transport cages for poultry as a main route for transmitting the strain between different farms. Similar scenarios with large outbreaks related to one or two single strains (ST-50 and ST-257) had also been observed in previous years and this raised questions about certain strains being especially adapted to survive outside the intestine. The aim here was to examine whether outbreak strains and other strains of C. jejuni have different potential to resist different stress conditions that may be encountered between the poultry farm and the human intestine.

The epidermis is composed of multiple cell strata where viable keratinocytes, in the basal layer (stratum basale (SB)), go through a range of steps with the final stage of being dead corneocytes in the outer most layer (stratum corneum (SC)). The differentiation, which can be thought of as programmed cell death, include several key processes that are essential for an intact skin barrier. The route from SB to SC is accompanied by changes, such as osmotic pressure and pH, that are believed to trigger some of these processes. In this project, HaCaT cells were incubated with, commonly used, skin care substances (urea, glycerol, transcutol and salicylic acid) to assess their impact on cell viability, by MTT-assay, and gene expression, by qPCR. Further, the relationship between osmotic pressure, viability and gene expression was studied. The excipients showed a dose-dependent decrease of keratinocyte viability which also was explained by elevated osmotic pressure when concentration was increased. Exceptions were however observed for transcutol, which showed protective features against osmotic stress. Upregulation of the genes were mainly observed when cells were treated with high concentrations. Involucrin was affected by the substances to a greater extent when compared to other markers. The upregulation of involucrin was however seen to be driven by the osmotic pressure rather than biological effects of the molecules. The project conclude that the viability and gene expression of the keratinocytes are highly related to the osmotic pressure and probably influences the differentiation to a greater extent than the molecules themselves.

Natural products and their derivatives have and are still used by humans for various health ailments due to their rich sources of drug discovery. New biologically active compounds from natural products play a key role in drug development. Marine sponges and their associated microbes contain a lot of bioactive compounds that are potential for drug development. These compounds produce chemical compounds with useful pharmaceutical properties such as antitumor, anti-infective, anti-inflammatory, and antibacterial properties. The main focus of this project was on the antibacterial activity of six different sponge specimens. The aim was to screen the antibacterial activity of the sponge specimen’s extracts. In order to do so, a Minimum Inhibitory Concentration assay was performed to screen the sponge's antibacterial activity against E. coli and S. aureus. Analytical HPLC was used for separation and Solid Phase Extraction (SPE) was used for determining the effect of salts towards the inhibition of anti-bacterial activity for two selected extracts. Ethanolic extract of Stylissa massa showed antibacterial activity against S. aureus. SPE would be a rapid purification step to remove the salts present in sponges at a high concentration but it has not shown a significant effect on the inhibition of antibacterial activity. However, further separation and purification need to be done to be able to completely screen for all the six different sponge specimens.