Browsing by department "Elintarvike- ja ympäristötieteiden laitos"

The development of new antibiotics is very challenging work. However, it has become less attractive area of research for the pharmaceutical industry due to the rapid development of antibiotic resistance among bacteria. As a result, nowadays there are less new antimicrobial medicines appearing on the market. Increase in antibiotic resistance challenges the effective treatment of infectious diseases. Therefore, it is important to know about the existence and prevalence of antibiotic resistant bacteria and resistance genes. Waste water has been found to contain substantial amounts of antibiotics or their derivatives. Furthermore the bacterial density of the waste water is high. These factors may cause the selection pressure that assists the evolution, preservation and spread of antibiotic resistance in bacterial population. Wastewater treatment plants are believed to act as a reservoir of antibiotic resistant bacteria and antibiotic resistance genes. In this study, from samples taken from Viikinmäki wastewater treatment plant it was determined the quantity of resistance genes making bacteria resistance to a wide range of beta-lactam antibiotics. The samples were taken at the different stages of wastewater treatment process during three sampling days in June, September and December 2010. The samples were taken from the incoming wastewater and, from the final effluent and from the dried sludge. The DNA from wastewaters and sludge was isolated and the gene copy numbers of blaCTX-M-32 and blaOXA-58 antibiotic resistance genes were measured by quantitative PCR method. Antibiotic resistance gene copy numbers were normalized with 16S ribosomal RNA gene copy numbers. The antibiotic resistance genes investigated in this work make bacteria resistant to broad-spectrum penicillins, different groups of cephalosporins, monobactams, and carbapenems. It was found that blaOXA-58 and blaCTX-M-32 gene copies do exist in both incoming wastewater and dried sludge. From the incoming wastewater we found on average 3x10-3 blaOXA-58 gene copies/16S rRNA gene and 7x10-5 blaCTX-M-32 gene copies/16S rRNA gene. From dried sludge we found on average 3x10-3- blaOXA-58 gene copies/16S rRNA gene and 2x10-2 blaCTX-M-32 gene copies/16S rRNA. Both antibiotic resistance genes were also found in the final effluents. The amounts of antibiotic resistance genes /16S rRNA genes were found to decrease during of the wastewater treatment. In the final effluent the amount of blaOXA-58 genes was found to be two orders of magnitude less than in the incoming wastewater and for, blaCTX-M-32 gene copies it was found the decrease by one order of magnitude. Although the amount of antibiotic resistance genes decreased during of the wastewater treatment, the amount left in the final effluent is still notable and when it ends up in the water or soil it might have an effect to the antibiotic resistance in the nature.

Psychrotrophs, which are mostly gram negative bacteria, can produce heat stable proteases and lipases. Even though these bacteria cannot survive pasteurization of milk, the enzymes can. This can cause problems for the dairy industry. Psychrotrophic milk isolates also have multidrug-resistant traits for antimicrobials and may act as reservoir for resistance genes. The aims of the study were to test denaturing gradient gel electrophoresis (DGGE) for PCR products obtained from organic and conventional raw milk bacteria, to optimize DNA-extraction protocols and PCR-conditions for the raw milk samples and study the bacterial population changes during the cold storage. The aim was also to study the antimicrobial susceptibility of the bacterial isolates. The DNA was extracted from the raw milk samples, when received and after 4 days cold storage, using commercial kits. Nested-PCR was performed and samples were analysed using DGGE. Susceptibility to antimicrobials was determined by growing bacteria on plates that contained two different concentrations of five antibiotics. From the five antibiotics two were used as a combination. The composition of the bacterial population changed during the cold (4ºC) storage: the difference in DGGE profiles was clear between 0 and 4 days cold storage. Fingerprint profile analysis showed that irrespective of the origin of the raw milk, the sample profiles were clustered according to the sampling date (day 0 or day 4). There was no clear difference between DGGE-profiles from conventional and organic milk. Proportion of psychrotrophic bacteria increased and antimicrobial resistance seemed to be more prevailing in conventional than in organic raw milk. Antimicrobial resistance decreased after four days storage at 4ºC, in most of the cases. We showed that the PCR-DGGE-method is an efficient tool to analyse the changes in bacterial populations in raw milk and that cold storage has an evident effect in population composition.

Dynamics of raw milk associated bacteria during cold storage of raw milk and their antibiotic resistance was reviewed, with focus on psychrotrophic bacteria. This study aimed to investigate the significance of cold storage of raw milk on antibiotic-resistant bacterial population and analyse the antibiotic resistance of the Gram-negative antibiotic-resistant psychrotrophic bacteria isolated from the cold-stored raw milk samples. Twenty-four raw milk samples, six at a time, were obtained from lorries that collected milk from Finnish farms and were stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Antibiotics representing four classes of antibiotics (gentamicin, ceftazidime, levofloxacin and trimethoprim-sulfamethoxazole) were used to determine the antibiotic resistance of mesophilic and psychrotrophic bacteria during the storage period. A representative number of antibiotic-resistant Gram-negative isolates retrieved from the cold-stored raw milk samples were identified by the phenotypic API 20 NE system and a few isolates by the 16S rDNA gene sequencing. Some of the isolates were further evaluated for their antibiotic resistance by the ATB PSE 5 and HiComb system. The initial average mesophilic counts were found below 105 CFU/mL, suggesting that the raw milk samples were of good quality. However, the mesophilic and psychrotrophic population increased when stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Gentamicin- and levofloxacin-resistant bacteria increased moderately (P < 0.05) while there was a considerable rise (P < 0.05) of ceftazidime- and trimethoprim-sulfamethoxazole-resistant population during the cold storage. Of the 50.9 % (28) of resistant isolates (total 55) identified by API 20 NE, the majority were Sphingomonas paucimobilis (8), Pseudomonas putida (5), Sphingobacterium spiritivorum (3) and Acinetobacter baumanii (2). The analysis by ATB PSE 5 system suggested that 57.1% of the isolates (total 49) were multiresistant. This study showed that the dairy environment harbours multidrug-resistant Gramnegative psychrotrophic bacteria and the cold chain of raw milk storage amplifies the antibioticresistant psychrotrophic bacterial population.

Long QT syndrome, LQTS, is a congenital or acquired cardiac disorder characterized by prolonged cardiac repolarization phase. It is observed as a prolonged QT period in electrocardiodiagraph and can cause life-threatening specific ventricular tachycardia, torsades de pointes. Hundreds of mutations in 15 genes (LQT1-15) are linked to congenital LQTS. Worldwide prevalence of congenital LQTS gene mutations is from 1:2000 to 1:5000. However, the prevalence in Finland is much higher due to four founder mutations that alone occur in one out of 250 individuals. Acquired LQTS is often drug-induced and the most common cause for the withdrawal of drugs on the market. Carriers of LQTS mutations are more susceptible to acquired LQTS than normal population. LQTS-specific cardiomyocytes can thus provide a thorough model for drug cardiotoxicity screening, better insight into disease mechanisms and assist in drug development. This thesis was a part of a bigger project concentrating on validation of LQT2-specific cell lines that could be used for the purposes mentioned above. Induced pluripotent stem (iPS) cell technology enables creation of disease-specific pluripotent stem cell lines, which can be differentiated into any cell type. In this thesis, two LQT2-specific iPS cell lines derived from a clinically symptomatic 44-year-old female were used. She is heterozygous for Finnish founder mutation L552 in KCNH2 gene, which encodes the α-subunit of the cardiac rapidly activating potassium rectifier channel. iPS cells were first verified to express pluripotency markers and to form embryoid bodies containing all germ layers. iPS cells were then differentiated into cardiomyocytes by culturing them with END-2 cells and mechanical beating of the cardiomyocytes was assessed from video recordings. Single LQT2-specific cardiomyocytes showed LQT2-related phenotype in vitro with 43% of single LQT2 cardiomyocytes showing abnormal beating patterns and prolonged contraction time. This phenotype was rescued in LQT2-specific cardiomyocyte clusters. Finally, the expression ratios of wild type and mutated KCNH2 alleles were compared between cardiomyocytes derived from the female and her son, a carrier of the same mutation but with asymptomatic phenotype. Cardiomyocytes from both individuals expressed KCNH2 alleles with the ratio between 1:2 and 1:1 (wt:mut), thus allelic imbalance does not explain differences in the clinical phenotypes. All in all, the results of this thesis suggest that after further validation, mainly electrophysiology studies, these cell lines are most likely suitable to be applied for disease modeling, cardiotoxicity screening and finding new therapies for LQT2.

Neuropathic pain is pain caused by injury or damage to the nervous system. This adverse condition affects millions of people in all parts of the world, and no known cure has been developed. Existing treatments are mainly anti-depressants or opioids that alleviate symptoms instead of repairing damaged neurons. Glial cell line-derived neurotrophic factor (GDNF) and artemin, belonging to GDNF family ligands, have been shown to restore damaged neurons. However due to the poor pharmaceutical properties of these proteins, such as difficult administration and expensive production, their transition to clinics is complicated. That is why we have been developing small molecule GFL-mimetics as an alternative. One of these mimetics is a compound named BT44. Characterization of BT44 began with in vitro experiments, where we tested the compound’s ability to activate luciferase reporter gene in cells expressing GDNF (GFRalpha1 and RET) and artemin (GFRalpha3 and RET) receptors, as well as ability to induce RET phosphorylation and activate intracellular MAPK/ERK and Pi3-K/Akt pathways. Furthermore, we tested stimulation of neurite outgrowth by the compound from cultured dorsal root ganglion neurons. In a similar manner to GDNF and artemin, BT44 was shown to activate GFRalpha1/RET and GFRalpha3/RET receptors and induce RET phosphorylation and intracellular signaling, in addition to stimulating neurite outgrowth from cultured DRG neurons. Because of the promising in vitro results, we moved on to in vivo testing in rat spinal nerve ligation (SNL) model of neuropathic pain. Similarly to artemin, BT44 was able to alleviate mechanical nociception and cold allodynia in SNL rats. In addition, we found that BT44 normalized to a certain degree nociception-related markers influenced by SNL in the tissues of experimental animals, which emulates previously published results for artemin. To summarize, our results indicate that BT44 is effective in neuronal restoration and pain alleviation, suggesting it for further development as innovative neuropathic pain treatment.

The literature review elucidates the mechanism of oxidation in proteins and amino acids and gives an overview of the detection and analysis of protein oxidation products as well as information about ?-lactoglobulin and studies carried out on modifications of this protein under certain conditions. The experimental research included the fractionation of the tryptic peptides of ?-lactoglobulin using preparative-HPLC-MS and monitoring the oxidation process of these peptides via reverse phase-HPLC-UV. Peptides chosen to be oxidized were selected with respect to their amino acid content which were susceptible to oxidation and fractionated according to their m/z values. These peptides were: IPAVFK (m/z 674), ALPMHIR (m/z 838), LIVTQTMK (m/z 934) and VLVLDTDYK (m/z 1066). Even though it was not possible to solely isolate the target peptides due to co-elution of various fractions, the percentages of target peptides in the samples were satisfactory to carry out the oxidation procedure. IPAVFK and VLVLDTDYK fractions were found to yield the oxidation products reviewed in literature, however, unoxidized peptides were still present in high amounts after 21 days of oxidation. The UV data at 260 and 280 nm enabled to monitor both the main peptides and the oxidation products due to the absorbance of aromatic side-chains these peptides possess. ALPMHIR and LIVTQTMK fractions were oxidatively consumed rapidly and oxidation products of these peptides were observed even on day 0. High rates of depletion of these peptides were acredited to the presence of His (H) and sulfur-containing side-chains of Met (M). In conclusion, selected peptides hold the potential to be utilized as marker peptides in ?-lactoglobulin oxidation.

Microbial cellulases, e.g. cellobiohydrolases, are able to degrade cellulose and lignocellulosic biomass to smaller glucose-containing monomers and oligomers. Cellulases are often multi-domain enzymes comprised of different protein domains (i.e. modules), which have different functions. The main two components, which often appear in cellulases, are the cellulose-binding module (CBM) and the catalytic domain. The CBMs bind to cellulose, bringing the catalytic domains close to their substrate and increasing the amount of enzymes on the substrate surface. The catalytic domain performs the cleavage of the substrate, e.g. in the case of cellobiohydrolases hydrolyses or “cuts” the crystalline cellulose chain into smaller soluble saccharides, mainly cellobiose. Unlike aerobic fungi, which utilize free extracellular enzymes to break down cellulose, anaerobic microbes often use a different kind of strategy. Their cellulases are organized and bound to the cell surface in a macromolecular protein complex, the cellulosome. The core of the cellulosome is formed of a scaffolding protein (the scaffoldin) consisting mainly of multiple consecutive cohesin domains, into which the catalytic subunits of enzymes attach via a dockerin domain. This creates a protein complex with multiple different catalytic domains and activities arranged in close proximity to each other. Dockerins and cohesins are known to bind each other with one of the strongest receptor-ligand -pair forces known to nature. Dockerin containing fusion proteins have also been successfully combined in vitro with proteins containing their natural counterparts, cohesins, to create functional multiprotein complexes. In this Master’s thesis work the goal was to 1) produce fusion proteins in which different CBMs were connected to dockerin domains, 2) combine these fusions with cohesin-catalytic domain fusion proteins to create stable CBM and catalytic domain containing enzyme complexes, 3) to characterize these enzyme complexes in respect of their thermostability and cellulose hydrolysis capacity and 4) to ultimately create a robust and fast domain shuffling method for multi-domain cellobiohydrolases (CBH) to facilitate their faster screening. The hypothesis of the experiments was that different CBMs fused with a dockerin domain and the cellobiohydrolase catalytic domain fused with a cohesin domain could be produced separately and then be combined to produce a functional two-domain enzyme with a dockerin-cohesin “linker” in between. In this way time and work could be saved because not every different CBM- catalytic domain -pair would have to be cloned and produced separately. Several CBM-dockerin fusion proteins (in which the CBM were of fungal or bacterial origin) were tested for expression in heterologous hosts, either in Saccharomyces cerevisiae or Escherichia coli. The purified proteins were combined with a fungal glycoside hydrolase family 7 (GH7) cellobiohydrolase-cohesin fusion protein produced in S. cerevisiae. The characterization of the catalytic domain-CBM -complexes formed through cohesin-dockerin interaction included thermostability measurements using circular dichroism and activity assays using soluble and insoluble cellulosic substrate. The results were compared to enzyme controls comprising of the same CBM and catalytic domain connected by a simple peptide linker. The results showed that the cohesin-dockerin –linked cellobiohydrolase complex performed in the cellulose hydrolysis studies in a similar manner as the directly linked enzyme controls at temperature of 50˚C and 60 ˚C. At temperatures of 70 ˚C the complex did not perform as well as the control enzymes, apparently due to the instability of the dockerin-cohesin interaction. The thermostability measurements of the enzymes, together with the previously published data supported the hydrolysis results and this hypothesis. The future work should be aimed at enhancing the thermostability of the cohesin-dockerin interaction as well as on verifying the results on different cellulase fusion complexes.

Infant undernutrition with associated diseases is a leading cause of under-five deaths globally, causing 45% of child deaths. A critical point for the nutritional status of the infant is the time when the first foods are introduced in addition to breastfeeding, i.e. complementary feeding. Based on prior research, complementary feeding practices are inadequate in East Africa, including Uganda. Particular concerns are the not timely introduction of complementary foods and low dietary diversity of children under two years. Complementary feeding is a complex set of behaviours. Good complementary feeding comprises, in addition to nutritious food itself, the feeding moment, technique and style. The promotion of good complementary feeding practices therefore demands multiple approaches. The aim of this study was to explore complementary feeding perceptions and practices in the context of the Health Belief Model (HBM) and to gain understanding on how to promote health behaviour change for better complementary feeding. The study was carried out in the rural area of Kirewa, Uganda. All together 9 focus group discussions (FGD) were held for caretakers of children under two: mothers, fathers, grandfathers and grandmothers. A set of educational videos on infant care and feeding practices, the GloCal-videos, were used as a projective tool in the FGDs. In addition, one individual interview with the local health care worker was conducted. The data from the FGDs and the interview were analysed with a deductive content analysis method based on the HBM. The findings from this research demonstrate that complementary feeding practices among the study participants were suboptimal in relation to timing, dietary diversity, consistency and feeding frequency. The importance of complementary feeding as a health behaviour preventing malnutrition and stunting was not understood among these Kirewan caretakers. The findings from the HBM based analysis suggest messages about the susceptibility of children to detrimental consequences of poor feeding and their seriousness should be targeted to caretakers. Based on this study, the GloCal-videos may work as cues to action for better complementary feeding practices.

Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.

Oat protein is a valuable cereal protein with high protein content and a good balance of amino acids profile. However, the inflexible molecular structure and poor watersolubility of oat protein limit its application in food industry. To exploit the food use of oat, the functionalities of oat proteins should be improved. ?-chymotrypsin, a proteolytic enzyme, has been proven as being able to deamidate some food proteins without severe hydrolysis under alkaline pH conditions, hence to improve the protein functionalities. The aim of this research was to test whether oat protein could be deamidated by ?- chymotrypsin under alkaline pH conditions. The extent of ?-chymotrypsin-induced proteolysis on oat protein was also studied. Oat protein was treated by ?-chymotrypsin under alkaline conditions from pH 8 to 11 at room temperature for 2 hours. The deamidation degrees of the proteins were measured by ammonia quantification. The hydrolysis degrees of proteins were analyzed by trichloroacetic acid precipitation and protein quantification method. The changes of molecular weights were analyzed by SDS-PAGE and SE-HPLC. The action of ?-chymotrypsin under alkaline pH conditions induced slight deamidation of oat protein to low deamidation degrees (9-12%). On the other hand, ?-chymotrypsin mainly caused hydrolysis of oat protein to hydrolysis degrees between 49% and 62%. SDS-PAGE and SE-HPLC analysis also revealed that significant hydrolysis of oat protein occurred during the reaction. The hydrolysis mainly caused the shift of oat protein fractions from molecular weight above 20 kDa to molecular weight below 15 kDa. Change in pH did not cause significant differences on deamidation degrees and hydrolysis degrees of oat protein In conclusion, oat protein could not be effectively deamidated by ?-chymotrypsin under alkaline pH conditions, while the proteolysis was severe.