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(2021)Despite their immobile nature, their ability for adaptation allows plants to face harmful conditions from the environment to successfully survive and reproduce. Plant cells sense and integrate signals from the environment and activate response mechanisms. Participants in these mechanisms are the receptor-like protein kinases (RLKs) and a subgroup of RLKs, the cysteine-rich receptor-like kinases (CRKs). Members of this family have been associated with functions related to environmental stress responses in plants. CRK2 is one interesting member of the CRK clade of RLKs. While roles of CRK2 in the response to biotic and abiotic stimuli have been recently described, many aspects of the diverse functions of CRK2 remain elusive. The reduced size of the crk2 mutant suggests that developmental processes are affected by the absence of the protein. One of the objectives of this work was to analyse potential reasons for the smaller size of crk2. The difference in plant size could be due to a reduced number of cells. Results from the analysis of young cotyledons showed that the smaller plant size is not due to a reduced cell number in leaves when compared to Arabidopsis thaliana (Arabidopsis) ecotype Columbia (Col-0). Another way to understand the processes in which a protein is involved is to target possible interaction partners. Therefore, genotyping and analysis of growth phenotypes of T-DNA insertion mutant lines for candidate interaction partners for CRK2 was performed. The results revealed smaller phenotype for a nitrate transporter (NRT1.7) mutant in fresh weight and rosette area whereas for a protein kinase (QSK1) mutant, higher fresh weight but reduced rosette area was observed compared to Col-0. Generation of constructs for fusion protein expression and purification revealed the possibility of expressing tagged cytoplasmic regions of these proteins for further analysis of protein-protein interaction through kinase assays due to the kinase activity of CRK2. Generation of fluorescent-tagged proteins from the candidate interaction partners allowed for localization studies via confocal microscopy to determine the co-localization to the plasma membrane of these proteins with CRK2, which is located to plasma membrane under standard growth conditions. The co-localization results suggest that the proteins NRT1.7 and QSK1 colocalize with CRK2, which is a step forward in the verification of their possible interaction in planta. The smaller size of the nrt1.7 and qsk1 mutants indicates that the lack of these proteins affects plant development.
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(2016)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.
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(2023)In recent years, exceptionally large bacteriophages with genome sizes over 500 kilobase pairs (kbp), called megaphages, have been discovered from sequence data, but no previous publications discussing megaphage isolates have been published. In 2011, a phage infecting a Flavobacterium strain was isolated from the Kymijoki river. The phage, named FKy-1, was determined to have a genome size of 643 kbp, based on yet unpublished results, making it the first described megaphage isolate. In this study, we focused on characterizing megaphage FKy-1, by observing the virus morphology, determining the type and length of its life cycle, and measuring its stability in different temperatures and conditions. Purification of the phage by precipitation and ultracentrifugation in a sucrose density gradient resulted in separation of both virion and phage subcomplexes. Based on transmission electron microscopy and cryogenic electron microscopy, FKy-1 was observed to have typical myovirus morphology, with a large icosahedral head of around 160 nm in diameter, and a tail of around 180 nm in length. Molecular masses of the major proteins present in the virion and phage subcomplexes were estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 50-70 kDa for the major capsid protein, 60-70 kDa and 150-200 kDa for the major tail proteins. Digestion attempts with restriction endonucleases proved unsuccessful, indicating possible phage genome modifications or other defensive mechanisms. The phage was determined to have a lytic life cycle which takes over 3 h to cause cell lysis, resulting in the release of around 10 progenies per infected host cell. The phage proved to be quite stable, with minimal impact on infectivity measured at a temperature range of -20 °C to +40 °C, and in minimal buffer conditions. In summary, we proved that the purification method used here is well suited for megaphages, and that FKy-1 is of myovirus morphology, produces a low number of progenies per host, and is relatively stable. As no other publications regarding megaphages exist, this study acts as a good basis for future research regarding megaphage morphology, infection cycle and stability.
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(2020)This study aimed to investigate the microbiological quality of the whole and gutted Baltic herring at different seasons by traditional culture-dependent methods combined with the identification of bacterial isolates by MALDI-TOF MS. Additionally, the microbiome of the herring was characterized by culture-independent 16S rRNA gene amplicon sequencing. Bacterial counts were within acceptable limits at all seasons although the H2S-producing bacteria levels were above the recommended level of 5 log10 CFU g-1 at two sampling points. With the culture-dependent methods and the sequencing of the 16S rRNA gene, the microbiome of the whole and gutted herring was dominated by the bacterial class Gammaproteobacteria. Shewanella, Pseudomonas, and Aeromonas were the most frequently isolated genera among the viable population identified with MALDI-TOF MS. With the culture-independent approach, Shewanella followed by Psychrobacter were the most abundant genera. Additionally, a high relative abundance of the phyla Firmicutes and Actinobacteria and, in some samples, Epsilonbacteriaeota represented by the genus Arcobacter, was detected. Variances in the microbiological quality of different herring batches observed in this study could not be attributed to the season. Therefore future research through a longer period was proposed, including data on the environmental factors, such as the fishing location and the water quality, possibly affecting the quality of the herring.
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(2020)Chloroplasts are essential plant photosynthetic organelles evolved from a prokaryotic endosymbiont many years ago. A vast majority of chloroplast proteins are encoded in the nucleus and then imported post-translationally by multiprotein translocases located in the membrane of the organelle. It was identified that outer envelope membrane (OEM) components are subject to ubiquitin-proteasomal degradation, governed by a recently established proteolytic system called CHLORAD (chloroplast-associated degradation). It has been suggested that this machinery is involved in regulation of plastid biogenesis and stress tolerance in plants by protein import regulation and remodelling of the organellar proteome. In this study, to further investigate factors involved in chloroplast protein import regulation, we aimed to characterize two putative regulators SKIP6, an F-box/kelch repeat protein, and ASK1, a component of CUL1-based SCF E3 ligase, identified by tandem affinity purification of TOC components and SP1. We performed physiological analyses on skip6-1 and ask1-1 single mutant Arabidopsis plants to identify whether these factors are required for degradation of OEM translocase components (TOC machinery). To identify an association of these factors with the TOC machinery and CHLORAD components, we employed subcellular localization and co-immunoprecipitation (IP) assays in protoplasts. Double mutant sp1 ppi1 and sp2 ppi1 plants were previously shown to specifically supress an atToc33 mutation (specific suppression of ppi1 chlorosis phenotype), resulting in greener and larger plants. Following, for second-site specific suppressor analyses of the atToc33 mutation, we generated ask1-1 ppi1 double mutant plants and provided their initial characterization. As the CHLORAD system was shown to be vital for plant development and to contribute to stress tolerance, therefore, in this study the involvement of SKIP6 in stress tolerance in mutant plants was analysed by implementing osmotic and salt stresses. Physiological analyses revealed an early-senescence phenotype in the skip6-1 single mutant plants, which could be attributed to degradation of TOC components and subsequent decrease in chlorophyll level. Interestingly, an opposite effect was observed after dark treatment, in which SKIP6 knockout mutants remained greener with higher abundance of TOC proteins and chlorophyll level in comparison to wild-type plants. Stress-induced experiments did not show the involvement of SKIP6 in stress tolerance at early developmental stages. Subcellular localization and co-IP experiments revealed cytosolic localization of SKIP6 and its physical interaction with the TOC machinery, respectively. Obtained double mutant ask1 ppi1 plants presented male sterility as well as growth suppression followed by greener leaves at late developmental stages. In summary, our results provide initial characterization of unknown SKIP6 protein suggesting its involvement as a component of SCF E3 ligase (CUL1-ASK1-SKIP6) in the reorganization of the TOC machinery and CHLORAD components at early and late developmental stages, respectively. These initial data represent one of the first steps towards broadening our knowledge on the regulatory network of chloroplast biogenesis in plants, as well as important advance in the development of new strategies for crop improvement.
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(2022)Hexanal is a highly volatile liquid that can prolong the shelf life of fruits and vegetables (Paliyath and Subramanian 2008). Previously, Lehtonen et al. (2020) introduced a freeze-dried active packaging material which was continuously releasing hexanal through the naturally occurring lipid oxidation reactions of sunflower oil. In this study, nine different sunflower oil-based matrices were characterized at different stages of their preparation process. They contained crystallization modifiers (acylglycerols, phytosterols, or their blend), coating materials (gum Arabic and maltodextrin), and were produced by spray-drying of an emulsion. The crystallization modifiers in the matrix may enable the control over the start of the reaction for hexanal release. The emulsions with crystallization modifiers were significantly unstable, their viscosity increased with an increasing proportion of gum Arabic, and they contained crystalline regions from the first day of preparation. All tested emulsions formed a white powder after spray drying and the powder particle sizes were characterized. The differential scanning calorimetry (DSC) did not reveal any melting point of the lipid phase in majority of the powder samples. However, the DSC results showed that the melting temperatures of corresponding oleogels with acylglycerols and acylglycerol/phytosterol blend were 64 and 56.6 °C, respectively. In this thesis, emulsions containing acylglycerols and/or phytosterols were characterized and shown to be suitable for the encapsulation by the spray drying technique. Further studies focusing on the hexanal release from the matrix need to be performed since no hexanal release was detected from the powder samples.
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(2023)Abstract Faculty: Faculty of Agriculture and Forestry* and Faculty of Medicine *coordination Degree programme: Master′s program in Microbiology and Microbial Biotechnology Author: Johanna Potila Title: Characterization of potentially therapeutic bacteria from a healthy fecal donor. Level: Master′s thesis Month and year: August 2023 Number of pages: 40 Keywords: Clostridioides difficile, dysbiosis, FMT, next-generation probiotics, adhesion, anti-inflammatory Supervisors: PhD Kaisa Hiippala, PhD, Docent Reetta Satokari and PhD Pauliina Lankinen Where deposited: E-thesis University of Helsinki Abstract: Recurrent Clostridioides difficile infection (rCDI) is a healthcare-associated infection related to antibiotic use, that causes significant morbidity and mortality. Fecal microbiota transplantation (FMT) is the most effective treatment for rCDI and it is successful in nearly 90% of patients. However, there are some risks related to FMT use such as the potential risk of transferring pathogens or other phenotypes despite donor screening. Defined bacterial mixtures consisting of endogenous commensal gut microbes with beneficial properties could be used instead of FMT to mitigate the risks and improve the availability of the treatment. 12 bacterial strains previously isolated from a healthy fecal donor were characterized in this study. At first, oxygen tolerance and culturability of the isolates in several different media were examined. The second aim was to investigate if these isolates are safe for bacteriotherapeutic use by testing hemolytic properties, antibiotic susceptibilities and proinflammatory properties. The third objective was to investigate potential beneficial properties such as adherence of the isolates to mucus and epithelial cell lines and anti-inflammatory effects on epithelial cells. Caco-2 and HT-29 cell lines were used as a model of intestinal epithelial cells. Growth was abundant on standard brain heart infusion (BHI) medium supplemented with 0,5% yeast extract and more than half of the isolates tolerated the 4-hour oxygen exposure. These results suggest that many of the strains have good production characteristics. All 12 isolates were non-hemolytic and most of them were susceptible to many commonly used anti-microbials such as amoxycillin/clavulanic acid and piperacillin/tazobactam. Low induction of interleukin-8 (IL-8) release from HT-29 cells was observed for all the isolates which indicates no pro-inflammatory effect. These safety tests suggest that the isolates are safe for therapeutical use. Adhesion to mucus and intestinal epithelial cells (HT-29, Caco-2) was low to moderate (2-7%), which can potentially promote their colonization in the gut. No attenuation of Escherichia coli lipopolysaccharide (LPS)-induced IL-8 release from HT-29 cells was observed, which indicates that characterized strains do not have anti-inflammatory effects on epithelial cells. However, it is likely that they have some other important roles in the gut e.g., in cross-feeding networks and can thus help with restoration of a healthy, diverse gut ecosystem. In conclusion, the characterized isolates could be suitable for bacteriotherapeutic use in the treatment of rCDI.
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(2019)Antibiotic-resistant bacteria are an increasing threat to global health, caused by the excessive use of antibiotics and the lack of new antimicrobial agents being introduced to the market. New approaches to prevent and cure bacterial infections are needed to halt the growing crisis. One of the most promising alternatives is phage therapy which utilizes bacteriophages to target and kill pathogens with specificity. Pseudomonas aeruginosa is a common opportunistic pathogen that is intrinsically resistant to antibiotics, making it one of the most heavily studied targets of phage therapy. In this study, I characterized four P. aeruginosa phages, fHo-Pae01, PA1P1, PA8P1 and PA11P1, and evaluate their potency in therapeutic applications. Bioinformatic analysis of the genomes revealed the phages to be genetically highly similar and belonging to the Pbunavirus genus of the Myoviridae family. No genes encoding harmful toxins, antibiotic-resistance, or lysogeny were predicted. On the other hand, many of the predicted genes had unknown functions. The host ranges of the phages were assessed using 47 clinical P. aeruginosa strains and predicted host receptor binding tail proteins were compared. Some correlation between the host ranges and mutations in the tail proteins were observed but this alone was not sufficient to explain the differences in the host ranges. The recently isolated vB_PaeM_fHoPae01 (fHo-Pae01) phage was further characterized by a one-step growth curve and imaged with a promising atomic force microscopy method that had not been used before in the Skurnik group. Though the imaging results failed to provide any further knowledge of the phage, the 70-minute-long latent period of infection could be determined from the growth curve. Anion- exchange chromatography was found inefficient in purifying the fHo-Pae01 phage, so alternative methods such as endotoxin columns should be used when purifying these phages for patient use. In conclusion, all four phages appeared to be safe for therapeutic use based on current knowledge, and PA1P1 and PA11P1 were the most promising candidates due to their broad host ranges.
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(2024)Aspergillus niger is a well-studied filamentous ascomycete fungus and one of the most used fungal species in various biotechnological sectors. Recently the research focus on A. niger has shifted towards production of biochemicals and biomaterials from plant biomass residues. However, there are still many aspects of the plant biomass conversion process by A. niger that are not known in detail, including its sugar uptake systems. Sugar uptake in fungi relies on sugar transporter (ST) proteins that control the uptake of different sugar molecules. Fungal STs are abundant and diverse in their function, which is why many of them remain uncharacterized. Identification of fungal STs for substrates like xylose can improve the industrial production of lignocellulose-based bioproducts by e.g. improving the substrate uptake rate in fungal cell factories. In this thesis, four putative xylose STs from A. niger, XltD, XltE, XltG, and XltH, were characterized both physiologically and functionally in A. niger and Saccharomyces cerevisiae, respectively, together with two previously functionally characterized xylose transporters, XltA and XltB. In addition, a fifth putative xylose transporter XltF was characterized functionally in S. cerevisiae. For the physiological characterization, A. niger 6Δxlt strain was generated by deleting xltA, xltB, xltD, xltE, xltG, and xltH genes using CRISPR/Cas9 methodology. The physiological analysis of the 6Δxlt strain revealed the presence of additional xylose transporters in the A. niger genome, which still remain to be discovered. The functional characterization of the putative A. niger xylose STs was carried out by creating six recombinant S. cerevisiae IMK1010 strains producing the STs fused with a green fluorescent protein. Functional characterization confirmed the correct localization of the STs within plasma membrane, except for XltG which accumulated inside the yeast cells. This indicated potential localization of XltG within endoplasmic reticulum and a putative role in intracellular sugar transport. Growth assays of the recombinant yeast strains demonstrated variable affinities of the A. niger STs for hexoses. The results showed XltF not to be a xylose ST, but instead a hexose ST with low-affinity for fructose and dual-affinity for glucose. The STs were further tested for their substrate specificity and affinity for xylose in a competitive assay between xylose and glucose. XltE displayed a preference for xylose over glucose identifying it as a new low-affinity xylose transporter. Although further research is needed to elucidate the roles of the studied A. niger STs, XltE is a promising candidate for enhancing xylose uptake in fungal cell factories.
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(2015)Recently, a new muscular disorder has been reported in chicken M. pectoralis major called Wooden Breast that might be linked to intensive growth selection. The purpose of this study was to find the influence of Wooden Breast condition on protein composition of the breast muscle with special emphasis on myofibrillar and sarcoplasmic protein fractions. For fulfilling the aims of the study, a total number of 34 pectoralis major muscles from both Wooden Breast and normal chickens (Ross 508, Aviagen Ltd, Scotland) were used to evaluate protein composition, protein profile of sarcoplasmic and myofibrillar proteins and their changes, by doing one-dimensional SDS-PAGE analysis. M. Pectoralis major defected by Wooden Breast demonstrated significant decline in sarcoplasmic (P?0.001) and myofibrillar (P?0.05) protein content, in comparison with normal breast muscles. Furthermore, gel electrophoresis indicated significant changes in the intensity of 18 sarcoplasmic protein bands and 7 myofibrillar protein bands. Majority of affected sarcoplasmic proteins were glycolytic enzymes. Moreover, impacted myofibrillar proteins play a role in muscle fibre integrity (e.g. desmin) or calcium signalling. Results of the present study also revealed desmin overexpression with coexistence of a desmin fragment in Wooden Breast which was confirmed by Western-blot technique. In conclusion, the findings of this study indicated defected chicken breast with Wooden Breast contained less protein and the composition differed compared to normal chicken breast muscle, which proves the influence of Wooden Breast condition on protein characteristics of chicken breast muscle. Further studies are needed to interpret the protein changes in the Wooden Breast and the possible role of these changes on metabolic and structural status of the muscle.
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(2017)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.
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(2021)To determine the role of metalloprotease EGY1 and hormone signalling pathways in PSII repair cycle, a mutant named white because of early senescence was identified having 4bp deletion in EGY1. To further characterize the growth responses in white mutant, two suppressors (white suppressor 1 and white suppressor 2), mutated in STAY GREEN1 (SGR1) which prevents chlorophyll degradation, restored the normal white phenotype was identified upon suppressor mutant screens. This study investigated the effect of chloroplast translation inhibitors (lincomycin/chloramphenicol) and MV (methyl viologen) on photosynthesis in Arabidopsis thaliana single and double white mutants. Furthermore, a second goal was to verify the correct identification of the mutations in white suppressor 1 and white suppressor 2. Western blotting and pulse amplitude modulated fluorimeter (PAM) was used to quantify the D1 protein (reaction core of PSII) levels and photochemical efficiency (Fv/Fm) respectively. Immunoblotting revealed a pronounced decrease in D1 levels for both white and egy1. PAM results showed a high tolerance of white mutant towards lincomycin/chloramphenicol. The white suppressors complemented the lincomycin/chloramphenicol tolerance of white mutant. The white mutant was highly MV sensitive. This MV response was altered in white double mutants (white ein2-1, white sr1-4D and white rcd1-4), suggesting that hormone signalling was involved in the response to MV. The decreased abundance of D1 in the white mutant suggests a role for EGY1 in PSII assembly and D1 turnover under light stress. In all assays (immunoblotting and PAM), the white mutant and egy1-2 gave the same results, this confirms the correct identification of the white mutant as a new egy1 allele. The successful restoration of lincomycin /chloramphenicol tolerance by white suppressors (S1 and S2), implicates that chlorophyll breakdown impacts on correct photosynthesis function. The suppressors S1 and S2 were transformed with wildtype SGR1, which restored the white mutant phenotype. Thus, the suppressor phenotype was caused by mutations in SGR1.
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(2022)The coronavirus disease 19 (COVID-19) pandemic currently poses a challenge to the healthcare system and global public health. The upsurge of new SARS-CoV-2 variants, the uneven vaccine distribution worldwide, and the documented reinfections raise a concern about the protective immunity of COVID-19 recoverees. In this context, reliable methods for the detection of SARS-CoV-2 neutralizing antibodies are needed. Considering the methodological complexity and limitations of traditional virus neutralization tests, surrogate enzyme-linked immunosorbent assays (sELISA) constitute a promising alternative allowing high-throughput testing. However, there is still a need of assessing the specificity and sensibility of these assays so that they can be clinically applied. In this thesis, two goals were pursued; the detection of neutralizing antibodies in COVID-19 recoverees plasma samples using an in-house microneutralization assay and the comparison of these results with those obtained with two sELISA; SARS-CoV-NeutraLISA surrogate neutralization (Euroimmun) and cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript). The SARS-CoV-2 microneutralization assay was performed with VERO E6 cells and the Fin-1 strain of the SARS-CoV-2 virus. The plasma samples were provided by the Helsinki University Hospital and were previously screened with commercial IgG-ELISA targeting the anti-SARS-CoV-2 spike subunit 1 (Euroimmun) and nucleocapsid (Abbott) proteins. A total of 111 samples were tested, 74% of them presented a detectable NAb titer with at least two of the methods. The neutralizing antibody titer obtained with the microneutralization assays resulted in an overall proportion of positives lower than expected. Therefore, the in-house microneutralization assay needs further optimization or a different neutralization assay should be selected instead for future analysis. The combined data from the three tests was used to determine the sensitivity (99%, 83%, 81%) and specificity (72%, 100%, 100%) of cPass, Neutralisa and microneutralization assays respectively. This data suggests the use of cPass (GenScript) in primary screenings, in combination to Neutralisa (Euroimmun) to confirm secondary tests.
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(2021)As the number of vegetarians and flexitarians is increasing, the demand for meat alternatives is following the same trend. Faba bean is a promising crop for environmental, health, and social reasons. From its seeds, several products can be produced, such as flour, protein concentrate, and protein isolate. These materials can be further processed for the development of texturized vegetable proteins. In this context, high moisture extrusion (HME) is an innovative technology, which requires more water, but milder temperatures than the more studied low moisture extrusion. The present thesis aimed at covering the current knowledge gap on this technology, by focusing on the flavor aspect of faba bean-based meat alternatives. The volatile profile of faba bean flour, protein concentrate, protein isolate, and mixtures was studied before and after high moisture extrusion by means of HS-SPME-GC-MS. Comparisons were made between raw materials (flour, protein concentrate, protein isolate, mixtures) and extrudates. The effect of the origin (Canadian and Finnish) of the protein concentrate was also investigated. Materials were also characterized for the following aspects: moisture, ash, protein, lipids, lipid-degrading enzymes (lipase and lipoxygenase), free amino acids, and free mono-, di-, and oligosaccharides. Several classes of volatile compounds were observed in both raw materials and extrudates, namely organic acids, alcohols, aldehydes, alkanes, alkenes, aromatic compounds, furans, esters, ketones, nitro compounds, and terpenes. Many compounds were characteristic of only one material, demonstrating that the formation of certain volatiles was product dependent. However, some other compounds were found in all materials, such as 1-hexanol, hexanal, and 2-pentylfuran. Literature addresses these compounds as responsible for the beany flavor. HME caused the inactivation of lipase and lipoxygenase but did not prevent the formation of lipid oxidation products. Products from Maillard reaction were not found in the extrudates. After HME, 1-hexanol levels decreased, whereas the abundance of 2-pentylfuran significantly increased. The behavior of hexanal after HME was less stable and predictable than the two beforementioned compounds. Not only these novel findings could have application in product development, but they also laid the basis for further research aimed at improving the flavor of faba bean.
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(2022)Inflorescence meristem development in plants generally falls into two types: one is the indeterminate type when the main axis keep growing and new flowers are initiated on its flank; while the other is the determinate type when the main axis terminates as a flower and growth continues sympodially. The Asteraceae plant family harbors a unique type of inflorescence, called the flower head. It combines up to hundreds of individual florets into a single structure, but its infloresecence meristem has a determinate fate and can only produce a certain number of florets before getting consumed. The genetic regulation of such determinacy has been recently brought into attention in Gerbera hybrida, a model system used for studying inflorescence and flower development in Asteraceae. So far, several genetic regulators have been identified regulating the determinacy of inflorescence meristem in Gerbera. This thesis aims to characterize new Gerbera transgenic lines to study the function of the Gerbera homolog of TERMINAL FLOWER 1 (GhTFL1). In Arabidopsis and other species, TFL1 is known to counteract with the flower meristem identity genes and maintain the indeterminacy of the inflorescence meristem. Previous results by overexpressing GhTFL1 under constitutive 35S promoter in Gerbera convert the determinate fate of inflorescence meristem into indeterminate. To better study the GhTFL1 function, an inducible overexpression system pOpON2-pOp6/LhGR-N was introduced, and the transgenic lines have been made for GhTFL1. In this this thesis, in total of 19 candidate lines were screened with GUS staining assay and RT-PCR. Two promising lines (TR4 and TR7) with strongest responses in GUS staining and high expression of the target gene were identified. Further treatment of dexamethasone was conducted in these two lines to the growing rosette; however, no clear phenotypes was observed in these lines. The treatment results suggested that further optimization should be made, in particular, the timing of treatment shall be the determining factor for a successful treatment.
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(2021)Semliki Forest virus (SFV) is an enveloped virus with positive-sense single-stranded RNA genome that encodes nine proteins, of which four non-structural proteins, nsP1-4, form the replication/transcription complex (RTC) along with several host proteins, which play an important role in the replication of the virus. To establish the interactome of SFV RTC, a promiscuous biotin ligase capable of biotinylating proximal endogenous proteins in the presence of exogenous biotin was genetically fused to nsP3. After establishing the stability, kinetics and functionality of this virus, BHK-21 cells were infected with this mutant SFV at multiplicity of infection of 50 plaque forming units per cell. At an early time point of 2.5 hours post infection, 50 μM biotin was added to medium for 15 minutes. Cells were lysed, and biotinylated proteins were enriched with streptavidin beads, and analyzed through tandem mass-spectrometry. We were able to identify several key host protein interactions, some of which were already established before, but also a several new ones. Many of the host proteins detected were involved with the formation of stress granules, including G3BP’s, or contained a SH3-binding domain (SRC homology 3) like CD2AP, SH3KBP1 and BIN1, and some of them also had RNA binding motifs. In future, we wish to study the role of these identified host proteins in the replication of SFV through gene silencing as well as their co-localization with the RTC and nsP3 with the help of Immunofluorescence.
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(2014)Forest biomass is considered as one of the most important alternative energy sources across the globe. Growing attention has been given to the studies concerning biomass and related bioenergy and biofuel, and their potential for future development. This study takes higher education as unique aspect, focusing on the awareness of Chinese university students of Forest Based Bioenergy (FBB) development and how education background / awareness may influence the FBB development in China. Since FBB is relatively a new concept in China, its development and further utilization are believed to largely relay on the matters of education, social trend and awareness. Students in higher education are considered as a special group: they may be educated related to FBB and will become the future consumers and even decision-makers. This make awareness, attitude and opinions about FBB from the students` point of view significant. A literature review was made for the background study and quantitative research, plus surveys and interviews were conducted as data collection methods. Objectives of the thesis are to study the awareness of and attitudes towards FBB among Chinese university students and if those opinions were influenced by their studies. Results indicate that education strongly affects students´ attitudes. FBB development is seen as a positive signal and students are likely to support FBB development. FBB is believed as a new trend of renewable energy development. However, FBB in China will not see a rapid booming in the near future and it has only limited impact towards the traditional fossil fuel domination, but due to its characteristics, governmental recognition and growing awareness, it certainly shall be seen as strong supporter of China´s sustainable development. It also has to be holistically utilized considering environmental, social and economic aspects, to reach its full potential and to support China´s target of sustainable energy development.
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(2018)The literature review dealt with the chemical and physical properties of chitin polymer and its degradation product glucosamine. Particular emphasis was given to studies on the analytical methods of determination of chitin from various products including gravimetrical, spectrophotometric and chromatographic methods. The aim of this study was to optimize an analytical method to determine insect chitin and to apply the method to quantify chitin in whole insects and their soluble protein fractions. Two species of insects were selected: mealworm larvae (Tenebrio molitor) and cricket (Acheta domesticus). Whole insects and their protein fractions were treated with alkaline to remove protein. Chitin in de-proteined insect material was then hydrolysed into glucosamine using 6M HCl. Glucosamine from the hydrolysate was determined using chromatographic and spectrophotometric methods, where chromatographic method was optimized and validated. UPLC-FLR method was specific for glucosamine and the UPLC system could separate two target peaks (glucosamine isomers: α and β). Glucosamine content was calculated using a calibration curve which showed excellent linearity in range 0.0033~24.0 ng/inj. with the determination coefficient more than 0.999 during the study period. The instrumental limit of detection and limit of quantification were 0.00095 ng/inj. (2 µL) and 0.0033 ng/inj. (7 µL), indicating a satisfied sensitivity. Recovery of glucosamine spiked to sample matrix (de-proteined cricket flour) following chitin hydrolysis was not satisfactory (~75%) using HPLC-FLR method, which indicated that spectrophotometric method gave chitin amount closer to the true value due to a higher recovery (>90%). Major findings on chitin amount was ~5% on dry matter basis in both mealworm and cricket. A small percentage of chitin was found in insect protein fractions extracted by 0.1M NaCl.
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(2010)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.
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(2024)As the impacts of climate change have become more tangible and severe, it has become clear that changing the approach to resource consumption is crucial. The housing and construction sector has a major role to play in the fight against climate change, as it accounts for around 50% of all material consumption, and the construction sector is also responsible for over 35% of the EU's waste generation. The circular economy (CE) is commonly viewed as a substitute for the traditional linear economic model and therefore it is widely seen as one of the possible solutions for decarbonizing the housing and construction sector and, at the same time, as one of the solutions for tackling climate change. This thesis focuses on exploring the sustainability and circular economy activities of stakeholders in the Finnish housing and construction sector. A particular focus is on the circular economy actions, which will be analyzed with the ReSOLVE framework. Also, the suitability of the framework for mapping circular economy actions in the housing and construction sector will be explored. The thesis thus aims to answer the following research questions: how sustainability and the circularity are visible in the activities of stakeholders in the Finnish housing and buildings sector, and how suitable the ReSOLVE framework is for assessing circularity in this sector. The qualitative research data consists of fourteen stakeholder interviews, which were also complemented by background documents provided by the stakeholders. By utilizing content analysis and the ReSOLVE framework, it was found that the self-designated sustainability activities of the stakeholders were mostly either knowledge generation, dissemination or influencing activities, but the ReSOLVE framework also revealed a wide range of circularity activities already present in the stakeholders’ actions. The framework also worked as a tool to bring forward different aspects of circularity that would not necessarily have been discovered with other more traditional CE frameworks. The qualitative results suggest that within the stakeholder group there are different ways of implementing the circular economy across the different CE dimensions, and that the circular economy is therefore not a one-dimensional activity that is either implemented by an actor or not. The results also support previous literature that a holistic and systemic approach is needed to achieve sustainability transition in the construction and housing sector.
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