Browsing by Subject "biotechnology"

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  • Kiviniemi, Eero A. (Helsingin yliopisto, 2018)
    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.
  • Peltoniemi, Olli; Björkman, Stefan; Oropeza-Moe, Marianne; Oliviero, Claudio (2019)
    This review aims to describe changes in production environment, management tools and technology to alleviate problems seen with the present hyperprolific sow model. Successful parturition in the pig includes the possibility to express adequate maternal behaviour, rapid expulsion of piglets, complete expulsion of placenta, elimination of uterine contamination and debris, neonatal activity and colostrum intake. We focus on management of large litters, including maternal behaviour, ease of parturition, colostrum production, piglet quality parameters and intermittent suckling. There are also some interesting developments in technology to assess colostrum and immune state of the piglet. These developments may be utilized to improve the success rate of reproductive management around farrowing, lactation and after weaning. We also discuss new insights in how to examine the health of the mammary gland, uterus and ovaries of hyperprolific sows. Finally, we assess the latest developments on breeding and technology of hyperprolific sows, including artificial insemination (AI), real-time ultrasound of the genital tract and embryo transfer (ET). We conclude that 1) for the sow to produce sufficient colostrum, both the behavioural and physiological needs of the sow need to be met before and after parturition. Furthermore, 2) new ultrasound and biopsy technology can be effectively applied for accurate diagnosis of inflammatory processes of the udder and uterus and timing of AI regarding ovulation to improve insemination efficiency. Finally, 3) developments in cryopreservation of germ cells and embryos appear promising but lack of valid oocyte collection techniques and nonsurgical ET techniques are a bottleneck to commercial ET. These latest developments in management of parturition and reproductive technology are necessary to cope with the increasing challenges associated with very large litter sizes.
  • Gabryelczyk, Bartosz (Helsingin yliopisto, 2015)
    The possibility of controlling interactions at interfaces and surfaces of solid materials is highly interesting for a wide range of materials-related nanotechnological applications, for example, colloidal systems, adhesives, biosensors, biomimetic composite and biomedical materials. In Nature, many proteins and peptides possess the ability to recognize, specifically bind, and modify the surfaces of solid materials through sophisticated mechanism of molecular recognition. These properties have been developed during evolution via successive cycles of random mutations and selection. The natural evolution processes can be mimicked in the laboratory scale with the use of a directed evolution approach, for instance, based on the selection of short material-specific peptides from the combinatorial libraries displayed on the surface of bacteriophages or bacterial cells. Selected from billions of different variants, material-specific peptides can be studied by experimental and computational methods to define their sequence, structure, and binding properties. Subsequently, they can be engineered in order to improve their binding affinity and tailor their function for practical applications. The studies presented in this thesis show how phage display was used to identify peptides binding to diamond-like carbon (DLC). DLC is an amorphous form of carbon, with chemical and physical properties resembling natural diamond. It is used as a coating material in many industrial and biomedical applications. Peptides binding to DLC were selected form a combinatorial phage display library. Their binding and molecular basis of the function were investigated in different molecular contexts (when displayed on the phage surface, forming fusion proteins, or present in free soluble form), using multiple independent methods. It was also demonstrated that the peptides can be used in nanotechnological applications, i.e., as a self-assembling coating on the DLC surface, and for controlling properties of a colloidal form of DLC. Besides finding and characterizing peptides binding to DLC, the thesis also highlights different challenges of the directed evolution techniques, for example, selection of target unrelated peptides during biopanning, and the necessity of multiple independent ways of analyzing the functionality of selected peptides.
  • El Sayed Bashandy, Hany (Helsingin yliopisto, 2016)
    The flavonoid biosynthetic pathway and flavonoid metabolites have been extensively studied because of their biological roles in plant and animal kingdoms. Flavonoid metabolites are involved in plant resistance, UV-protection, pollinator attraction and have antioxidant effects. Plant species synthesise a large number of secondary metabolites, including flavonoids, carotenoids and others. Flower colour is an important feature for marketing of the ornamental model plant gerbera (Gerbera hybrida). In gerbera, flavonoid metabolites accumulate in the adaxial epidermal layer of flower petals and lead to different flower colours and patterns. These metabolites consist of three subgroups, flavones, flavonols and anthocyanins in gerbera, which have also been used to characterize different cultivars. The cultivars show variation in flower colour according to their anthocyanin type. The acyanic gerbera cultivars have flavonoid profiles comparable to the cyanic cultivars, except for the synthesis and accumulation of specific anthocyanins. Metabolite profiles indicated that all analysed acyanic cultivars have a block in a late stage of the anthocyanin pathway. Ivory, a sport of the pelargonidin-cultivar Estelle, has white flowers. Ivory has flavones and flavonols, but no anthocyanin. Gene expression of all flavonoid pathway genes was similar in Estelle and Ivory. However, both cultivars have two different alleles encoding dihydroflavonol 4-reductase and in Ivory one of them (GDFR1-2) was found to have a point mutation resulting in inactivation of the encoded enzyme. Still, Ivory expresses the second allele (GDFR1-3) and accumulates active DFR enzyme. The cyanidin cultivar President expresses only the GDFR1-3 allele, but cannot synthesize pelargonidin. Therefore, GDFR1-2 contributes specifically to pelargonidin biosynthesis and GDFR1-3 to cyanidin biosynthesis. This could be explained by a coordinated biosynthesis of anthocyanins in multi-enzyme complexes, metabolons. Gerbera chalcone synthases (GCHSs) belong to the superfamily of Type III polyketide synthase enzymes. GCHS1, 3 and 4 have different contributions to the flavonoid pathway, according to the tissue specific and post-transcriptional regulation. RNA interference of CHS encoding genes was used in different gerbera cultivars to show that GCHS1 has the main contribution to anthocyanin accumulation in petal tissues. GCHS4 was strongly expressed in petals but did not lead to anthocyanin accumulation. Still, GCHS4 is expressed and encoded a functional enzyme in the vegetative tissues.
  • Kabiersch, Grit (Helsingin yliopisto, 2013)
    Endocrine disrupting compounds are synthetic or natural compounds that mimic the action of hormones and thus disrupt or alter functions of the endocrine system usually through direct interactions with nuclear receptors. The main objective of this work was to develop strategies of how to degrade endocrine disrupting compounds and how to monitor the removal of the endocrine disrupting effect with focus on the estrogenic compound bisphenol A and on the virilizing compound tributyltin. Bisphenol A is used as plasticizer of polycarbonate plastics, which are used for food packaging, coatings of metal cans and baby bottles. It can leach out from these materials during washing and sterilization processes or after landfilling. The endocrine disrupting effects of bisphenol A are exerted through binding and activating the estrogen receptor, a nuclear receptor. As a consequence, bisphenol A acts as a potential risk factor for the development of prostate and breast cancer and is suspected to reduce the number of sperm cells in men. Tributyltin has been widely used in antifouling paints and is found in high concentrations in the vicinity of shipping routes and in harbor sediments. Through binding and activation of the retinoic X receptor, also a nuclear receptor, tributyltin induces a phenomenon called imposex in female gastropods, which means that in addition to female sex organs a penis and vas deferens are formed. The occurrence of both compounds, bisphenol A and tributyltin, can be monitored and quantified by the use of analytical methods such as HPLC, LC-MS and GC-MS. However, they are time-consuming, require high amounts of environmentally hazardous solvents and eluents as well as specific technical equipment and competence. In contrast, microbial bioassays offer inexpensive, easy-to-use and small-scale measurements. Furthermore, they can be employed to assess bioavailability. In this work, a receptor-based bioluminescent yeast assay was developed that has the advantage of qualitatively monitoring the endocrine disrupting effect of the compound as well as its metabolites. Furthermore, the lignin-decomposing abilities of litter-degrading fungi were exploited. These fungi produce highly active extracellular oxidative enzymes such as laccase and manganese peroxidase. These enzymes degrade recalcitrant substances such as plant lignin but also persistent environmental chemicals. A novel bioluminescent yeast assay was constructed and characterized to detect organotin compounds such as tributyltin, triphenyltin and their metabolites. A chimeric human retinoic X receptor alpha is expressed from a plasmid. Upon binding of tributyltin or another ligand, expression of a luciferase reporter gene is triggered. After addition of D-luciferin, light is emitted and detected luminometrically. The assay has proven to be highly specific towards organotin compounds and natural ligands of the retinoic X receptor. Tributyltin was detected in nanomolar concentrations. At these low concentrations, tributyltin already exerts an endocrine disrupting effect in nature. Experiments were performed in small-scale and high-throughput manner and results of one analysis were obtained within one working day. Next, a novel sampling method to determine extracellular fungal enzymes in agar was developed. Small pieces of growing solid-state fungal cultures were placed in a centrifugation tube filter, containing a cellulose acetate membrane. Centrifugation recovered water-soluble material including many enzymes. The recovery of two added model enzymes, laccase and manganese peroxidase, was in the range of 50 to 75 %. This method allowed the collection of spatial data from very small and defined areas of solid fungal cultures. It is also very well suitable for screening approaches, i.e. it can combine toxicity tests and the investigation of the influence of toxic/endocrine disrupting compounds on enzyme production of the fungus under study. Finally, the degradation of the endocrine disrupting compound bisphenol A was studied in detail. Cultures of the litter-degrading fungi Stropharia rugosoannulata and Stropharia coronilla as well as a partially purified neutral manganese peroxidase from the latter were used to successfully degrade the estrogenic compound bisphenol A in culture medium or cell-free reaction solution, respectively. A bioluminescent yeast assay, expressing the estrogen receptor alpha, was employed to follow the removal of estrogenic activity. S. coronilla was shown to be the most efficient fungus; the estrogenic activity was reduced by 100 % in liquid cultures as well as during treatment with manganese peroxidase. In cultures of S. rugosoannulata, the estrogenic activity declined as well but temporarily re-appeared. Selected samples from this cultivation were additionally investigated for potential metabolites using LC-MS analysis and a pathway of bisphenol A conversion was hypothesized.
  • Ramakrishnan, Muthusamy; Yrjälä, Kim; Vinod, Kunnummal Kurungara; Sharma, Anket; Cho, Jungnam; Satheesh, Viswanathan; Zhou, Mingbing (2020)
    Sustainable goals for contemporary world seek viable solutions for interconnected challenges, particularly in the fields of food and energy security and climate change. We present bamboo, one of the versatile plant species on earth, as an ideal candidate for bioeconomy for meeting some of these challenges. With its potential realized, particularly in the industrial sector, countries such as China are going extensive with bamboo development and cultivation to support a myriad of industrial uses. These include timber, fiber, biofuel, paper, food, and medicinal industries. Bamboo is an ecologically viable choice, having better adaptation to wider environments than do other grasses, and can help to restore degraded lands and mitigate climate change. Bamboo, as a crop species, has not become amenable to genetic improvement, due to its long breeding cycle, perennial nature, and monocarpic behavior. One of the commonly used species, moso bamboo (Phyllostachys edulis) is a potential candidate that qualifies as industrial bamboo. With its whole-genome information released, genetic manipulations of moso bamboo offer tremendous potential to meet the industrial expectations either in quality or in quantity. Further, bamboo cultivation can expect several natural hindrances through biotic and abiotic stresses, which needs viable solutions such as genetic resistance. Taking a pragmatic view of these future requirements, we have compiled the present status of bamboo physiology, genetics, genomics, and biotechnology, particularly of moso bamboo, to drive various implications in meeting industrial and cultivation requirements. We also discuss challenges underway, caveats, and contextual opportunities concerning sustainable development.
  • Elbadri, Khalil (Helsingin yliopisto, 2020)
    An increased attention has been drawn towards porous silicon (PSi) based materials for biomedical applications, due to their promising features demonstrated through several scientific studies. Here, we further investigated the biological responses of PSi nanoparticles (NPs) with different surface chemistries, including immunomodulatory effects, inflammation mitigation and biocompatibility. In this collaborative study, the PSi NPs were investigated both in vitro and in vivo, using different molecular biology and biochemistry techniques, e.g., qPCR, ELISA, cell sorting and cell viability assays. Our results showed the capabilities of these PSi NPs to relieve the inflammatory conditions, whereas significant decrease was recorded of pro-inflammatory cytokines: TNF-α, IL-1β and IL-6. Likewise, these PSi NPs revealed a considerable consumption aptitude of pro-inflammatory reactive oxygen species molecules. Administrating PSi NPs in an acute liver inflammation (ALI) model, showed no conspicuous influence on cellular viability. Thus, the outcome of this study demonstrates the potential biocompatibility of PSi nanomaterials, in addition to their outstanding features as potential candidates for further incorporating in ALI applications.
  • Blick, Amanda (Helsingin yliopisto, 2020)
    Patents may be refused in Europe on the grounds that the commercial exploitation of otherwise patentable inventions would be considered contrary to ordre public and morality. Whereas patent offices have applied these exclusions only infrequently in the past, recent developments in the research of arising gene editing technologies such as CRISPR/Cas9 and prime editing have given rise to varying opinions as to the approach that should be adopted in relation to patents. While genetics have been described as promising to offer an unprecedented contribution to improve health care, from the perspective of social sustainability, these advancements should be shared with society as a whole and with the international community. Despite the visions of a promising future, not much progress has been shown in advancing these goals – instead, considerable resources have been allocated for managing the perceived risks of gene editing rather than providing incentives to research new opportunities for its use. This is embodied in the restrictive policy that the European Union has adopted towards inventions involving germline gene therapy with the Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions which unambiguously prevents the patentability of processes for modifying the germline genetic identity of human beings. Patents act as a negative force that simply allow the patent holder to prohibit others from commercially exploiting the invention conferred by the patent; they do not, however, form any absolute positive right to use an invention. A strong patent position is nevertheless understood to be a prerequisite for investment, and although patent law is not considered the primary control mechanism for regulating innovation, exclusions on patenting de facto indirectly regulate the types of therapies for which capital may be attracted in order to fund the significant costs associated with bringing new therapies or medicinal products to the market. Given the justification of the patent system providing economic incentives for the technologies the development of which we want to promote in our society, as European Union Member States have played an important role in pioneering new technologies which have later transferred to developing countries, this approach may have effects on a global scale, preventing individuals suffering from severe hereditary illnesses from achieving the right to the highest attainable standard of health which along with the strengthened view on human rights is understood to extend to preventive medicine. Reports from more recent sources such as the Nuffield Council on Bioethics have opened the discussion on how the concept of human dignity is not helpful in the context of germline gene therapy, proposing alternative principles such as the welfare of the future person as well as social justice and solidarity to provide help with the assessment on the acceptability of human applications. While the preamble in the Directive 98/44 declares there to be a “consensus within the Community” that interventions in the human germline offend against ordre public and morality, through balancing these divergent legal-ethical values, this thesis argues how these unequivocal prohibitions to patenting in fact fail to respect the social and cultural context of each Member State. Influenced through time and historical events, the basic European bioethical principles are differently reflected in the legal culture of each Member State – and as ordre public and morality should correspond in particular to ethical or moral principles recognized in each Member State, considering all human applications of these technologies unequivocally contrary to ordre public and morality does not adequately reflect the values and ethical positions of all Member States. From the perspective of social sustainability, as ordre public and morality patenting exclusions discourage investing in the research and development of these technologies, they also work as to hinder the right to benefit from scientific and technological progress. The policy decisions that the European Union adopts around the economic incentives on investing in further research has consequences on the global level, as these technologies would eventually spread to developing nations that are disadvantaged under current conditions of scientific research and innovation. In terms of benefit sharing and solidarity, it is just as important to ensure that effective policies exist to secure that patents do not end up acting as limiters of welfare and that the growing developments in knowledge and technology will not widen the existing social inequities but rather act as to reduce them.
  • Häyry, Matti (Yliopistopaino, 2001)
    Doctors, nurses and scientists have often been accused of ‘playing God’ in life-and-death decisions, reproductive medicine and genetics. The tenor of this book is that the same accusation can be extended to ethicists and public decision-makers who refuse to examine the is­ sues analytically before they pass their judgements. In many cases, they are the jealous gods who obstruct the voyage of honest philoso­phers and bioethicists who desperately try to find their home island of intellectual zeal and emotional security in the stormy sea of high feeling and firmly held prejudice.
  • Zhu, Lingping (Helsingin yliopisto, 2022)
    The vast ranges of chemically diverse plant polyketides are well-known for their important functions in plants and to human health. Plant polyketides consist of the most widely distributed pollen wall structural element sporopollenin, the ubiquitous and chemically diverse flavonoids, and various taxa-specific natural products. Despite the vast diversity, plant polyketides are synthesized in a common logic. Type III polyketide synthases (PKSs) synthesize the backbones and post-PKS tailoring enzymes provide accurate modifications to determine the final chemical diversity. Compared with plant PKSs that have been intensively investigated, the important tailoring enzymes are largely unknown. The ornamental plant Gerbera hybrida is rich in two defense-related polyketides gerberin and parasorboside, in addition to sporopollenin and flavonoids. Gerberin/parasorboside biosynthesis has been shown to be initiated by gerbera 2-pyrone synthase 1 (G2PS1), while the accessory polyketide reductases (PKRs) have been missing. In this thesis, by coexpression analysis, we identified two PKR candidates in this pathway, gerbera reductase1 (GRED1) and GRED2. They were shown to be the second PKR required for parasorboside biosynthesis by expression and metabolite analysis of different gerbera tissues, cultivars, and transgenic gerbera plants and in vitro enzyme assays. We also showed that PKRs act on the linear triketide intermediate prior to its lactonization in gerberin/parasorboside biosynthesis. This updated pathway indicates that plant polyketide biosynthesis shares tailoring strategies with fungi and bacteria. Sporopollenin has been uncovered to be synthesized through a conserved anther-specific PKS pathway. We showed that GRED1 and GRED2 are orthologues of Arabidopsis tetraketide α-reductase 2 (AtTKPR2), a minor PKR involved in sporopollenin biosynthesis. Their important role in parasorboside biosynthesis indicates that the duplicate gerbera TKPR2s have been recruited from the ancient sporopollenin biosynthesis to a defense-related PKS pathway. The functional diversification of gerbera TKPR2s is an example of how plants get metabolic innovation for adaption during evolution. We showed that GRED1 and GRED2 still sustained the minor role in pollen wall formation. Moreover, we identified and characterized GTKPR1, the gerbera orthologue of AtTKPR1, the predominant PKR in sporopollenin biosynthesis. GTKPR1 was shown to play a conserved predominant role in pollen wall formation. Cultivar-specific pigmentation of gerbera has been shown to be a result of substrate preferences of dihydroflavonol 4-reductases (DFRs). Substrate specificity of DFRs from different species have been investigated for decades. However, which amino acids are responsible for it remains unknown. Two major technical challenges hindering the investigation are: difficult to make heterologous DFRs and lack of a reliable assay for the determination of substrate preferences. In this thesis, we established a highly efficient DFR expression system in tobacco and refined the traditional BuOH-HCl assay into a reliable and robust assay for the determination of DFR substrate preferences.
  • Chong, Sun-Li (Helsingin yliopisto, 2014)
    O-acetylglucuronoxylans (AcGX) are the major hemicelluloses found in the secondary cell wall of dicotyledon species. The backbone is formed by β(1→4)-linked xylopyranosyl (Xylp) residues, which are substituted by α(1→2)-linked (4-O-methyl)glucopyranosyluronic acid ((Me)GlcpA). The AcGX are also highly acetylated on the 2-O or 3-O; or both positions of Xylp units. Notably, acetylation patterns in AcGX are not well understood since they are typically destroyed during the alkaline isolation. Accurate quantitation of MeGlcpA is also challenged by the lack of commercially available MeGlcpA sources, thus the accuracy of MeGlcpA content determined with the GlcpA standard is unknown. The current thesis established new procedures of detailed characterization of AcGX. The xylan OLIgosaccharide Mass Profiling (OLIMP) method encompassed endoxylanase hydrolysis and mass spectrometry detection. The endoxylanase cleaves the xylan backbone into acetylated xylooligosaccharides (AcXOS). As the action is hindered by the side groups, endoxylanase can be a selective tool to liberate AcXOS from plant tissues for fingerprinting the acetylation pattern in AcGX. Additionally, mass fragmentation analyses were performed to elucidate the spatial distribution of acetyl residues. The accuracy of MeGlcpA quantitation using the GlcpA standard was examined by comparing it to the in-house purified MeGlcpA in acid methanolysis and gas chromatography (GC) analysis. Several of the genes responsible for the biosynthesis of AcGX were previously identified using Arabidopsis thaliana as the model plant. Herein, the structures of AcGX in Arabidopsis wild-type and biosynthetic mutant plants that are defective in reducing end tetrasaccharide sequence or backbone synthesis, irx7, irx9, irx10 and irx14; and (Me)GlcpA addition, gux1gux2 were analyzed using methods established for structural characterization of AcGX. Mono-acetylations (2-O or 3-O position) were reduced in irx7, irx9 and irx14, whereas 2-O acetylation was elevated in the (Me)GlcpA deficient mutant, gux1gux2, indicating that the addition of (Me)GlcpA residues is taking place before acetylation of xylans. Structural elucidation on the major AcXOS liberated from wild-type plant suggests that the acetyl residues are added in every other Xylp residue in AcGX. Interestingly, a novel pentose substitution on the GlcpA side group in AcGX was identified. In acid methanolysis, the GlcpA standard was partially lactonized, thus yielding six derivatives in GC chromatogram. When all six GlcpA derivatives were used in the calibration curve, the MeGlcpA content was underestimated by nearly 30%. The MeGlcpA content can be closely estimated by choosing the appropriate GlcpA derivatives in the calibration curve. The method was used to investigate the impact of Schizophyllum commune glycoside hydrolase family 115 α-glucuronidase (AGU) transgene expressed in Arabidopsis. The (Me)GlcpA content in the ScAGU115 expressing plants was surprisingly unchanged despite the active recombinant enzyme present within the cell walls. In this work, the acetylation pattern in the AcGX of Arabidopsis wild-type and mutant plants was studied in detail. The methods developed herein revealed that the acetylation of AcGX was reduced in Arabidopsis lines that encode a defective biosynthetic gene related to backbone or reducing end sequence synthesis, suggesting pleiotropic effect of a single gene mutation in xylan biosynthesis. The MeGlcA substituents in AcGX can be effectively reduced by disruption of the glucoronyltrasnferases; however, the substitution in the AcGX of gux1gux2 was compensated by acetylation. On the other hand, constitutive transgenic expression of ScAGU115 α-glucuronidase did not remove the (Me)GlcpA substituents in planta. The reason may be ascribed to the shielding by neighbouring acetyl substituents, or limited accessibility of the recombinant enzyme to cell wall substrates. Therefore, a viable approach for the effective tailoring of AcGX substituents in planta could be co-expression with an acetyl xylan esterase to obtain synergism between these side-group removing enzymes.
  • Tupasela, Aaro (2000)
    This paper proposes to discuss the shortcomings of three popular science and technology study models - the Triple Helix, Mode 2 and Entrepreneurial Science. It also focuses on the development of supra-national and national science and technology policies and the role they have played in the commercialization of university research. The three models and innovation policies are analyzed through a case study of Helsinki University Licensing Ltd. (HUL), a technology transfer company founded in 1992 by the University of Helsinki and the Finnish National Fund for Research and Development (Sitra). The purpose of the company is to patent and license innovations developed at the University of Helsinki. The paper identifies contradictions that emerge in the patenting of research developed at the University of Helsinki, Finland's largest public research university. It deploys the concepts of technological systems, diffusion of innovations and national ideologies to problematize the ubiquity of current models and policies. The study draws on interview material, policy documents, HUL documents, patenting statistics and newspaper articles. These sources are used to argue that the three models and current innovation policy lack a critical approach in looking at the technological development of the biosciences in Finland. Innovation policies are analyzed on the supra-national and national levels and compared to the strategies that have been adopted at the University of Helsinki during the 1990s. The assumptions set forth by the models are critiqued in light of obstacles HUL has encountered with patenting and licensing. The study concludes that current innovation policies are contradictory because they encourage the broad diffusion of university research and its patenting at the same time. The patenting of research confers monopoly rights over innovations and is an effective tool for the private industry to minimize risk. At the same time, however, competition and the dissemination of information are compromised. The role of individual actors is identified as an important component of the innovation process in Finland and the success of HUL. Professional know-how is a key factor in attracting business from researchers. Finally, the models are too descriptive and neglect to account for variation among different technological systems.
  • Nagy, Kinga K.; Skurnik, Mikael; Vertessy, Beata G. (2021)
    Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA-protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes.