Towards the characterization of a novel thermohalophilic antioxidant Thioredoxin from the metagenome of the Red Sea; LCL of Atlantis II Brine pool

Due to the crucial antioxidant role of Thioredoxin (Trx) system in various vital cellular processes; DNA synthesis, oxidative stress defense, protein folding, apoptosis and cell growth, this fundamental system is widely expressed in mostly all life\u27s kingdoms. Thus using metagenomic approaches to characterize novel Trx system in unexplored harsh environment will open the window for understanding the evolution of Trx system and its unique adaptation in extreme habitations. One of the unexplored unique ecosystems is the Red Sea\u27s Atalntis II brine pool, specifically, the lower convective layer (LCL). The exceptional harsh conditions of the LCL; anoxic condition, high temperature around 70°C, high salinity (26%), and high metal content, have a significant contribution for being a unique infrequent ecosystem. The objective of this study is to characterize a novel Trx isolated from the LCL. Successfully, in the experimental part, Trx was expressed in E. coli and purified, where the purified Trx has shown a clear antioxidant activity with a unique thermohalophilicity. In conclusion, we have characterized a unique antioxidant Trx protein

Mapping, isolation and characterization of genes responsible for late blight resistance in potato

Late blight (LB), caused by the oomycete Phytophthora infestans, is one of the most devastating diseases on potato. Resistance (R) genes from the wild species Solanum demissum have been used by breeders to generate late blight resistant cultivars, but resistance was soon overcome by the pathogen. A more recent screening of a large number of wild species has led to the identification of novel sources of resistance, many of which are currently being characterized. R-gene based resistance to any plant pathogen has been conceptualized according to a model known as gene-for-gene interaction. When matching avirulence (Avr) and resistance (R) proteins are produced by the pathogen and the plant respectively, a resistance response is triggered resulting in a hyper-sensitive response (HR) causing necrosis and cell death at the infection site. If one of these components is missing the plant-pathogen interaction is compatible and will result in completion of the life cycle of the pathogen. This thesis describes the cloning and the characterization of the resistant alleles Rpi-vnt1.1, Rpivnt1.2 and Rpi-vnt1.3 from Solanum venturii and their counterpart Avr-vnt1 from Phytophthora infestans. The Rpi-/Avr- genes pair Rpi-vnt1/Avr-vnt1 along with R3a/Avr3a and Rpi-blb3/Avr2 have been used to study the genetic and molecular mechanisms behind tuber blight resistance. The cloning of Rpi-vnt1 alleles (Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3) was achieved by the combination of long range PCR (Chapter 2) and a classical map based cloning strategy (Chapter 3). The long range PCR made use of Tm2 homologous PCR primers, upon identification of Tm2 sequence homology in associated markers generated with an NBStargeted fingerprinting technique. Rpi-vnt1 alleles belong to the CC-NBS-LRR class of plant R genes and encode predicted peptides of 891 and 905 amino acids, respectively, which share 75% amino acid (a.a.) identity with the ToMV resistance protein Tm-22 from tomato. Compared to Rpi-vnt1.1, the allele Rpi-vnt1.3 harbors a 14 amino acid insertion in the Nterminal region of the protein and two different amino acids in the LRR domain. Despite these differences, Rpi-vnt1.1 and Rpi-vnt1.3 genes have the same resistance spectrum. An allele mining study of Rpi-vnt1 alleles across Solanum section Petota showed that the three functional alleles were confined within S. venturii as only two accessions from the closely related species S. weberbaueri and S. mochiquense carried Rpi-vnt1.1 (Chapter 4). Subsequent alignment of Rpi-vnt1-like homologs with Rpi-vnt1 alleles revealed the presence of illegitimate recombination (IR) signatures suggesting that two successive deletion events might have occurred in the CC domain. Meanwhile, the construction of a Neighbor Joining tree, based on AFLP data from all the accessions carrying Rpi-vnt1 alleles or Rpi-vnt1-like homologs showed that Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 alleles belong to a monophyletic clade. Signatures of illegitimate recombination and the monophyletic grouping of Rpi-vnt1 alleles suggested how Rpi-vnt1.1, Rpi-vnt1.2 and Rpi-vnt1.3 could have evolved. Extensive phenotyping with various Phytophthora isolates identified another Rpi gene in S. venturii named Rpi-vnt2, complementing the Rpi-vnt1 allelic resistance spectrum. The genetic position of this second independent locus is not yet identified. The identification of the matching avirulence factor from the pathogen, Avr-vnt1, was achieved by using an efficient and high throughput effector screen of resistant wild potato species (Chapter 5). Avr-vnt1 encodes a typical RXLR-EER effector which expression is induced 2 days post inoculation. Avr-vnt1 is located on a single locus in the reference strain T30-4. Among nine isolates, four alleles were identified. The virulent strain EC1 carries a functional coding sequence of Avr-vnt1 but fails to express the gene. In Chapter 6, the genetic and molecular mechanisms of tuber late blight have been investigated. Using transgenic cv. Desiree plants transformed with Rpi-vnt1.1, R3a or Rpiblb3 tuber blight resistance could be studied in an identical genetic background. First, we demonstrated that transient co-expression of the matching Avr- genes in these transgenic tuber slices trigger a hypersensitive responses (HR), showing that the presence and interaction of both proteins is sufficient to establish tuber blight resistance. Second, phenotypic and molecular analysis of a panel of transformants for Rpi-vnt1.1, R3a and Rpi-blb3, and transcriptional analysis of the corresponding effectors (Avr-vnt1, Avr3a and Avr2 respectively) during leaf and tuber infection showed that the expression level of a given Rgene should equal or exceed the expression level of the matching effector in order to trigger an efficient resistance response in the tuber. Therefore, foliar and tuber late blight resistance are controlled by similar genetic mechanisms. The perceived lack of phenotypic correlation between foliage and tuber blight resistance is thus solely due to the tissue specific expression level of the Rpi-gene. In the general discussion (Chapter 7), results from the experimental chapters are discussed in a broader perspective. <br/

Engineering nonphosphorylative metabolism for the biosynthesis of sustainable chemicals

University of Minnesota Ph.D. dissertation. December 2016. Major: Chemical Engineering. Advisor: Kechun Zhang. 1 computer file (PDF); xii, 211 pages.Lignocellulosic biomass is one of the largest sources of organic carbon on Earth with the potential to replace fossil fuels for the production of transportation fuels and chemicals. The two biggest challenges facing biosynthesis is the limited natural metabolic capacity of microorganisms and the effective utilization of lignocellulosic biomass. To overcome the first obstacle, over the past several decades researchers have successfully expanded the natural metabolic pathways of microorganisms to allow biosynthesis of a wide array of compounds with applications as advanced biofuels, industrial chemicals, and pharmaceuticals. Most industrial fermentations convert glucose, the major sugar present in biomass, into a value added chemical but are unable to utilize pentose sugars which make up ~30% of a typical biomass feedstock. To improve the overall economics of fermentation process, it is important to ensure that all major sugars present in the feedstock are efficiently converted to target chemicals. This work addresses both these challenges by establishing a novel alterative pathway called nonphosphorylative pathway in Escherichia coli which enables the utilization of underutilized pentose sugars such as D-xylose and L-arabinose using fewer steps and with higher theoretical yields than conventional glycolysis and pentose phosphate pathways (PPP). This nonphosphorylative pathway can convert D-xylose and L-arabinose to 2-ketoglutarate (2-KG), an important TCA cycle intermediate, using less than 6 steps. A growth selection platform based on 2-ketoglutarate (2-KG) auxotrophy was designed in E. coli to confirm the functionality of nonphosphorylative metabolism in host organism. The growth selection platform was also used to mine nonphosphorylative gene clusters from other organisms with improved activity. The pathway was then expanded to allow biosynthesis of two commercially important chemicals, 1,4-butanediol (BDO) and γ-aminobutyric acid (GABA). To improve production titers and yields of the process, protein engineering was used to reduce by-product formation and metabolic engineering was used to eliminate competing pathways and increase carbon flux towards the target compound. Furthermore, to improve uptake of pentoses by E. coli, pentose transporter was overexpressed to allow better carbon utilization. This nonphosphorylative metabolism serves as an efficient platform for biosynthesis and can be extended to produce a variety of compounds derived from TCA cycle including, but not limited to, L-glutamate, mesaconate, 5-aminolevulinic acid, and glutaconate. While the nonphosphorylative pathway has been successfully used for conversion of simple pentose sugars into important chemicals like BDO and GABA, the breakdown of biomass into these pentoses is the bigger challenge. This work also briefly addresses this challenge by comparing different acid hydrolysis treatment conditions to breakdown arabinoxylans in wheat bran into sugars - glucose, D-xylose, and L-arabinose - which can then be used in fermentation via nonphosphorylative metabolism

Environmental Technologies

This book on Environmental Technology takes a look at issues such as air, soil and noise pollution problems, environmental quality assessment, monitoring, modelling and risk as- sessment, environmental health impact assessment, environmental management and envi- ronmental technology development. It represents institutional arrangements, financial mechanisms and some sustainable technologies. The user can always count on finding both introductory material and more specific material based on national interests and problems. The user will also find ample references at the end of each chapter, if additional information is required. For additional questions or comments the user is encouraged to contact the author

Environmental Effects of Stratospheric Ozone Depletion, UV Radiation, and interactions with Climate Change: 2022 Assessment Report

The Montreal Protocol on Substances that Deplete the Ozone Layer was established 35 years ago following the 1985 Vienna Convention for protection of the environment and human health against excessive amounts of harmful ultraviolet-B (UV-B, 280-315 nm) radiation reaching the Earth’s surface due to a reduced UV-B-absorbing ozone layer. The Montreal Protocol, ratified globally by all 198 Parties (countries), controls ca 100 ozone-depleting substances (ODS). These substances have been used in many applications, such as in refrigerants, air conditioners, aerosol propellants, fumigants against pests, fire extinguishers, and foam materials. The Montreal Protocol has phased out nearly 99% of ODS, including ODS with high global warming potentials such as chlorofluorocarbons (CFC), thus serving a dual purpose. However, some of the replacements for ODS also have high global warming potentials, for example, the hydrofluorocarbons (HFCs). Several of these replacements have been added to the substances controlled by the Montreal Protocol. The HFCs are now being phased down under the Kigali Amendment. As of December 2022, 145 countries have signed the Kigali Amendment, exemplifying key additional outcomes of the Montreal Protocol, namely, that of also curbing climate warming and stimulating innovations to increase energy efficiency of cooling equipment used industrially as well as domestically. As the concentrations of ODS decline in the upper atmosphere, the stratospheric ozone layer is projected to recover to pre-1980 levels by the middle of the 21st century, assuming full compliance with the control measures of the Montreal Protocol. However, in the coming decades, the ozone layer will be increasingly influenced by emissions of greenhouse gases and ensuing global warming. These trends are highly likely to modify the amount of UV radiation reaching the Earth\u27s surface with implications for the effects on ecosystems and human health. Against this background, four Panels of experts were established in 1988 to support and advise the Parties to the Montreal Protocol with up-to-date information to facilitate decisions for protecting the stratospheric ozone layer. In 1990 the four Panels were consolidated into three, the Scientific Assessment Panel, the Environmental Effects Assessment Panel, and the Technology and Economic Assessment Panel. Every four years, each of the Panels provides their Quadrennial Assessments as well as a Synthesis Report that summarises the key findings of all the Panels. In the in-between years leading up to the quadrennial, the Panels continue to inform the Parties to the Montreal Protocol of new scientific information

New Trends in Environmental Engineering, Agriculture, Food Production, and Analysis

This Special Issue presents the latest advances in agriculture, aquaculture, food technology and environmental protection and engineering, discussing, among others, the following issues: new technologies in water, stormwater and wastewater treatment; water saving, lake restoration; new sludge and waste management systems; biodiesel production from animal fat waste; the microbiological quality of compound fish feeds for aquaculture; the role of technological processes to improve food quality and safety; new trends in the analysis of food and food components including in vitro, in vivo, and in silico analyses; and functional and structural aspects of bioactivities of food molecules

Mikrobioomi väärtus terviseuuringutes

Väitekirja elektrooniline versioon ei sisalda publikatsiooneTehnoloogia areng on andnud inimesele võimaluse uurida ümbritsevat maailma nurkade alt, mille jaoks veel mõned kümnendid tagasi võimalused puudusid. Üks selliseid teadusvaldkondi on inimese mikrobioomi ehk meie kehal ja kehas elavate mikroorganismide nagu näiteks bakterite ja viiruste uurimine. On teada, et mikrobioomil on oluline funktsioon inimese tervisele ning mikrobioomi kooslust omakorda mõjutavad suurel määral meie elustiil, toitumisharjumused, ümbritsev keskkond ning tervislik seisund. Just seosed haigustega on tekitanud huvi mikrobioomi kasutamiseks meditsiinilistes rakendustes. Doktoritöö eesmärk oli uurida, millised faktorid lisaks teadaolevatele on seotud meie soolestiku mikrobioomi kooslusega ning kuidas on mikrobioomi andmeid võimalik kasutada haiguste diagnoosimiseks ning haigusriskide hindamiseks. Esiteks uurisime teist tüüpi diabeeti ning näitasime, et mikrobioom aitab senisest täpsemalt ennustada muutusi veresuhkru regulatsiooni kirjeldavates parameetrites, milleks olid eelkõige insuliini eritamisega seotud näitajaid. Järgmiseks eesmärgiks oli kirjeldada Eesti populatsiooni soolestiku mikrorbioomi profiiili ning tuvasatada mikrobioomi kooslust mõjutavad faktorid. Eesti Geenivaramu terviseandmestikku kasutades tuvastasime, et antibiootikumide pikaajalisel korduval kasutamisel on akkumuleeruv mõju mikrobioomi kooslusele olenemata sellest, kas antibiootikume on kasutatud viimase kuue kuu jooksul. Analüüsides pikaajalise antibiootikumide mõju arvesse võtmine võimaldas omakorda täpsustada haigusspetsiifilisi muutusi mikrobioomis. Lisaks uurisime, kas soolestiku mikrobioomi abil inimeste grupeerimine võimaldaks ka kasutust kliinilistes rakendustes. Selgus, et selliselt mikrobioomi kooslust lihtsustades on võimalik küll anda hinnang inimese üldisele elustiilile, kuid tõendid haiguste diagnoosimisel või haiguste riski hindamiseks pole piisavalt tugevad. Kokkuvõttes on mikrobioomi uurimisel meditsiinis suur potentsiaal, mis võimaldab täiendada olemasolevaid võimalusi haiguste diagnoosimiseks ning riskide hindamiseks, kuid see eeldab täiendavaid teadmisi ja uuringuid.The technological revolution allows us to study the world beyond the limits that were holding us back only a couple of decades ago. One of such fields is the study of the human microbiome. Tiny microorganisms making up the microbiome such as bacteria and viruses have been known to intervene with our health for centuries, but the whole microbial ecosystem has turned out to be more complex than previously thought. The extent of the role of the microbiome to our own functioning and well-being is just starting to unravel. Nevertheless, microbiome has been associated with a large variety of intrinsic and extrinsic factors, including various complex diseases. This evidence is leading a slow but steady progress towards clinical applications such as using microbiome for improving disease diagnostics or estimating the risk of developing a condition. This thesis aimed to expand the understanding of the factors influencing our gut microbiome composition and assess the possibility and challenges in using the microbiome composition for the clinical applications. Firstly, we identified novel microbial biomarkers for identifying the progression of type 2 diabetes (T2D), which can be used to improve the current risk estimation. Secondly, using the comprehensive health data available in the Estonian Biobank, we characterized the profile of the gut microbiome in the Estonian population and identified various factors affecting the microbiome. Our study indicated that the long-term antibiotics usage has an accumulative effect on the gut microbiome composition independent of recent usage. The novelty of this result has a significant impact on the microbiome field and the future analysis need to account for such drug effects. Lastly, we considered dividing the subjects into a few distinct clusters based on their microbiome composition and evaluated the clinical applicability of such representation. We showed that although this approach is desirable in its simplicity, it is not sufficient for clinical applications. In conclusion, the microbiome science is heading towards clinical applications, but exploratory analysis is still needed. Nevertheless, the challenges ahead do not overshadow the enthusiasm.https://www.ester.ee/record=b551831

Rakku sisenevate peptiidi/nukleiinhappe komplekside kirjeldamine ja nende rakku sisenemise mehhanismid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Kõrge spetsiiflisuse ja madala kõrvalmõjude tekkeriski tõttu peetakse nukleiinhappeid väga suure ravipotentsiaaliga molekulideks. Bioloogilise aktiivsuse saavutamiseks on vajalik nukleiinhapete sisenemine rakkudesse ning jõudmine sihtkohta kas rakutuumas või tsütoplasmas. Suure molekulmassi ja negatiivse laengu tõttu pole aga nukleiinhapped võimelised ise rakke ümbritsevat plasmamembraani läbima. Seetõttu on arendatud mitmesuguseid meetodeid, mille abil nukleiinhappeid efektiivsemalt rakkudesse suunata, ning üheks neist on rakku sisenevate peptiidide (RSP; i.k. cell-penetrating peptide, CPP) kasutamine. RSP-d on lühikesed, enamasti katioonsed ja/või amfipaatsed aminohappelised järjestused, mis on võimelised läbima plasmamembraani ning transportima rakkudesse erinevaid bioaktiivseid molekule. Selleks, et RSPd saaksid nukleiinhappeid rakkudesse transportida, on vajalik peptiidse vektori sidumine lastmolekuliga. Lihtsaim võimalus on RSP siduda lastmolekuliga kompleksi moodustamise teel, millega saadakse RSP/nukleiinhappe nanopartiklid, mida hoiavad koos laengulised ja hüdrofoobsed interaktsioonid. RSP/nukleiinhappe nanokompleksid sisenevad rakkudesse efektiivselt ning tagavad kõrge lastmolekuli bioaktiivsuse, samas ei ole antud süsteem veel jõudnud kliiniliste katsete või kasutuseni. Üheks põhjuseks siinjuures on tekkivate nanokomplekside omaduste (nt suurus, kuju, laeng) komplitseeritud kirjeldamine. Teine probleem, mis takistab mittekovalentse strateegia rakendamist, on vähene arusaam nanokomplekside rakku sisenemise mehhanismidest ja rakusisesest suunamisest. Nii RSP/nukleiinhappe komplekside omaduste selgitamine kui nende rakku sisenemise mehhanismide ja rakusisese suunamise mõistmine on aga äärmiselt vajalikud, et parendada transportpeptiidide omadusi ja tagada lastmolekulide võimalikult kõrge aktiivsus nende kasutuselevõtuks biomeditsiinis, vältides sealjuures soovimatuid kõrvalmõjusid. Antud töös kirjeldasime hiljuti väljatöötatud PepFect ja NickFect tüüpi peptiidide ja nukleiinhapete vaheliste nanokomplekside suurust, kuju ja laengut. Lisaks sellele identifitseerisime uuritud RSP/nukleiinhappe komplekside peamised rakku sisenemise mehhanismid ning analüüsisime rakku viidud nukleiinhappe paiknemist ja selle muutumist ajas.Nucleic acids are highly promising candidates for the treatment of various diseases. In order to achieve biological functionality, nucleic acids need to be internalized by cells and reach their action site in cytoplasm or nucleus. However, due to the large size and negative charge, naked nucleic acids are not capable of traversing the plasma membrane of cells. A wide variety of delivery vectors have been designed to facilitate the cellular uptake of nucleic acids. One class of such vectors are cell-penetrating peptides (CPPs), short sequences of 5-40 amino acid residues, which are capable of gaining access to the interior of cells, and importantly, mediate the internalization of coupled cargo molecules. The simplest way to couple CPPs to nucleic acids is to mix the peptide and cargo. The co-incubation of CPPs and nucleic acids leads to the formation of nanocomplexes due to electrostatic and hydrophobic interactions between the peptide and cargo. Co-incubation strategy has been shown to yield high bioactivities of cargos in numerous studies. However, this approach has not reached clinical trials yet. One reason behind this is the complicated physicochemical characterization of the forming nanocomplexes. However, in order to be considered for implementation in biomedicine the properties of CPP/nucleic acid complexes such as size, morphology and charge need to be characterized in detail. Another bottleneck which impedes the implementation of non-covalent strategy for nucleic acid delivery is the poor knowledge of the cellular uptake mechanisms and intracellular trafficking of CPP/nucleic acid nanocomplexes. However, detailed characterization of the cell internalization pathways and cellular trafficking of CPP/cargo complexes are essential for avoiding undesired side effects and refining their properties in order to yield higher activities of delivered cargo. The main objectives of the current thesis were to characterize the physicochemical properties of CPP/nucleic acids nanocomplexes formed by PepFect and NickFect type carrier peptides, and to examine the cellular uptake mechanisms and intracellular trafficking of nanocomplexes and nucleic acids

Bolaamphiphiles as a novel drug delivery system in the treatment of diseases of the brain

The incidence of central nervous system (CNS) diseases, such as glioblastoma, Alzheimer’s disease and Parkinson’s disease, will increase substantially in the next few decades. However, treatment for diseases of the brain is limited due to the restrictive physical and functional blood-brain barrier (BBB) dividing the brain and the vascular system. Bolaamphiphilic (BA) vesicles, produced from vernonia oil, encapsulate a wide range of therapeutic molecules, and offer an alternative drug delivery system to penetrate the brain to treat diseases of the CNS. We present novel anionic BA vesicles that cross biological barriers including the blood-brain barrier. BA vesicles were characterised by dynamic light scattering, transmission electron microscopy, zeta potential analysis and size exclusion column chromatography. In vitro studies were performed on numerous CNS-representative and other cell lines - BV2 microglia, SH-SY5Y neurones, LN229 glioblastoma, HEK-293T epithelia, hCMEC/D3 endothelia and HASTR/ci35 astrocytes. In vivo studies were performed using C57BL/6 male mice. A novel methodology was developed to permit synthesis of the novel BAs first described here, directly from vernonia oil, the starting material. This study has shown that a novel preparation of anionic BA form vesicles that encapsulate a range of different cargoes including tracer dyes and antibody fragments albeit with a low encapsulation efficiency. They do not influence cell viability or cause an acute immune response. They have been shown to penetrate the BBB in vivo. Analysis of the original BA material has shown to consist of two compounds, both of which have been synthesised and characterised. The original material synthesised from vernonia oil and used to produce BA vesicles was thought to be cationic. However, after profiling the vesicles synthesised were found to have a negative zeta potential demonstrating that they are novel. They were further tested and found to cross biological barriers in vitro. Newly synthesised vesicles require further characterisation and optimisation to improve stability and encapsulation efficiency. The original anionic BA material has been shown to cross the BBB within 30 minutes of intravenous injection. These results demonstrate that whilst further studies are required this is a candidate drug delivery system to treat diseases of the brain