A trealase solúvel foi purificada a partir do intestino de S. frugiperda. Os pKas dos grupos catalíticos determinados por inativação química são similares aos pKas determinados por analise cinética, indicando que a enzima tem um grupo carboxila que atua como um nucleófilo e um grupo guanidina que atua como doador de prótons. Dietil pirocarbonato não afeta a enzima, exceto na presença de MalfaGlu (inibidor competitivo). A modificação com DPC diminui a atividade enzimática da trealase e muda o valor do pKa do resíduo de Arg, indicando que o resíduo de His modula o pKa do doador de prótons. Trealase tem dois subsítios para a ligação de glicose e baseado na proteção por MalfaGlu durante a modificação química é possível inferir que o subsítio que liga MalfaGlu contém o grupo carboxila, e o outro subsítio possui o resíduo de Arg que atua como grupo catalítico e o resíduo de His. Usando diferentes estratégias nós obtivemos uma seqüência parcial do cDNA que aparentemente codifica para trealase (denominada trealase 1) e clonamos e expressamos a enzima denominada trealase 2. A trealase 2 foi expressa em Bl21 DE3 e purificada, sendo que suas propriedades são similares a enzima solúvel. O cDNA da trealase 1 provavelmente codifica para a trealase de membrana encontrada no intestino de S. frugiperda. A trealase 2 tem 587 aminoácidos, um pepitideo sinal com 23 aminoácidos e seis possíveis sítios para glicosilação. A enzima apresenta alta identidade e similaridade (61% e 76%, respectivamente) com a trealase digestiva de B. mori. Foi determinada a atividade de trealase presente na carcaça, Túbulos de Malpighi, corpo gorduroso, intestino e hemolinfa de Tenebrio molitor, Musca domestica, Spodoptera frugiperda e Diatraea saccharalis na presença e na ausência de ß-glicosideos tóxicos produzidos por plantas. Os glicosídeos usados foram amigdalina, prunasina, florizina e o aglicone mandelonitrila. E a atividade das trealases de T. molitor e S. frugiperda foi determinada também na presença de esculina. Prunasina é o melhor inibidor das trealases de T. molitor, já para as trealases (ligadas a membrana) de D. saccharalis o melhor inibidor é florizina e esculina é o melhor inibidor das trealases de S. frugiperda. Nós alimentamos S. frugiperda com uma dieta contendo 0,1% de esculina e sua presença nós tecidos foi detectada por fluorescência. Esculina foi encontrada no corpo gorduroso, Túbulos de Malpighi e hemolinfa e não foi encontrada na carcaça. A maior quantidade de esculina foi registrada na hemolinfa (0,2 mM) e as larvas alimentadas com uma dieta contendo esculina são 40% menor que as larvas alimentadas numa dieta controle. A inibição das trealases pode ser um dos fatores que leva a diminuição de peso das larvas experimentais. As larvas de S. frugiperda criadas numa dieta com 0,1% de amigdalina apresenta em alguns tecidos um aumento na atividade especifica de trealase o que não é observado quando as larvas são alimentadas com uma dieta com 0,1% de esculina. O aumento na atividade especifica de trealase pode ser uma das razões pela qual o desenvolvimento de S. frugiperda não é afetado pela amigdalina presente na dieta. / A soluble trehalase was purified from Spodoptera frugiperda midgut. The pKas of the catalitical groups determined by chemical inactivation agrees with the ones determined by kinetical analysis, indicating that the enzyme has a carboxyl group that acts as a nucleophile and a guanidine group that is the proton donor. Diethyl pyrocarbonate (DPC) does not affect to the enzyme, except in the presence of MalphaGlu (a competitive inhibitor). DPC modification decreases trehalase activity and changes the pKa value of the catalytical Arg residue, indicating that pKa of the proton donor His residue modulates. Trehalase has two subsites for glucose binding and based on the protection by MalphaGlu against chemical modification it is possible to infer that the subsite that binds MalphaGlu contains the catalytic carboxyl, whereas the other has the catalytical Arg residue and the His residue. Using different strategies we succeeded in obtaining a partial sequence of a cDNA that apparently codes for trehalase (called trehalase 1) and in molecular cloning and expressing the enzyme named trehalase 2. Trehalase 2, expressed in Bl21 DE3 cells was purified and its properties are similar to the soluble enzyme. Trehalase 1 cDNA probably codes for a membrane-bound trehalase found in S. frugiperda midgut. Trehalase 2 has 587 amino acids, a signal peptide with 23 amino acids and six possible sites for glycosilation. The enzyme present higher identity and similarity (61% and 76%, respectively) to digestive trehalase of Bombyx mori. Trehalase from body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor, Musca. domestica, Spodoptera frugiperda and Diatraea saccharalis were assayed with and without the presence of toxic glucosides produced by plants. The glucosides used were amygdalin, prunasin, phlorizin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with esculin. Prunasin is the best inhibitor in T. molitor and M. domestica, phlorizin in D. saccharalis (only membrane-bound activity) and esculin in S. frugiperda. We fed S. frugiperda with a diet containing 0.1 % esculin and followed its fate by fluorescence. Esculin is recovered from fat body, Malpighian tubules and haemolymph. No esculin was found in body wall. The majority of esculin was recovered in haemolymph (0.2 mM) and larvae fed on esculin-containing diet weigh 40 % less than control ones. Trehalase inhibition by esculin may account for at least part of the observed decrease in larval weight. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activities in several tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why S. frugiperda development is affected by esculin, but is not impaired by amygdalin present in the diet.

The genetic systems involved in the utilisation of aromatic β-glucosides in E. coli consist of the bgl, asc, and chb operons and the locus bglA encoding phospho-β-glucosidase A. The bgl and asc operons are known as cryptic or silent systems since their expression is not sufficient for utilisation of these sugars in wild type strains of E. coli. Their transcriptional activation by different classes of mutations confers a Bgl+ phenotype to the mutant. The maintenance of cryptic genes without accumulating deleterious mutation in spite of being silent is an evolutionary puzzle. Several observations have suggested the possibility that these genes may be expressed under specific physiological conditions conferring a fitness advantage to the organism. The main aim of this study was to investigate the possible role of aromatic β-glucoside catabolic systems of E. coli in combating nutrient stress and microaerobic growth conditions. The results presented in Chapter 2 address the evolution of aromatic β-glucoside catabolic systems when exposed to a novel β-glucoside as the sole substrate. The results indicate that the bgl opeon, the primary system involved in the utilisation of the aromatic β-glucosides arbutin and salicin, is also involved in esculin utilisation. In the absence of bglB encoding the enzyme phospho-β-glucosidase B, activation of the silent asc operon enables esculin utilisation. The bglA gene encoding phospho-β-glucosidase A specific for arbutin, can undergo successive mutations to evolve the ability to hydrolyse esculin and salicin sequentially when bglB and ascB are absent. The Esc+ and Sal+ mutants retain their arbutin+ phenotype, indicating that the mutations enhance the promiscuity of the enzyme. Sequencing data indicate that the first step Esc+ mutant carries a four base insertion within the promoter of the bglA gene that results in enhanced transcription of bglA. RT-PCR studies confirm that both the steady-state levels as well as the half-life of the bglA mRNA are enhanced in the mutant. This is further corroborated by the observation that overexpression of wild type bglA in the parent strain using a multicopy plasmid confers an Esc+ phenotype. The second step Sal+ mutant carries a point mutation within bglA ORF, a thymine to guanine transversion at position 583 (T583G) of the bglA gene, resulting in an amino acid change from cysteine to glycine at position 195 (C195G) of the BglA ORF close to the active site. Presence of a plasmid carrying the T583G mutation, introduced by site-directed mutagenesis, results in a Sal+ phenotype, confirming the role of the transversion in conferring the Sal+ phenotype. Based on docking studies, the positioning of salicin into the substrate binding site of the mutant BglA enzyme is different compared to wild type BglA due to the loss of stearic hindrance for the binding of salicin when C195 is replaced by the smaller amino acid glycine in the mutant protein. These observations indicate that under conditions of nutrient deprivation, exposure to novel substrates can result in the evolution of new metabolic capabilities by the sequential modification of a pre-existing genetic system. In the case of one novel substrate, the mutation results in the overexpression of the hydrolytic enzyme, while in the case of the second substrate, a mutation close to its active site increases its substrate specificity. Results presented in Chapter 3 specifically deal with the involvement of the bgl operon under low levels of oxygen. Earlier observations have shown that there is a 22 fold enhancement in the expression of the bgl operon under anaerobic condition. The present results provide evidence that bgl expression has a physiological role under low levels of oxygen and in addition suggest a possible mechanism for the overexpression of the bgl operon that involves the ArcAB two component system known to mediate regulation under microaerobic and static conditions. Transcription studies using a lacZ reporter fused to the wild type bgl promoter show that there is enhanced transcription from the bgl promoter under microaerobic and static conditions in the presence of arcA encoding the response regulator compared to that in its absence. The positive effect of arcA on the expression of the bgl operon is dispensable in the absence of H-NS since presence or absence of arcA does not change the expression of the bgl operon in an hns-null background, implying that the involvement of ArcA is via antagonizing H-NS. Competition experiments indicate that there is growth advantage associated with the activated allele of the bgl operon under low levels of oxygen since Bgl+ strains carrying the activated allele of the bgl operon as well as strains expressing BglG constitutively can out-compete wild-type strains. Presence of the wild type arcA allele results in a strong growth advantage compared to its absence under static conditions but not aerobic condition. The bgl operon seems to be one of the possible downstream targets of ArcA under static condition since absence of the bgl operon results in a modest reduction of the growth advantage (GASP) phenotype conferred by arcA. The up-regulation of the bgl operon is likely to enable the cells to scavenge available nutrients from their niche more efficiently. These experiments also show that the GASP phenotype associated with BglG constitutive strains under static conditions involves downstream genes that are different from oppA known to be one of the downstream targets during aerobic growth. It is possible that under low level of oxygen, the bgl operon is regulating a different set of downstream genes involving a different mechanism. In summary, the results of this investigation show that the aromatic β-glucoside catabolic systems in E. coli play a role in the generation of new metabolic capabilities via mutations in pre-existing genetic systems as well as through changes in gene expression patterns. The mechanisms outlined in this study are likely to be of broader significance applicable to microbial evolution under stress in general.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries. / AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.

SummarySummarySummarySummary Cytokinins (CKs) are important group of plant hormones involved in a wide range of physiological and developmental processes. Endogenous levels of CKs as well as proportions of individual CK forms and derivatives are not constant and differ among plant species. The amounts of biological active CK forms (free bases and ribosides) are regulated through tangled machinery of metabolic conversions including biosynthesis, conjugation and degradation pathways. The main object of this thesis was to characterize the metabolic pathways involved in the regulation of bioactive CK levels in plants especially via CK biosynthesis with aspect to the environmental stimuli and via N- glucosylation pathway. It was shown, that light signal is an important input for modulating some CK-related genes and CK levels in Arabidopsis plants. The complex diurnal expression profiles of CK-biosynthetic genes (AtIPT1 - AtIPT9) in Arabidopsis plants indicated a strong dependence of AtIPT1 and AtIPT5 on light/dark phase in leaf rossetes. In contrast, no diurnal oscillation of AtIPT transcript levels was recorded in roots. Although the content of endogenous CKs was not constant in plants and varied during a day, no statistically significant correlation between light/dark cycle and oscillation in CK levels was...

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No description available.

Bacteria are ubiquitous in all ecosystems and are often challenged by multiple stresses such as extreme temperatures, high salt concentrations, nutrient limitation, pH variations, radiation, predation and the presence of antibiotics/toxins. The most challenging among them is predation pressure which is one of the major causes of their mortality in different niches. Bacteria have evolved different adaptive measures to counter predation. Some of them include change in shape, size, motility, and unpalatable aggregate formation. Aromatic β-glucosides such as salicin, produced by plants as secondary metabolites, play a significant role in protecting them from herbivores. Members of the family Enterobaceriaceae primarily present in soil, e.g. Erwinia chrysanthemi (a phytopathogen) and Klebsiella aerogenes, can utilize the aromatic β-glucosides salicin and arbutin (likely to be present in soil derived from decomposing plant materials) as a carbon source unlike their fellow members such as Escherichia coli, Shigella sonnei, and Salmonella present in the gut environment. Bacteria can obtain energy by metabolizing β-glucosides in the form of glucose. Whether they can also use these molecules as defense tools in a manner similar to plants is an intriguing possibility. In such an event, Bgl+ bacteria could derive a dual advantage in terms of energy generation and protection from predation. The current study was initiated to investigate a possible link between β-glucoside metabolism and self-defense in Enterobacteriaceae. Different members of Enterobacteriaceae comprising of both laboratory strains and natural isolates were considered as prey. Predators included were laboratory strains and soil isolates of bacteriovorous nematodes of the Rhabditidae family, the amoeba Dictyostelium discoidium and a bacteriovorous Streptomyces sp. The predator-prey interaction was analyzed by performing viability and behavioral assays in the context of β-glucoside metabolism Results presented in Chapter 2 show that active catabolism of aromatic β¬glucosides like salicin, arbutin and esculin by Bgl+ bacteria decreases the viability of their predators. The aglycone products released during β-glucosides metabolism, e.g. saligenin in the case of salicin, are the causative agents of the mortality of the predators. The lethality is reversible up to a specific threshold of exposure. Saligenin acts as a chemo-attractant that lures and kills Caenorhabditis elegans N2. In the case of nematodes that succumb, bacteria can derive nutrition from the dead predators indicating a conversion of prey to predator. Experiments with mutant strains of Caenorhabditis elegans suggest that the dopaminergic receptor dop-1 is involved in mediating saligenin toxicity. Studies mentioned in Chapter 3 revolve around the relevance of the predator-prey interaction discussed in Chapter 2 in the natural environment. Members of Enterobacteriaceae and their predator amoebae (cellular slime molds) and nematodes were isolated from soil. They show coexistence in most of the soil samples analyzed. All the predators isolated from soil and other natural isolates of Caenorhabditis succumb to saligenin as their laboratory counterparts with higher sensitivity in some of the strains. Soil nematodes belonging to genera Oscheius and Mesorhabditis avoid saligenin unlike the members of Caenorhabditis genus which are attracted towards saligenin. This indicates that the soil nematodes are often exposed to saligenin or saligenin-like compounds, resulting in the evolution of a genetic machinery to avoid these toxic compounds. Studies with quasi-natural environments like soil and fruit indicate that β-glucoside metabolism have similar effects on predator prey interaction in these environments, reinforcing the relevance of these observations to the natural ecology of the organisms. The studies reported in Chapter 2 and 3 shed light on a novel defense strategy of otherwise non-pathogenic members of Enterobacteriaceae which comes with a dual advantage. These results have also brought into focus issues such as the benefit derived by bacterial populations that are genetically heterogeneous, consisting of both Bgl+ and Bgl-strains. The broad implications and future directions of the work are discussed in Chapter 4. Work presented in Appendix deals with the investigation of the pattern of cellobiose utilization in Shigella sonnei. As mentioned in Chapter 1, it is known that members of Enterobacteriaceae exhibit diversity in their pattern of β-glucoside utilization. Wild type strains of both E. coli and Shigella sonnei are unable to utilize Arbutin, Salicin and Cellobiose. While E. coli can acquire cellobiose utilizing ability directly from the wild type state (Arb-Sal-Cel-), Shigella sonnei strains, though closely related to E. coli, have to undergo a series of mutations in a specific sequence to become capable of utilizing these sugars. Characterization of a few Shigella sonnei Cel+ mutants showed a different mode of activation of the chb operon (known to be involved in cellobiose utilization in E. coli). Considering the ecological significance of the ability to hydrolyze aromatic β-glucosides, a detailed understanding of the metabolic capability of different strains and the molecular mechanism involved becomes significant.

The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

Given the rising demand for diverse cell types in regenerative and transfusion medicines, such as human induced pluripotent stem cell-derived neurons (iPSC-Ns), human T/chimeric antigen receptor (CAR) T cells, and human natural killer (NK) cells, the ability to cryopreserve cells has become increasingly important. In regenerative medicine, iPSC-Ns are powerful tools for treating and modelling neurodegenerative diseases. Moreover, transplants/transfusions of T/CAR T cells or NK cells offer promising treatment for numerous types of tumors, such as leukemia and multiple myeloma. Cryopreservation of cells at sub-zero temperatures (-80 to -196 °C) allows for the development of master cell banks that can be used for clinical applications. Conventional cryoprotective agents (CPAs), such as dimethylsulfoxide (DMSO) and glycerol, are utilized to protect cells from cryoinjuries associated with the freezing process. However, the use of high concentrations of DMSO (i.e., 10 to 20%) has been shown to be accompanied with toxic effects on patients receiving cell therapies if it is not removed or diluted prior to transfusion. Moreover, DMSO does not prevent the occurrence of the cryoinjury associated with ice recrystallization, which is one of the major causes of cell death/damage during cryopreservation. As a result, there is a surge of attention toward developing new non-toxic cryo-additives that inhibit ice recrystallization during cryopreservation to permit future advancement in regenerative and transfusion medicines. Moreover, the use of ice recrystallization inhibitors (IRIs) as novel CPAs has become a promising strategy to improve cell viability and function post-thaw. The Ben laboratory heavily invested in synthesizing several classes of carbohydrate-based small molecule IRIs (i.e., O-linked alkyl and aryl glycosides, and N-aryl-D-gluconamides), and studying the correlation between their IRI activity and molecular properties, such as polar surface area to molecular surface area (PSA/MSA) ratio. Moreover, compounds that belong to the O-linked aryl glycosides and N-aryl-D-gluconamides classes of IRIs have been shown to enhance the viability and functionality of red blood cells (RBCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) after thawing. Part of the research presented throughout this thesis focuses on structure-activity relationship (SAR) studies of alkyl pyranoses with modified alkyl chain lengths to explore any correlations between the IRI activity and the net polarity (i.e., PSA/MSA ratio) of the IRI candidates. O- and C-linked alkyl pyranose derivatives with different alkyl chain lengths were synthesized and their IRI activity was assessed using the modified splat cooling assay. While the IRI activity of the O- and C-linked alkyl glucosides did differ as the length of the alkyl chain increased, no correlation between the PSA/MSA ratios and their IRI activity was observed. In addition, this work allowed for investigation into the effect of the type of the glycosidic bond (i.e., C-O and C-C bonds) at the anomeric position, on the IRI activity of the different compounds. The O-linked alkyl glucosides appeared to be more IRI active than the C-linked compounds, suggesting the nature of the glycosidic bond is important for IRI activity. The second part of the research presented in this thesis focuses on examining the potential for IRIs to cryopreserve iPSC-Ns, T/CAR T cells, and NK cells. 2-fluorophenyl-D-gluconamides (2FA), which is one of the most active IRIs from the N-aryl-Dgluconamides, has shown promising results in maintaining a high number of viable and functional HSCs and iPSCs post-thaw, and therefore it was employed in the cryopreservation protocol of iPSC-Ns, human-derived T/CAR T cells, and human-derived NK cells. The efficacy of the cryopreservation protocol being constructed was evaluated by assessing the post-thaw viability and recovery rate, as well as the functionality of iPSCNs, T/CAR T cells, and NK cells post-thaw. These studies showed that protecting against ice recrystallization during cryopreservation with IRIs increases the number of viable and functional iPSC-Ns, and T/CAR T cells. It was also observed that employing IRI technology in the cryopreservation protocol of NK cells does not compromise their functionality compared to fresh, non-frozen NK cells. Overall, inhibition of ice recrystallization using IRIs appeared to enhance the cryopreservation outcomes of the different cell types, which will allow for the development of off-the-shelf cell therapy products and improvement of the delivery of efficacious cell products to clinics and hospitals.

Mit den vorgestellten Untersuchungen sollten das Potential der in Europa vorkommenden Echten Goldrute für den Anbau evaluiert und der Einfluß von Erntetermin, Schnitthöhe und Düngungsmaßnahmen sowie der abiotischen Umweltfaktoren Licht und Wasser auf den Ertrag und den Inhaltsstoffgehalt von Solidago virgaurea L. geprüft werden. In vier Feldversuchen wurden Inhaltsstoffgehalte, Ertragsparameter, Entwicklungsstadien sowie morphologische Merkmale untersucht. Die Gehalte an Flavonolglycosiden, Phenolglucosiden und Kaffeesäurederivaten wurden mittels HPLC und nachfolgender DAD-Detektion bestimmt. 45 Akzessionen aus dem europäischen Verbreitungsgebiet wurden anhand ihres Blühtermines gegliedert, den Unterarten virgaurea und minuta zugeordnet, und mit Hilfe von 33 morphologischen Merkmalen charakterisiert. Die Analyse von 452 Einzelpflanzen ergab Spannweiten von 0.4 bis 2.4 % Flavonolglycosiden, 0.7 bis 4.6 % Kaffeesäurederivaten und 0 bis 1.6 % Phenolglucosiden. Die phytochemische Zusammensetzung war innerhalb der einzelnen Akzessionen, insbesondere in der Unterart virgaurea hoch variabel. In einer anbaubezogenen Gesamtbewertung der Akzessionen erwiesen sich Herkünfte der Unterart virgaurea aufgrund hoher Drogenerträge von maximal 109 dt/ha und eines ausgewogenen Inhaltsstoffspektrums als die Wertvollsten. Im Lauf der Ontogenese stieg der Drogenertrag der Echten Goldrute an, während die Inhaltsstoffgehalte schwankten (Kaffeesäurederivate, Flavonolglycoside) oder relativ kontinuierlich absanken (Phenolglucoside). Eine gehaltsorientierte Ernte müßte daher zum relativ frühen Zeitpunkt des mittleren Knospenstadiums erfolgen. Hohe Masse- und Inhaltsstofferträge sind in einem Ernteverfahren mit niedrigen Schnitthöhen von 15 bis 30 cm erreichbar. In voll besonnten Pflanzen wurde im Vergleich zu schattierten eine höhere Anreicherung von Flavonolglycosiden und Kaffeesäurederivaten festgestellt, die durch die UV-Schutzfunktion dieser Stoffgruppen erklärbar ist. Daher muß angenommen werden, daß ontogenetisch bedingte Gehaltsveränderungen der Flavonolglycoside und Kaffeesäurederivate durch unterschiedliche Strahlungsverhältnisse überlagert werden können. Stickstoffdüngung verursachte neben der erwarteten ertragssteigernden Wirkung Unterschiede im Inhaltsstoffgehalt. In der ungedüngten Variante wurden jeweils die höchsten Flavonolglycosidgehalte bestimmt, gefolgt von der organischen Düngung sowie der Mehrnährstoff- und Stickstoffdüngung. Während die ermittelten Stickstoff- (maximal 96 kg/ha N) und Phosphorentzüge (maximal 39 kg/ha P2O5) vergleichsweise gering waren, wurden Kaliumentzüge (maximal 172 kg/ha K2O) festgestellt, die auch im Vergleich zu anderen Blatt- und Krautdrogen relativ hoch ausfielen. Im Gesamtüberblick aller Einflüsse wurde deutlich, daß Flavonolglycoside, gefolgt von Kaffeesäurederivaten am empfindlichsten auf Umwelteinflüsse wie Lichteinstrahlung und Nährstoffverfügbarkeit reagierten, während die Gruppe der Phenolglucoside nur geringe Schwankungen zeigte. Die Ergebnisse aller Versuchsfragen spiegeln das Spannungsfeld von landwirtschaftlichem Ertrag und pharmazeutisch geforderter Qualität wider, die häufig negativ korreliert sind. Bei der Produktion der Droge von Echter Goldrute muß daher eine Balance zwischen beiden Zielgrößen gefunden werden. / The aim of the presented studies was to evaluate the potential for cultivation of the European Goldenrod (Solidago virgaurea L.) and to examine the influences of harvesting date, cutting height, fertilization and of the abiotic environmental factors light and water on drug yield and compound content of the plant. In four field trials a fingerprint of 10 constituents, several parameters of drug yield, development stages and morphological traits were examined. The contents of flavonol glycosides, phenolic glucosides and caffeic acid derivatives were determined by HPLC and UV-detection by DAD. 45 european accession were sorted by their dates of anthesis and subdivided into the subspecies virgaurea and minuta and thereafter characterized by 33 morphological traits. The analysis of 452 individual plants showed ranges of 0.4 to 2.4 % flavonol glycosides, 0.7 to 4.6 % caffeic acid derivatives and 0.0 to 1.6 % phenolic glucosides. Within the examined accessions, especially subspecies virgaurea, a high variability of the phytochemical composition was observed. In an evaluation, accessions of subspecies virgaurea turned out to be most suitable for cultivation due to high drug yields up to 106 dt per ha and well-balanced spectra of constituents. During ontogenesis the drug yield of S. virgaurea increased, while compound contents varied (caffeic acid derivatives, flavonol glycosides) or decreased continuously (phenolic glucosides). Therefore a harvest aiming at high compound contents has to be carried out at a early development stage of mid budding. High mass and constituent yields can be obtained by a harvesting method with low cutting heights of 15 to 30 cm above ground. Plants in a sunny environment accumulated significantly higher amounts of flavonol glycosides and caffeic acid derivatives than shaded plants, which can be explained by the UV-protective function of these compound groups. Therefore it can be concluded that phytochemical variations during ontogenesis could be modified by different light conditions. Beside the expected drug yield increase, fertilization with nitrogen caused differences in the compound contents. The highest flavonol glycosides contents were observed in the unfertilized treatment, followed by treatments with organic fertilization, multinutrient fertilizer and calcium ammonium nitrate. While the nitrogen uptake by the plant (maximal 96 kg/ha N) and phosphorus (maximal 39 kg/ha P2O5) were relatively low, contrary potassium uptake (maximal 172 kg/ha K2O) was relatively high compared to other herbal drugs. In an overview of all influences it became obvious that the reaction of flavonol glycosides and caffeic acid derivatives were relatively sensitive to environmental influences such as light conditions and nutrient availability, while the group of phenolic glucosides showed low variations. The results of all field trials reflect the conflict between agricultural yield and pharmaceutical quality which are frequently negatively correlated. Therefore a balance between these two goals has to be found during drug production of S. virgaurea.