This thesis concerns the development of surface ligation techniques for the preparation of carbohydrate biosensors. Several methodologies were developed based on efficient photochemical insertion reactions which quickly functionalize polymeric materials, with either carbohydrates or functional groups such as alkynes or alkenes. The alkyne/alkene surfaces were then treated with carbohydrate azides or thiols and reacted under chemoselective Cu-catalyzed azide-alkyne cycloaddition (CuAAC) or photo-radical thiol-ene/yne click chemistry, thus creating a range of carbohydrate biosensor surfaces under ambient conditions. The methodologies were evaluated by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) flow through instrumentations with recurring injections of a range of lectins, allowing for real-time analysis of the surface interactions. The developed methods were proven robust and versatile, and the generated carbohydrate biosensors showed high specificities and good capacities for lectin binding.  The methods were then used to investigate how varying the glycan linker length and/or a sulfur-linkage affect the subsequent protein binding. The survey was further explored by investigating the impact of sulfur in glycosidic linkages on protein binding, through competition assays with various O/S-linked disaccharides in solution interactions with lectins. / QC 20120309

The aromatic beta-glucosides of plant origin, salicin and arbutin, serve as carbon sources for the sustenance of bacteria when ‘preferred’ sugars are absent in the environment. In the family Enterobacteriaceae, there are varied patterns for utilization of these beta-glucosides, wherein, in some members the ability to utilize salicin or arbutin is cryptic while in others it is completely absent. Escherichia coli harbors silent or cryptic genetic systems for the utilization of arbutin and salicin, which are activated by spontaneous mutation(s). Of these systems, the bgl operon of E.coli has been used as a paradigm for silent genes and extensive studies have been carried out to understand its silencing and activating mechanisms. Mutational activation of the wild type bgl operon in E.coli leads to the acquisition of the ability to utilize both arbutin and salicin. Preliminary studies have shown that aromatic beta-glucoside utilization in Shigella sonnei, which is evolutionarily related to E.coli, shows a two-step activation process wherein the wild type strain first becomes Arb+, which subsequently mutates to Sal+. The genetic systems responsible for beta-glucoside utilization, including the bgl operon, are conserved in S.sonnei to a large extent. A major difference is that the bglB gene encoding the phosphor-β-glucosidase B is insertionally inactivated in S.sonnei. As a result, activation of the bgl operon in the first stage leads to expression of the permease, BglF, which along with the phosphor-β-glucosidase A expressed from an unlinked constitutive gene, bglA, confers an Arb+phenotype. Salicin is not a substrate for the enzyme BglA and therefore a second mutational event is required for the acquisition of the Sal+ phenotype. Interestingly, the insertion within bglB is retained in AK102, the Sal+ second step mutant of S.sonnei. Therefore, the locus involved in conferring salicin utilization ability is unknown. However, S.sonnei is not amenable to routine genetic echniques and an E.coli bglB model was generated by creating an insertion in the bglB gene to identify the locus involved in conferring the Sal+ phenotype. Like S.sonnei, this E.coli strain, SD-1.3, also showed a two-step activation process for the utilization of salicin. Utilization of salicin in the Sal+ second step mutant of SD-1.3 could require activation of other silent genetic systems such as the asc operon and the chb operon or mutation in loci such as bglB or bglA. Linkage analysis by P1 transduction showed that activation of the asc operon is required for conferring a Sal+ phenotype in the second step mutant. The asc operon comprises of two genes, ascF encoding a PTS permease and ascB encoding a phosphor-β-glucosidaseB.The Precise mechanism of activation of the asc operon is not known but, it has been speculated that AscG, encoded by an upstream gene, acts as a repressor. Results presented in this thesis show that BglF is responsible for the transport of salicin and AscB provides the phosphor-β-glucosidase B in the Sal+ second step mutant of the E.coli strain SD-1.3. Analysis of the expression of the ascFB operon by measuring the transcripts as well as the activity of phosphor-β-glucosidase B showed that it is enhanced in the Sal+ second step mutant of SD-1.3 in the presence of the inducer. The expression of the ascFB operon is also increased constitutively when ascG is replaced by an antibiotic cassette in the parent strain SD-1.3 and the Arb+ first step mutant, indicating that AscG acts as a repressor for the asc operon. Moreover, inactivation of ascG in the parent leads to utilization of salicin in a single step by the activation of the bgl operon to provide the transport function, indicating that the inactivation of ascG is sufficient to activate the expression of ascB. Similarly, loss of AscG–mediated repression of the asc operon confers salicin utilization ability to the Arb+ first step mutant of SD-1.3. Interestingly, measurement of phosphor-β-glucosidase B activity in a Sal+ second step mutant derivative deleted for ascG showed a constitutive increase in the expression of the ascFB operon. Thus, AscG mediates the induction of the asc operon in response to salicin. In order to study the mechanism of activation of the asc operon, the ascB gene was cloned from the Arb+ first step mutant and the Sal+ second step mutant of SD-1.3 in a low copy number vector. Both these constructs were able to confer a Sal+ phenotype to the Arb+ first step mutant indicating absence of any genetic change in ascB in the Sal+ second step mutant. This was also confirmed by sequencing of ascB gene from the strains that showed no changes in the nucleotide sequence. Absence of any insertions within ascG showed that activation of the ascoperon is not achieved through disruption of ascG in the Sal+ second step mutants analyzed. AscG belongs to the GalR family of repressors in which some members require a mutation to enable the binding of sugar to mediate induction. Nucleotide sequence analysis showed that there was no change in the ascG gene in the Sal+ mutants analyzed. However, when the upstream regulatory region of the ascFB operon was analyzed a mutation was found in the -10 sequence of the putative promoter of the ascFB genes. This change leads to a stronger promoter as it brings the -10 sequence closer to the consensus sequence. Therefore, salicin utilization is achieved in the Sal+ second step mutant analyzed by an increase in expression of the asc operon by a promoter-up mutation. The negative effect of binding of AscG on expression of the ascFB operon is relieved in presence of the inducer, salicin. The possible role of the asc operon in salicin utilization in S.sonnei was tested by replacing the ascB gene by anantibiotic cassette in AK102, the Sal+ second step mutant of S. sonnei. This did not lead to loss of salicin utilization. By gene targeting approach it was also found that none of the phosphor-β-glucosidases known in E.coli are involved in degradation of salicin in AK102. A search of the S. sonnei genome database indicated the presence of two putative phosphor-β-glucosidases encoded by glvG and SSO1595. Replacement of glvG gene by anantibiotic cassette in AK102 did not lead to loss of salicin utilization. However, a similar replacement of SSO1595 in AK102 resulted in a Sal+ phenotype indicating that SSO1595 provides the phosphor-β-glucosidase in the Sal+ second step mutant of S. sonnei. A homolog of this enzyme is not present in E.coliorinany of the other members of the Shigella genus. Transcription alanalysis as well as measurement of phosphor-β-glucosidase B activity showed that expression of SSO1595 is enhanced constitutively in AK102. To study the mechanism of mutational activation for achieving salicin utilization in S. sonnei, SSO1595 was cloned from AK101, theArb+ first step mutant and AK102, the Sal+ second step mutant in a low copy numbe rvector. Both these constructs were able to confer a Sal+ phenotype to AK101 indicating an absence of genetic change in SSO1595 in AK102. This was also confirmed by sequencing of SSO1595 gene from the strains. Analysis of the upstream regulatory region of SSO1595 in AK102 indicated a deletion of around 1.0kbp sequence. This was also confirmed by nucleotide sequencing of the region. By primer extension analysis it was found that a new transcriptional start site is generated upstream to the deletion in the Sal+ second stepmutant of S.sonnei. Acquisition of the Sal+ phenotype in AK102 is therefore the resultof the SSO1595 gene being brought under a new promoter as a result of a DNA rearrangement. Overall, this study suggests that a high degree of similarity at the genomic level between organisms does not always ensure similarity in genetic mechanisms as two distinct pathways are responsible for conferring utilization of salicinin S. sonnei and E.coli.

During pulp and papermaking process a huge amount of water is used. The wastewater contains a large amount of pollutants and has to be treated before it reaches the recipient. In March 2004 a new bleaching plant was started up at SCA Graphic Sundsvall AB, Ortviken’s paper mill using peroxide. The production of bleached thermo mechanical pulp and thus the load to the wastewater plant increased. During bleaching substances dissolve from the pulp and the amount of COD in the wastewater increase. To keep the efficiency of chemical and biological oxygen demand (COD, BOD) removal, the aerated lagoon is supplied by liquid oxygen. In November the same year a new pre-step including a pre-aeration, carrier and selector step was added to the existing wastewater plant. The COD-loading to the lagoon decreased and the supply of liquid oxygen was reduced. This master thesis has been carried out at SCA Graphic Research AB, Sundsvall. The aim was to chemically characterize the wastewater with focus on the composition of carbohydrates, lignin and extractives and its influence on the biodegradability. Measurements have been carried out on total samples, suspended solids, colloidal and dissolved substances using GF/A and ultra filtration. Measurements were performed on process water from the bleaching plant and on water during the wastewater treatment process. The results show that the chemical composition of dissolved substances has a larger impact on the biodegradability than the total amount of COD. A large amount of lignin will make the wastewater harder to degrade. Suspended solids have lower biodegradability compared to the dissolved fraction. Analyzes of suspended material from the bleaching plant show a relative composition of around 41 % lignin. Analyzes done before on the dissolved fraction show a composition of 29 %. It seems that a larger amount of lignin is represented in suspended solids compared to the dissolved fraction. This can explain the low biodegradability, combined with the theory that the suspended solids are less accessible for the microorganisms. To reduce the outgoing COD the incoming flow of dissolved lignin and suspended solids to the active sludge plant has to decrease. COD in the outgoing wastewater to the recipient consists of around 90 % lignin. Extractives as saturated fatty acids, palmitic and stearic acid plus lignans (which is interpreted as a byproduct from lignin degradation) do not degrade remarkable during wastewater treatment. Flocculation of COD is one way to reduce the discharge to the recipient. Addition of 2,6 g/l PAX-18 (a high charged aluminum complex) to the wastewater from the bleaching plant gives a reduction of 40 % COD and 50 % lignin in laboratory scale and it is recommended to study further. Time related studies of BOD show that the microorganisms need five days to degrade organic material and the hydraulic retention time in the aerated lagoon has to be guaranteed. / Vid massa- och pappersframställning åtgår stora mängder vatten. Det processberörda avloppsvattnet innehåller mycket föroreningar varför det måste renas innan det släpps ut till recipienten. I mars 2004 startades ett nytt väteperoxidblekeri vid SCA Graphic Sundsvall AB, Ortvikens pappersbruk varvid produktionen av blekt termomekanisk massa ökade och den biologiska reningen belastades hårdare. Blekning löser ut vedämnen från massan och bidrar således till ökad mängd syreförbrukande ämnen i avloppet. För att upprätthålla reduktionen av syreförbrukande ämnen (COD, BOD) tillförs ren syrgas till den luftade dammen. I november samma år utökades den externa reningsanläggningen med ett biologiskt försteg, en s.k. multibioanläggning bestående av ett frisimmarsteg, ett bärarsteg samt ett selektorsteg. COD-belastningen in till den luftade dammen minskade och tillförseln av ren syrgas kunde reduceras. Examensarbetet har utförts vid SCA Graphic Research AB, Sundsvall med syftet att kemiskt karaktärisera avloppsvattnets sammansättning av kolhydrater, lignin och extraktivämnen och dess inverkan på vattnets grad av biologisk nedbrytbarhet. Analyser har gjorts på totalprov, suspenderande ämnen samt kolloidalt och löst material via fraktionering med hjälp av GF/A- och ultrafiltrering. Analyser gjordes dels på processvatten från blekerierna och dels på avloppsvatten under reningsprocessen. Resultaten visar att den kemiska sammansättningen hos löst material har större inverkan på avloppsvattnets grad av nedbrytbarhet än den absoluta halten av syreförbrukande ämnen. En hög andel lignin ger ett mer svårnedbrytbart vatten. Suspenderat material är vidare mer svårnedbrytbart än de lösta fraktionerna. Analys av suspenderande substans hos blekpressat visar att andelen lignin är omkring 41 % medan arkiverade analysresultat på den lösta fasen visar ett innehåll på omkring 29 %. Det tyder alltså på en betydligt högre andel lignin i den partikulära fasen jämfört med den lösta, vilket i kombination med att materialet inte är lika lättillgängligt för mikroorganismerna kan förklara den låga bionedbrytbarheten. Om COD-utsläppet ska minskas bör inkommande flöde av löst lignin och suspenderande substans till den biologiska reningen reduceras. COD-halten i utgående avloppsvatten till recipienten utgörs av omkring 90 % lignin. De extraktivämnen som inte bryts ned är mättade fettsyror, exempelvis palmitin- och stearinsyra samt lignan, vilket antas bildas som restprodukt vid degradering av lignin. Flockning av syreförbrukande ämnen är ett sätt att gå till väga för att minska utsläppet av dessa till recipienten. Genom tillsats av 2,6 g/l PAX-18 (ett högladdat aluminiumkomplex) till blekeriavloppet kunde 40 % COD och 50 % lignin reduceras. Flockningsförsöket är gjort i laboratorieskala vid rumstemperatur och ytterligare utvärdering rekommenderas. Tidsstudier av BOD visar att mikroorganismerna behöver fem dagars uppehållstid för att hinna bryta ned organiskt material i avloppsvattnet, varför omloppstiden i den luftade lagunen inte bör understiga denna tidsrymd.

No description available.

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

<p>Dynamic Combinatorial Chemistry (DCC) is a recently introduced supramolecular approach to generate dynamically interchanging libraries of compounds. These libraries are made of different building blocks that reversibly interact with one another and spontaneously assemble to encompass all possible combinations. If a target molecule, for instance a receptor is added to the system and one or more molecules show affinity to the target species, these compounds will, according to Le Châtelier´s principle, be amplified on the expense of the other non-bonding constituents. To date, only a handful of different systems and formats have been used. Hence, to further advance the technique, especially when biological systems are targeted, new reaction types and new screening methods are necessary. This thesis describes the development of reversible sulfur reactions, thiol/disulfide interchange and transthiolesterification (the latter being a new reaction type for DCC), as means of generating reversible covalent bond reactions. Two different types of target proteins are used, enzymes belonging to the hydrolase family and the plant lectin Concanavalin A. Furthermore, two new screening/analysis methods not previously used in DCC are also presented; the quartz crystal microbalance (QCM)-technique and catalytic self-screening.</p>

Throughout their life cycle, plants adjust growth in response to their developmental and environmental situation within the limits of their energetic capacities. This capacity is defined by the local sugar availability, which is constantly modulated through synthesis, transport and consumption of sugar. The monitoring of sugar presence is carried out by a complex signalling network in which simple sugars (e.g. glucose, fructose and sucrose) act as metabolic signals for the modulation of physiological processes. However, often it remains unclear whether the regulation is induced by the simple sugars themselves or by their derivatives generated during sugar metabolism. This thesis focuses on the dissection of distinct sugar signals, their generation, perception and impact on the modulation of gene expression and cell division both in cell culture and young seedlings. Based on a stem-cell-like A. thaliana cell culture, which could be sustained in a hormone-free media, a new biological system, supplied with Xyl as the only carbon source was developed. The performance of a variety of sugar and sugar analogue treatments in this novel system allowed for the identification of sugar-responsive candidate genes, which were specifically regulated by glucose, fructose and sucrose. For several genes (e.g. bZIP63, AT5g22920, TPS9, MGD2 and BT2), this regulation required both sugar transport into the cytosol and metabolisation for the generation of the signal. Furthermore, gene expression analyses in young A. thaliana seedlings indicated the requirement for the catalytic activity of hexokinase 1 in the regulation of bZIP63, Atg22920 and BT2 under conditions of a perturbed carbohydrate balance. These findings have been combined in a proposed model for the transcriptional regulation of bZIP63, AT5g22920, TPS9, MGD2 and BT2, which further proposes a function of those genes in the regulation of cell division. The optimisation of a protocol for long-term real-time live-cell imaging provided a valuable tool to show that, similar to gene expression, the progression of cell division depended on a sugar-type-specific regulation at the single-cell level; this regulation was most likely caused by prolongation of the interphase. Together with the observation of cell death and growth arrest of the primary root in intact seedlings in response to the glucose analogue 2dog, this led to the conclusion that sugar signals themselves were sufficient to induce cell division. However, the continuation of cell cycle progression and consequently organ growth over long-time required the availability of the energy contained in the sugar.

Syftet med denna studie var att undersöka familjers upplevelser av att minimera intaget av socker/snabba kolhydrater. Detta genomfördes med kvalitativa ostrukturerade intervjuer där föräldrarna i familjen fritt fick berätta utifrån olika teman. Dessa teman täckte områden som anledning, tillvägagångssätt, reaktioner, svårigheter, beteenden och eventuella hälsovinster relaterade till kostomläggningen. Utöver dessa familjer valdes tre bloggar ut inom området lågkolhydratkost. Dessa följdes och analyserades som komplement till intervjuerna. Resultaten tolkades utifrån socialkonstruktivistiskt synsätt och teorin om symbolisk interaktionism. De mest framträdande resultaten var att samtliga som ingått i studien har upplevt förbättringar i sin hälsa, i de flesta fall förbättrades även barnens kognitiva förmåga. Vidare upplevde de att traditionen med sötsaker som både belöning och tröst, är djupt rotad vilket innebar att kostomläggningen inte var så enkel att genomföra utan att få höra epitet som "fanatisk" och "extrem". Ingen av intervjupersonerna uppger att de kan tänka sig att återgå till kolhydratrik kost. / The aim of this study was to examine families' experiences of minimizing the intake of sugar/highcarbs. This has been studied with a qualitative unstructured method with the parents in three families from different free to tell based themes. These themes covered areas like reason, procedures, reactions, problems, behaviors and potential health benefits related to their dietary change. In addition to these families, three blogs were selected in the field of low carbohydrate diets. These were followed and analyzed as a supplement to the interviews. The results were interpreted on the basis of social constructivist approach and the theory of symbolic interactionism. The most prominent findings was that all who entered the study have experienced improvements in their health, in most cases also improved children's cognitive ability . Furthermore , they felt that the tradition of sweets as both reward and comfort, made their dietary change implement difficult without hearing the epithet as " fanatical " and " extreme" . None of the interviewees stated that they would be willing to return to the high-carbohydrate diet.

Made available in DSpace on 2016-06-02T19:32:09Z (GMT). No. of bitstreams: 1 5910.pdf: 1437016 bytes, checksum: 8d574c7743c2d70359b23e9e449a0b53 (MD5) Previous issue date: 2014-06-16 / Financiadora de Estudos e Projetos / Phytoplankton cells are base of aquatic food webs, so supporting higher trophic levels and helping equilibrium maintenance in such ecosystems. As residues, nano-TiO2 particles reach aquatic environments where they pose risks to the biota. Literature is controversial about the toxicity of nanoparticles to phytoplankton in general and most data obtained so far has been based on nanoparticles concentrations well above those estimated to occur in natural aquatic environments. Because nanoparticles size vary according to its concentrations, and its toxicity and reactivity are largely dependent on its size, investigations of nanoparticles toxicity at environmentally relevant concentrations are important for the understanding of these compounds onto phytoplankton cells. On the contrary, investigations using higher concentrations can be meaningless. This study aimed at investigating the effects of titanium dioxide nanoparticles (nano-TiO2) at environmentally relevant concentrations (5.10-9 to 5.10- 6 mol L-1) on the freshwater Chlorophyceae Scenedesmus bijugus. We present data of a systematic investigation onto the effects of TiO2 nanoparticles to Scenedesmus bijugus focusing on its physiology under controlled laboratory conditions. We evaluated cell density, chlorophyll a concentrations, growth rates, photosynthetic quantum yield of PSII (continue...) / Células do fitoplâncton são a base de cadeias alimentares aquáticas, apoiando níveis tróficos superiores e ajudando na manutenção do equilíbrio desses ecossistemas. Na qualidade de resíduos, as nano-TiO2 alcançam os ambientes aquáticos onde podem impor riscos à biota. A literatura é controversa sobre a toxicidade de nanopartículas ao fitoplâncton e, em geral, a maioria dos dados têm sido obtidos com base em concentrações de nanopartículas superiores às estimadas para ambientes aquáticos naturais. Considerando que as nanopartículas variam de tamanho de acordo com sua concentração, em decorrência da formação de agregados e, que sua toxicidade e reatividade são dependentes de seu tamanho, investigações sobre a toxicidade desses materiais devem ser feitas empregando-se concentrações ambientalmente importantes, ou seja, aquelas que podem ser encontradas em ecossistemas naturais. Este estudo teve como objetivo investigar os efeitos das nanopartículas de dióxido de titânio (nano-TiO2) na clorofícea de água doce Scenedesmus bijugus em concentrações ambientalmente representativas. Nesta pesquisa, apresentamos dados de uma investigação sistemática sobre os efeitos de nano-TiO2 em amplitude de concentração de 5.10-9 a 5.10-6 mol L-1 em Scenedesmus bijugus, focando em sua fisiologia sob condições controladas de laboratório. Foram avaliadas a densidade de células, as concentrações de clorofila a, taxas de crescimento, rendimento quântico máximo do PSII (continua...)

O controle glicêmico intensificado pode prevenir e/ou retardar o aparecimento das complicações crônicas do diabetes melito (DM). O carboidrato da dieta é o principal determinante da glicemia pós-prandial, sendo o índice glicêmico (IG) e a carga glicêmica (CG) úteis para prever a resposta glicêmica aos alimentos. O objetivo deste manuscrito foi revisar criticamente o papel das dietas de baixo IG na prevenção e controle metabólico do DM tipo 2 (DMT2). O risco para desenvolvimento de DMT2 com dietas de alto IG variou de 1,21 a 1,59. A redução de 12 a 32 unidades no IG da dieta diminuiu em 0,39 a 0,50 pontos percentuais a HbA1c. Os efeitos dessas dietas no perfil lipídico e peso corporal no DMT2 permanecem controversos. Em conclusão, as evidências atuais indicam que a incorporação do IG no planejamento dietético de pacientes com DMT2 contribui para a melhora do controle glicêmico. / The tight glycemic control can prevent and/or delay the development of chronic complications of diabetes mellitus (DM). Dietary carbohydrates are the main determinant of postprandial blood glucose and glycemic index (GI) and glycemic load are used to predict blood glucose response to foods. The aim of this paper was to critically review the role of low GI diets in type 2 DM (T2DM) prevention and metabolic control. The risk for development of T2DM with high GI diets ranged from 1.21 to 1.59.The reduction from 12- 32 units in the GI of diets decreased 0.39-0.50% in HbA1c values. However, the effects of these diets on lipid profile and body weight in patients with T2DM remain controversial. In conclusion, the current evidence indicates that the inclusion of GI in the dietary planning for patients with T2DM contributes to the improvement of glycemic control.