Esse estudo tem como hipótese que a variação sazonal da fotossíntese de laranjeira ‘Valência’ está relacionada a fatores ambientais e endógenos. A menor atividade fotossintética durante os meses frios e secos está possivelmente associada à baixa temperatura e ao acúmulo de reservas, podendo ocorrer mesmo em condições de boa disponibilidade hídrica. A alta atividade fotossintética durante os meses quentes e chuvosos é possivelmente induzida pela maior disponibilidade de recursos naturais (água, energia solar e temperatura) e maior demanda por carboidratos, haja vista que nessa ocasião ocorre fluxo vegetativo intenso. O objetivo foi testar a hipótese acima, avaliando a variação anual de algumas características fisiológicas relacionadas à fotossíntese (trocas gasosas, fluorescência da clorofila, potencial de água no ramo e conteúdo foliar de carboidratos) de laranjeiras ‘Valência’ em condições de campo, em Cordeirópolis (SP). A influência da baixa disponibilidade hídrica sazonal foi verificada comparando-se plantas em condições naturais com plantas sob irrigação. A produção e algumas características biométricas foram avaliadas. A maior atividade fotossintética de laranjeiras ‘Valência’ ocorre durante a primavera, sendo induzida pelo aumento da temperatura e disponibilidade hídrica, enquanto que a menor fotossíntese ocorre durante o inverno, quando há baixa temperatura e deficiência hídrica. Durante o inverno, a menor fotossíntese não é associada ao conteúdo total de reservas foliares. Os valores máximos de assimilação de CO2 são observados na primavera, quando as condições ambientais são intermediárias entre o verão e o inverno e há fluxos vegetativo e reprodutivo nas laranjeiras. A alta demanda de carboidratos por esses drenos causa decréscimos no conteúdo de reservas foliares entre outubro e novembro, possivelmente estimulando a fotossíntese na primavera. Embora a fotossíntese das plantas no verão seja maior que no inverno, a atividade fotossintética não alcança valores próximos aos da primavera por limitações de origem bioquímica e estomática. A menor atividade bioquímica é induzida pela maior temperatura do ar em relação à primavera, em especial durante o período vespertino, enquanto que a limitação de origem estomática é causada pela alta demanda evaporativa do ar no verão. Durante o período quente e chuvoso, a atividade fotoquímica se ajusta às condições ambientais e não limita a fixação de carbono. A menor atividade fotossintética durante o inverno é ocasionada pela menor condutância estomática, devida à baixa temperatura do ar e solo. A ocorrência de deficiência hídrica no inverno causa redução do potencial de água nos ramos, diminuindo ainda mais a condutância estomática. No inverno, há limitação bioquímica da fotossíntese devida à baixa temperatura do ar, indicada pela redução da eficiência instantânea de carboxilação. Em relação ao metabolismo fotoquímico, não há indícios de limitação da fotossíntese. / This study has as hypothesis that the seasonal variation of photosynthesis in ‘Valencia’ sweet orange plant is related to environmental and endogenous factors. The lower photosynthetic activity during cold and dry months is possibly associated to low temperature and accumulation of leaf reserves, occurring even in well-watered plants.The higher photosynthetic activity during warm and rainy months is probably induced by higher availability of natural resources (water, sunlight and temperature) and by carbohydrate demand, since there is intense vegetative flush during this period. The objective was to test the above hyphotesis, evaluating the annual variation of some physiological characteristics related to photosynthesis (gas exchange, chlorophyll fluorescence, stem water potential and leaf carbohydrate content) of ‘Valencia’ sweet orange plants under field conditions, in Cordeirópolis (SP). The influence of low seasonal water availability was verified by comparing plants under natural with irrigated plants. Fruit production and some biometric characteristics were evaluated. The highest photosynthetic activity in ‘Valencia’ sweet orange plants occurred in spring, being induced by increase of temperature and water availability, while the lowest photosynthesis occurred in winter, when there is low temperature and water deficiency. During the winter, the lowest photosynthesis is not associated to the total reserve content in leaves. The maximum CO2 assimilation values are observed in spring, when environmental conditions are intermediate between summer and winter and there are vegetative and reproductive flushes in sweet orange plants. The high demand for carbohydrates in those sinks causes reduction in leaf reserve contents between october and november, probably estimulating the photosynthesis in spring. Although the photosynthesis in summer is higher than in winter, the photosynthetic activity does not reach values close to ones observed in spring due to biochemical and stomatal limitations. The low biochemical activity is induced by higher air temperature when compared to spring, mainly during afternoon, while the stomatal limitation is caused by the high air evaporative demand in summer. During the warm and wet period, the photochemical activity adjusts itself to environmental conditions and does not limit the carbon fixation. The low photosynthetic activity during winter is caused by low stomatal conductance as a consequence of low air and soil temperature. The occurrence of water deficiency in winter causes reduction of stem water potential, which is an additional factor leading to reduced stomatal conductance. In winter, there is biochemical limitation of photosynthesis due to low air temperature, as indicated by reduced instantaneous carboxylation efficiency. Regarding the photochemical metabolism, there are not signs of limitation to photosynthesis.

As funções abrangentes e complexas de carboidratos nos sistemas biológicos oferecem muitas possibilidades para explorar essas estruturas no desenvolvimento de estratégias terapêuticas. Como consequência de sua estrutura e função, algumas glicosidases e glicosiltransferases envolvidas em patologias significativas como diabetes e doença de Chagas são alvos interessantes na busca por inibidores enzimáticos baseados em carboidratos. Assim, o conhecimento de características estruturais e mecanismo de ação de ?- e ?-glucosidases e trans-sialidases e as sólidas evidências de que glicosidases são sensíveis a efeito de multivalência sustentaram o planejamento de glycoclusters potencialmente inibidores dessas enzimas contendo três, quatro ou seis unidades correlatas aos seus substratos naturais (glicose e galactose/ácido siálico). Adicionalmente foram propostos glycoclusters baseados na estrutura do glicoaminoácido ?-GalNAc-O-Thr. Empregando reações de cicloadição CuAAC, treze dos dezesseis glycoclusters tetra- e hexavalentes inicialmente propostos foram obtidos em bons rendimentos, sendo 8 derivados de glicose, 4 de galactose e o derivado tetravalente de ácido siálico 7. Dez desses compostos são inéditos na literatura. Com a finalidade de gerar diversidade estrutural o derivado trivalente 29 foi preparado e permitiu o acoplamento de um imino-açúcar D-gluco ou L-gulo DNJ a estruturas trivalentes de glicose. A mesma estratégia levou à obtenção do glycocluster 85, contendo unidades de galactose e um resíduo de ácido siálico. Ensaios de cinética em ?-glucosidases de arroz e Saccharomyces cerevisiae e ?-glucosidase de amêndoas com os glycoclusters derivados de glicose 1-4, 9-12 e 63-66 mostraram atividade interessante apenas para os compostos mistos, evidenciando a importância do imino-açúcar para a interação com essas enzimas. No que diz respeito aos derivados multivalentes de galactose e/ou ácido siálico 5-7, 13, 14 e 85, inibição expressiva da enzima TcTS foi alcançada pelo derivado 7 (IC50 450 ?M). Ensaios de atividade tripanocida, bloqueio de invasão e citotoxicidade sobre fibroblastos de mamífero não-infectados cultivados in vitro evidenciaram atividade de todos esses compostos contra a forma amastigota e bloqueio de invasão celular por T. cruzi (diminuindo a infecção em concentrações de até 25?M). / The comprehensive and elaborate functions of carbohydrates in biological systems offer countless possibilities to apply these structures in the development of therapeutic strategies. Because of their structure and function, some glycosidases and glycosyltransferases involved in pathological conditions such as diabetes and Chagas disease are interesting targets in the search for carbohydrate-based enzyme inhibitors. Thus, the knowledge of structural features and the mechanism of action of ?- and ?-glucosidases and trans-sialidases, combined with strong evidences that glycosidases are sensitive to multivalent effect, have supported the design of substrate-based glycoclusters with potential glycosidases inhibitory properties. In addition, glycoclusters based on the structure of the ?-GalNAc-O-Thr glycoamino acid have been planned. Using CuAAC cycloaddition reactions, thirteen of the sixteen tetra- and hexavalent glycoclusters were obtained in good yields, being 8 glucose derivatives, 4 galactose, and the tetravalent sialic acid derivative 7. Ten of these compounds are unpublished. In order to generate structural diversity, the trivalent block 29 was prepared and allowed the coupling of trivalent glucose clusters to iminosugars D-gluco or L-gulo DNJ isomers. The same strategy afforded glycocluster 85, containing galactose units and a sialic acid residue. Kinetic assays in ? and ?-glucosidases with the glucose-derived glycoclusters 1-4, 9-12 and 63-66 showed interesting activity only for the heteroclusters compounds, evidencing the relevance of iminosugars for the interaction with these enzymes. Regarding galactose and/or sialic acid cluster 5-7, 13, 14 and 85, significant inhibition of the TcTS enzyme was achieved by derivative 7 (IC50 450 ?M). In vitro assays for trypanocidal activity and cytotoxicity showed good results for of all these compounds. They appear to block host cell invasion by T. cruzi (reducing infection index in concentrations up to 25?M).

The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives. The characteristics and specificity of CBM1s from theTrichoderma reeseiCel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The twoT. reeseiCBM1s as well as the CBM3 from theClostridium thermocellumCipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face ofValoniacellulose crystals. The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis. The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out inPichia pastoris. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, theT. reeseiCel6A CBM1 was expressed on the surface of thegram-positive bacteria,Staphylococcus carnosus. The engineeredS. carnosuscells were shown to bind cellulosefibers. To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of theS. carnosusand clearly enhanced the metal bindingability of the engineered bacteria. <b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,Trichoderma reesei, Staphyloccus carnosus.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

Carbohydrate recognition is a ubiquitous principle underlying many fundamental biological processes like fertilization, embryogenesis and viral infections. But how carbohydrate specificity and affinity induce a molecular event is not well understood. One of these examples is bacteriophage P22 that binds and infects three distinct Salmonella enterica (S.) hosts. It recognizes and depolymerizes repetitive carbohydrate structures of O antigen in its host´s outer membrane lipopolysaccharide molecule. This is mediated by tailspikes, mainly β helical appendages on phage P22 short non contractile tail apparatus (podovirus). The O antigen of all three Salmonella enterica hosts is built from tetrasaccharide repeating units consisting of an identical main chain with a distinguished 3,6 dideoxyhexose substituent that is crucial for P22 tailspike recognition: tyvelose in S. Enteritidis, abequose in S. Typhimurium and paratose in S. Paratyphi. In the first study the complexes of P22 tailspike with its host’s O antigen octasaccharide were characterized. S. Paratyphi octasaccharide binds less tightly (ΔΔG≈7 kJ/mol) to the tailspike than the other two hosts. Crystal structure analysis of P22 tailspike co crystallized with S. Paratyphi octasaccharides revealed different interactions than those observed before in tailspike complexes with S. Enteritidis and S. Typhimurium octasaccharides. These different interactions occur due to a structural rearrangement in the S. Paratyphi octasaccharide. It results in an unfavorable glycosidic bond Φ/Ψ angle combination that also had occurred when the S. Paratyphi octasaccharide conformation was analyzed in an aprotic environment. Contributions of individual protein surface contacts to binding affinity were analyzed showing that conserved structural waters mediate specific recognition of all three different Salmonella host O antigens. Although different O antigen structures possess distinct binding behavior on the tailspike surface, all are recognized and infected by phage P22. Hence, in a second study, binding measurements revealed that multivalent O antigen was able to bind with high avidity to P22 tailspike. Dissociation rates of the polymer were three times slower than for an octasaccharide fragment pointing towards high affinity for O antigen polysaccharide. Furthermore, when phage P22 was incubated with lipopolysaccharide aggregates before plating on S. Typhimurium cells, P22 infectivity became significantly reduced. Therefore, in a third study, the function of carbohydrate recognition on the infection process was characterized. It was shown that large S. Typhimurium lipopolysaccharide aggregates triggered DNA release from the phage capsid in vitro. This provides evidence that phage P22 does not use a second receptor on the Salmonella surface for infection. P22 tailspike binding and cleavage activity modulate DNA egress from the phage capsid. DNA release occurred more slowly when the phage possessed mutant tailspikes with less hydrolytic activity and was not induced if lipopolysaccharides contained tailspike shortened O antigen polymer. Furthermore, the onset of DNA release was delayed by tailspikes with reduced binding affinity. The results suggest a model for P22 infection induced by carbohydrate recognition: tailspikes position the phage on Salmonella enterica and their hydrolytic activity forces a central structural protein of the phage assembly, the plug protein, onto the host´s membrane surface. Upon membrane contact, a conformational change has to occur in the assembly to eject DNA and pilot proteins from the phage to establish infection. Earlier studies had investigated DNA ejection in vitro solely for viruses with long non contractile tails (siphovirus) recognizing protein receptors. Podovirus P22 in this work was therefore the first example for a short tailed phage with an LPS recognition organelle that can trigger DNA ejection in vitro. However, O antigen binding and cleaving tailspikes are widely distributed in the phage biosphere, for example in siphovirus 9NA. Crystal structure analysis of 9NA tailspike revealed a complete similar fold to P22 tailspike although they only share 36 % sequence identity. Moreover, 9NA tailspike possesses similar enzyme activity towards S. Typhimurium O antigen within conserved amino acids. These are responsible for a DNA ejection process from siphovirus 9NA triggered by lipopolysaccharide aggregates. 9NA expelled its DNA 30 times faster than podovirus P22 although the associated conformational change is controlled with a similar high activation barrier. The difference in DNA ejection velocity mirrors different tail morphologies and their efficiency to translate a carbohydrate recognition signal into action. / Kohlenhydraterkennung ist ein fundamentales Prinzip vieler biologischer Prozesse wie z.B. Befruchtung, Embryogenese und virale Infektionen. Wie aber Kohlenhydratspezifität und –affinität in ein molekulares Ereignis übersetzt werden, ist nicht genau verstanden. Ein Beispiel für ein solches Ereignis ist die Infektion des Bakteriophage P22, der drei verschiedene Salmonella enterica (S.) Wirte besitzt. Er erkennt und depolymerisiert die repetitiven Einheiten des O Antigens im Lipopolysaccharid, das sich in der äußeren Membran seines Wirtes befindet. Dieser Schritt wird durch die Tailspikes vermittelt, β helicale Bestandteile des kurzen, nicht kontraktilen Schwanzapparates von P22 (Podovirus). Das O Antigen aller drei Salmonella enterica Wirte besteht aus sich wiederholenden Tetrasacchariden. Sie enthalten die gleiche Hauptkette aber eine spezifische 3,6 Didesoxyhexose Seitenkette, die für die P22 Tailspikeerkennung essentiell ist: Tyvelose in S. Enteritidis, Abequose in S. Typhimurium und Paratose in S. Paratyphi. Im ersten Teil der Arbeit wurde die Komplexbildung von P22 Tailspike mit O Antigen Octasaccharidfragmenten der drei verschiedenen Wirte untersucht. S. Paratyphi Octasaccharide binden mit einer geringeren Affinität (ΔΔG≈7 kJ/mol) an den Tailspike als die beiden anderen Wirte. Die Kristallstrukturanalyse des S. Paratyphi Octasaccharides komplexiert mit P22 Tailspike offenbarten unterschiedliche Interkationen als vorher mit S. Enteritidis und S. Typhimurium Oktasaccharidkomplexen mit Tailspike beobachtet wurden. Diese unterschiedlichen Interaktionen beruhen auf einer strukturellen Änderung in den Φ/Ψ Winkeln der glykosidischen Bindung. Die Beiträge von verschiedenen Proteinoberflächenkontakten zur Affnität wurden untersucht und zeigten, dass konservierte Wasser in der Struktur die spezifische Erkennung aller drei Salmonella Wirte vermittelt. Obwohl die verschiedenen O Antigen Strukturen unterschiedliches Bindungsverhalten auf der Tailspikeoberfläche zeigen, werden alle vom Phagen P22 erkannt und infiziert. Daher wurde in einer zweiten Studie die multivalente Bindung zwischen P22 Tailspike und O Antigen charakterisiert. Die Dissoziationskonstanten des Polymers waren drei Mal langsamer als für das Oktasaccharid allein, was auf eine hohe Affinität des O Antigens schließen lässt. Zusätzlich wurde gezeigt, dass die Aggregate des Lipopolysaccharids in der Lage sind, die Infektiösität vom P22 Phagen zu reduzieren. Ausgehend davon wurde in einer dritten Studie die Bedeutung der Kohlenhydrat Erkennung auf den Infektionsprozess untersucht. Große S. Typhimurium Lipopolysaccharide Aggregate bewirkten die DNA Freisetzung vom P22 Kapsid. Dies deutet darauf, dass der P22 Phage keinen weiteren Rezeptor für die Infektion auf der Oberflächen seines Wirtes verwendet. Zusätzlich moduliert die P22 Tailspike Aktivität den Ausstoss der DNA vom P22 Phagen: Er ist langsamer, wenn der Phage Tailspikes besitzt, die weniger hydrolytisch aktiv sind und wurde nicht induziert, wenn Lipopolysaccharid eingesetzt wurde, dass zuvor mit Tailspike hydrolysiert wurde. Darüber hinaus wurde der Start der DNA Ejektion verzögert, wenn Tailspikes mit verminderter Affinität am Phagen vorhanden waren. Die Ergebnisse führten zu einem Modell für die Infektion von P22: Tailspikes positionieren den Phagen auf Salmonella enterica und ihre Aktivität drückt ein zentrales Strukturprotein des Phagen, das Stöpselprotein, auf die Membranoberfläche. Aufgrund des Membrankontaktes findet eine Konformationsänderung statt die zur Ejektion der Pilotproteine und zur Infektion führt. Vorhergehende Studien haben bisher nur die DNA Ejektion in vitro für Viren mit langen, nicht kontraktilen Schwänzen (Siphoviren) mit Proteinrezeptoren untersucht. In dieser Arbeit wurde das erste Mal die DNA Ejektion für einen Podovirus mit LPS Erkennung in vitro gezeigt. Die O Antigen Erkennung und Spaltung durch Tailspikeproteine gibt es häufig in der Phagenbiosphere, z.B. am Siphovirus 9NA. Die Kristallstrukturanalyse von 9NA Tailspike zeigt eine komplett gleiche Struktur, obwohl beide Proteine nur zu 36% Sequenzidentität besitzen. Zusätzlich hat 9NA Tailspike ähnliche enzymatische Eigenschaften. Diese ist für den DNA Ejektionsprozess im Siphovirus 9NA verantwortlich, der auch durch LPS Agreggate induziert wird. 9NA stößt dabei seine DNA 30 Mal schneller aus als Podovirus P22 obwohl die damit verbundene Konformationsänderung mit einer ähnlich hohen Aktivierungsbarriere kontrolliert wird. Daher spiegeln die Unterschiede in der DNA Ejektionsgeschwindigkeit der verschiedenen Tailmorphologien die Effezienz wieder, mit der die spezifische Kohlenhydraterkennung in ein Signal umgewandelt wird.

Pseudomonas aeruginosa and Candida albicans have shown antagonistic relationships, both in vivo and in vitro. The interaction between P. aeruginosa and C. albicans is one of the many prokaryotic-eukaryotic interactions existing in nature and needs more research due to their complex pathogenicity in humans. In this work, we have studied growth dynamics of Candida in a mixed population of Pseudomonas and Candida, and their receptor and ligand specificities, both from an ecological and a molecular point of view. Initially, growth, viability, and morphogenesis of Candida were studied in the presence of Pseudomonas and the conditioned medium of Pseudomonas using two Candida strains, namely CAF2 and tup1 mutant. The killing effect of Pseudomonas was more pronounced on the hyphal form of Candida. However, growth of Candida was inhibited by Pseudomonas irrespective of its morphological form. The conditioned medium had no effect on the growth rate of Candida. Nevertheless, it completely inhibited the germination of Candida. The attachment of Pseudomonas to Candida was studied using different strains of both, and with Pseudomonas cells harvested at different stages of its growth. The percent attachment varied with the age of the textit Pseudomonas culture. A lecB mutant of Pseudomonas showed a two-fold reduction in attachment compared to the wild type PAK strain. Carbohydrate inhibition studies confirmed that LecB is not directly involved in the attachment mechanism, but indirectly involves through regulating the expression of other proteins required for attachment. A genomic DNA library of Pseudomonas PAO1 was screened for clones that had acquired the ability to attach to C. albicans. A clone was isolated with a small increase in attachment and was subjected to genetic analysis. It contained nucleotides 458565 to 475917 of the Pseudomonas genome including some genes of the Pil-Chp gene cluster. Seven transposon mutants that represent mutations in ChpA,B,C,D,E operon and three other ORFs were selected, and their attachment ability was tested. All seven mutants showed a reduction in attachment indicating that this was a non specific effect, which could be attributed to the in vitro manipulation of the bacteria. We conclude that the attachment of Pseudomonas to Candida is multi-factorial.

Cell surface saccharides are involved in a variety of essential biological events. Fluorescent sensors for saccharides can be used for detection, diagnosis, analysis and monitoring of pathological processes. The boronic acid functional group is known to bind strongly and reversibly to compounds with diol groups, which are commonly found on saccharides. Sensors that have been developed for the purpose of saccharide recognition have shown great potential. However, they are very hydrophobic and this lack of essential water-solubility makes them useful in biological applications. The first section of this dissertation details the process of developing water-soluble saccharide sensors that change fluorescent properties upon binding to saccharides. The second section of the dissertation focuses on the development of DNA-minor groove binders as antiparasitical agents. Parasitical diseases comprise some of the world’s largest health problems and yet current medication and treatments for these parasitical diseases are often difficult to administer, costly to the patients, and have disruptive side effects. Worse yet, these parasites are developing drug resistance, thus creating an urgent need for new treatments. Dicationic molecules constitute a class of antimicrobial drug candidates that possess high activity against various parasites. The second section details the development of a series of di-cationic agents that were then screened in in vitro activities against parasitical species.

<p>The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives.</p><p>The characteristics and specificity of CBM1s from the<i>Trichoderma reesei</i>Cel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The two<i>T. reesei</i>CBM1s as well as the CBM3 from the<i>Clostridium thermocellum</i>CipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face of<i>Valonia</i>cellulose crystals.</p><p>The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis.</p><p>The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out in<i>Pichia pastoris</i>. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, the<i>T. reesei</i>Cel6A CBM1 was expressed on the surface of thegram-positive bacteria,<i>Staphylococcus carnosus</i>. The engineered<i>S. carnosus</i>cells were shown to bind cellulosefibers.</p><p>To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of the<i>S. carnosus</i>and clearly enhanced the metal bindingability of the engineered bacteria.</p><p><b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,<i>Trichoderma reesei, Staphyloccus carnosus</i>.</p>

Bakgrund: Diabetes typ 2 (DT2) är ett ökande hälsoproblem och sjukdomen innebär att kroppen producerar mindre insulin än kroppens aktuella behov. Övervikt, fysisk inaktivitet, ohälsosamma kostvanor, ärftlighet och stigande ålder är riskfaktorer för att utveckla sjukdomen. För att undvika komplikationer ska ett stabilt blodsocker eftersträvas. Sjuksköterskan ska vara ett stöd och ge kunskap om egenvården, som enligt Orem innebär upprätthållande av hälsa och livskvalitet. Fettsnål kost har under många år rekommenderat. Populariteten kring lågkolhydratskost har under de senaste åren ökat och det förekommer studier som visar att kosten kan vara fördelaktigt för personer med DT2. Syfte: Att beskriva effekter av lågkolhydratkost hos personer med DT2. Metod: Litteraturöversikten har kvantitativ design och innehåller 13 vetenskapliga artiklar. Fribergs analysmodell användes för att analysera artiklarna. Resultat: Lågkolhydratskosten visade en signifikant förbättring i glukos, kroppsmassa och blodfetter. Kosten visade inga stora skillnader mellan variablerna på lång sikt jämfört med andra koster. Det stöd som förekom till deltagarna i studierna visade sig vara betydelsefullt för att uppnå ovanstående effekter och framförallt då det gällde att bevara effekterna över tid. Slutsats: Lågkolhydratskosten medförde positiva effekter på kort sikt och effekterna samvarierade med omfattningen av stöd som ingick i studierna. Trots rädslan för att kosten medför ökad risk för hjärt- och kärlsjukdom påvisade studien inga risker med kosten på kort sikt. Nyckelord: diabetes typ 2, lågkolhydratskost, egenvård, Dorothea Orem, diet. / Background: Diabetes type 2 (DT2) is an increasing health problem. The disease means that the body produces less insulin than the body need. Obesity, physical inactivity, unhealthy diet, heredity and rising age are risk factors for the disease. To avoid complications, a stable blood sugar pursued. The nurse will provide support and give knowledge for self-care, that according Orem means to provide health and quality of life. Low fat diets have for many years recommended. The popularity around low carbohydrate diet has increased in recent years. Studies shows that low carbohydrate can be beneficial for persons with DT2. Aim: To describe the effects of low carbohydrate diets in people with DT2. Method: The literature review with a quantitative design, contains 13 scientific articles. Friberg analysis model was used to analyze the articles. Results: A significant improvement was observed in glucose, body mass and lipids. The diet showed no significant differences between the variables in the long term compared to other diets. The support that occurred to the participants in the studies were found to be significant in order to achieve the above effects and especially when it came to preserve the effects over time. Conclusion: The low carbohydrate diet resulted in short- term positive effects and these effects varied whit the extent of support the studies conducted. Despite fears that the diet increases the risk of cardiovascular disease, studies showed no short-term risks. Keywors: diabetes type 2, low carbohydrate diet, self care, Dorothea Orem, diet

Whole yellow pea flour (WPF) and fractionated yellow pea flour (FPF) are novel functional food ingredients that vary in nutritional composition. Consequently, the health benefits of WPF and FPF remain undefined. The purpose of this research was to identify the effects of WPF and FPF on risk factors and morbidities associated with cardiovascular disease, diabetes and obesity as well as the gastrointestinal microbiome. Using USDA recommended dosages of WPF and FPF, clinical endpoints and the colonic microbiome were investigated using a human clinical trial engaging a cross-over design and a diet and energy controlled paradigm. Humans were also utilized to investigate post-prandial glycemic responses and sensory characteristics of novel functional foods formulated with WPF. Finally, Golden Syrian hamsters were used to assess the impact of high doses of WPF and FPF on clinical endpoints and caecal microbial abundance. Results reveal that USDA recommended dosages of WPF and FPF in humans decreased (p<0.05) fasting insulin and estimates of insulin resistance compared to white wheat flour (WF). Android-to-gynoid fat ratios in women were lower (p=0.027) in the WPF group compared to the WF group. FPF decreased (p<0.05) post-prandial energy expenditure alongside a tendency (p<0.075) to reduce carbohydrate oxidation. Novel biscotti and banana bread formulated with WPF induced low post-prandial glycemic responses which were similar to boiled whole yellow peas and significantly lower (p<0.05) than white bread. Sensory analysis of novel WPF biscotti and banana bread demonstrated that WPF-based food products are palatable and acceptable for human consumption. Hamsters consuming diets containing 10% WPF and FPF induced similar reductions (p<0.05) in fasting insulin levels compared to controls. However, animals consuming WPF increased (p<0.05) oxygen consumption while FPF decreased (p<0.05) fasting glucose levels. In addition, terminal restriction fragment length polymorphism analysis revealed that WPF and FPF induced distinct shifts in caecal microbial populations within the phyla Firmicutes. Finally, pyrosequencing analysis of human fecal microbiota demonstrated that FPF and WPF induced shifts in bacterial genera, primarily within Bacteroidetes and Firmicutes. In conclusion, whole and fractionated yellow pea flours are functional food ingredients and can be utilized to manage risk factors for lifestyle-related diseases in humans.