Antibody combining sites are constructed from limited set of germ-line gene segments, yet are capable of both recognizing a broad range of common epitopes and eliciting an adaptive response to newly encountered pathogens. Carbohydrate antigens generally do not draw T cell help and concomitant affinity maturation in the humoral response. Therefore, anti-carbohydrate responses must rely more heavily on the primary germ-line gene repertoire. Antibodies are usually thought of as highly specific. It has been suggested that polyspecificity and cross-reactivity in germ-line antibodies is necessary to provide the protective mechanisms required to broaden the potential number of antigens recognized; however, the molecular mechanism underlying polyspecificity is poorly understood. To investigate the phenomena of specificity, cross-reactivity and polyspecificity in germ-line antibodies my thesis focuses first on the unique LPS inner core oligosaccharide of Chlamydiaceae, which contains variations within the conserved inner core trisaccharide Kdo(2→8)Kdo(2→4)Kdo (3-deoxy-D-manno-oct-2-ulosonic acid). Antibodies raised against this family-specific trisaccharide showed strong V-region restriction with two sets of heavy and light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones as the ‘S25-2 type’ and the ‘S25-23 type’. In contrast to the cross-reactive S25-2 and related antibodies, the S25-23 family of antibodies were shown to be specific for the Chlamydiaceae-specific trisaccharide antigen with no cross-reactivity to Kdo mono or disaccharides or to the Kdo(2→4)Kdo(2→4)Kdo trisaccharide antigen. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen. The structures of the antigen binding fragments of four S25-23-type mAbs have been determined to high resolution in complex with the Chlamydiaceae-specific epitope, revealing the molecular basis for their binding behaviour. The germ-line-encoded paratopes of these antibodies differ significantly from previously characterized S25-2-type mAbs. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the length of the trisaccharide antigen. Further S25-23-type antibodies are glycosylated on the variable heavy chain. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of a Gal(α1→3)Gal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic responses in patients receiving therapeutic antibodies. The second focus of my thesis research is the lipid A moiety of LPS, which is involved in septic shock. Though the lipid A epitope appears to be cryptic during infection with Gram-negative bacteria, there have been several reported instances of lipid A specific antibodies isolated from human sera. While these antibodies are strictly selective for lipid A, there are reports of polyspecificity of some anti-lipid A antibodies for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity has been reported to result in the unfortunate consequences of autoimmune disease. This thesis reports the first crystal structures of antibodies in complex with lipid A and single stranded nucleic acids, elucidating their mechanism for polyspecificity. Perhaps more importantly, the structures may yield clues to the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases. / Graduate / 2020-04-18 / 0487 / 0982

Bakgrund: Preoperativ fasta är nödvändigt innan anestesi för att reducera magsäckens innehåll och därmed minska risken för aspiration. Trots att internationella riktlinjer rekommenderar två timmars fasta för klar vätska är det många sjukhus som fortfarande använder sig av rutinmässig praxis att låta patienter fasta från midnatt. Detta kan innebära en onödigt lång fasta som ger patienten komplikationer i form av sekundära biverkningar och insulinresistens. Syfte: Att undersöka preoperativ omvårdnad i samband med fasta inför kirurgi. Metod: En litteraturstudie med beskrivande design bestående av 14 utvalda originalartiklar från databaserna Pubmed och Cinahl användes. Den teoretiska referensramen för denna studie var Katie Erikssons omvårdnadsteori gällande förståelse av lidande och lidandets drama. Resultat: Många patienter fastade längre än American Society of anesthesiologists (ASAs) rekommenderade riktlinjer. Att fasta längre än två timmar innan operation minskade inte risken för aspiration och gav inte någon mindre volym av magsäckens innehåll hos patienterna. Att ge patienter kolhydratrik dryck innan operation ökade patienternas pre- och postoperativa välbefinnande och innebar inga risker. Slutsats: Genom ett mer flexibelt arbetssätt vid planerad operation kan längden på fastan anpassas efter patientens individuella behov. I samband med planerad kirurgi kan kolhydratrik dryck vara ett bra komplement vid fasta för att minska patientens lidande. Sjuksköterskan är omvårdnadsansvarig och ska arbeta evidensbaserat samt se till patienternas bästa och tillgodose deras behov. Sjuksköterskan kan använda detta som underlag för att minska lidandet i samband med fastan. / Background: Preoperative fasting is necessary before anesthesia to reduce gastric contents and decrease the risk of aspiration. Although international guidelines recommend two hours fasting of liquids, many hospitals still practice nil-by-mouth after midnight. This might give an unnecessarily prolonged fasting which give the patient discomfort and insulin resistance. Aim:  To examine preoperative care in connection with fasting prior to surgery. Method: A literature study with descriptive design based on 14 original articles selected from the databases Pubmed and Cinahl was used. The theoretical frame of reference for this study was Katie Eriksson's nursing theory regarding understanding of suffering and the drama of suffering. Results: Many patients fast longer than American Society of anesthesiologists (ASAs) recommended guidelines. Fasting more than two hours before surgery did not decrease the risk of aspiration and did not decrease the gastric volume. To give patients a high carbohydrate drink before surgery increased the patient's pre- and postoperative comfort. Conclusion: Through a more flexible working method during the planned operation, the length of the fast can be adapted to the patient's individual needs. In conjunction with planned surgery, carbohydrate-rich beverages can be a good complement to fasting to reduce the patient's suffering. The nurse is responsible for nursing care and should work evidence-based and ensure the patients' best and meet their needs. The nurse can use this as a basis for reducing the suffering associated with fasting.

β-1,3-glucanases são enzimas encontradas em muitos organismos, como fungos, bactéria e plantas. Suas funções incluem remodelamento de parede celular, defesa e digestão. É um alvo interessante para o controle populacional de insetos-praga, porque é ausente em vertebrados. Em insetos, é encontrada no intestino de muitas ordens diferentes, e hidrolizam β-1,3 ou β-1,3(4)-glucanas ingeridas, mas pouco se sabe sobre as propriedades e a função dessas enzimas. Nós estudamos três espécies de insetos, de três ordens diferentes. Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) e Abracris flavolineata (Orthoptera) são insetos herbívoros e pestes de plantações, mas suas beta-1,3-glucanases diferem significativamente. A β- 1,3-glucanase de S. frugiperda (SLAM) foi purificada do intestino médio da larva. Ela apresenta uma massa molecular de 37,5 kDa, um pH ótimo alcalino de 9,0, é ativa contra β-1,3-glucana (laminarina), mas é incapaz de hidrolizar β-1,3-1,6-glucana de levedura ou outros polisacarídeos. SLAM é não-processiva (0,4), e não é inibida por altas concentrações de substrato. Diferente de outras β-1,3-glucanases digestivas de insetos, SLAM é incapaz de lisar células de Saccharomyces cerevisae. O cDNA correspondente à SLAM foi clonado e sequenciado, demonstrando que a proteína pertence à família 16 das Glicosídeo-Hidrolases. A modelagem tridimensional de SLAM, feito com base em homologia de sequência, sugere que o resíduo E228 possa afetar a ionização dos resíduos catalíticos, causando o deslocamento do pH ótimo da enzima. Anti-corpos específicos para SLAM foram produzidos, e estes reagem com uma única proteína oriunda do intestino médio da larva, responsável pela atividade β-1,3- glucanásica majoritária. A imunocitolocalização de SLAM demonstra que a enzima é encontrada em vesículas secretórias e no glicocálix das células colunares, e portanto não é originária de simbiontes. Nós clonamos e sequenciamos o cDNA correspondente à β-1,3-glucanase majoritária presente no intestino médio da larva de T. molitor (TLAM). Ela pertence à família 16 das Glicosídeo-Hidrolases e está relacionada com proteínas ligantes de β -glucanas, da mesma forma que a enzima de S. frugiperda. A modelagem tridimensional por homologia de sequência permitiu identificar alguns resíduos de amino-ácidos (E174, E179, H204, Y304, R127 e R2181) no sítio ativo da enzima, que podem ser importantes para a atividade de TLAM. A β-1,3-glucanase digestiva do gafanhoto Abracris flavolineata (Orthoptera) é diferente das enzimas já estudadas em insetos. Ela apresenta uma estratégia catalítica processiva, liberando glicose como maior parte dos produtos, e é inibida por altas concentrações de substrato. Para estudar as bases estruturais desse mecanismo, nós procuramos obter a sequência de cDNA correspondente à enzima já caracterizada. O alinhamento múltiplo das β-1,3- glucanases de insetos e proteínas ligantes de beta-glucanas indicou que uma duplicação gênica da enzima do ancestral comum de moluscos e artrópodes. Uma cópia originou as beta-1,3-glucanase de insetos, perdendo uma região N-Terminal com cerca de 100 pares de bases, enquanto a outra cópia originou as proteínas ligantes de beta-glucana, perdendo os resíduos catalíticos. / β-1,3-glucanases are widespread enzymes, found in all major groups of invertebrates, fungi, bacteria and plants. Since this enzyme is absent in vertebrates, it constitutes an interesting target for control of insect pests population. Its functions range from cell wall remodeling, defense and digestion. In insects, it is found in the gut of many different orders, hydrolyzing β-1,3 or β-1,3(4)-glucanas, but little is known about the properties and function of these enzymes. We studied three insect species each pertaining to a different order. Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) and Abracris flavolineata are herbivores and crops pests, but their β-1,3- glucanases differ significantly. S. frugiperda β -1,3-glucanase (SLAM) was purified from the larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3- glucanases from insects, SLAM is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLAM was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. SLAM homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLAM antiserum reacts with a single protein in the insect midgut. Immunocytolocalization reveals the presence of the enzyme in secretory vesicles and glycocalyx from columnar cells. We cloned and sequenced the cDNA of T. molitor β-1,3-glucanase. It belongs to glycoside hydrolase family 16, and is related to other insect glucanases and glucan-binding proteins. Sequence analysis and homology modeling allowed the identification of some residues (E174, E179, H204, Y304, R127 and R181) at the active site of the enzyme, which may be important for TLAM activity. The grasshopper A. flavolineata has a β-1,3-glucanase with a processive catalytic strategy. To study the structural basis of this property, we aimed to obtain its encoding sequence to better understand this catalytic mechanism. Multiple sequence alignment of insects β-1,3-glucanases and β-glucan-binding protein indicates that the β- 1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the insect β-1,3-glucanases after losing a 100 bp N-terminal portion and the arthropode β-glucan-binding proteins by the loss of the catalytic residues.

<p>Lignocellulose is an ubiquitous source of fixed carbon that is presently underexploited for renewable energy technologies. Currently, producing enzyme cocktails that robustly degrade these feedstocks is a significant economic bottleneck. Anaerobic gut fungi native to the digestive tracts of ruminants and hindgut fermenters are widely understudied despite their inherent ability to degrade a significant portion (~50%) of the lignocellulose in herbivorous animals. Challenges in cultivation due to their strict oxygen sensitivity, and the lack of a central repository to maintain axenic stocks substantially impede the progress with anaerobic fungi. Yet, these microbes have evolved elegant strategies and may harbor novel biomass degrading enzymes that could be used to more efficiently hydrolyze lignocellulose. Developing these organisms through characterization and genome engineering will yield significant contributions to the bioenergy community by improving hydrolysis technologies.</p> <p>In this work, we report the isolation of four novel species of anaerobic gut fungi. A more complete characterization of one of our four fungal isolates is investigated, whereby the effects of substrate composition and the corresponding fungal growth rates are compared. I also explore the growth of one of our fungal isolates on transgenic poplar to understand how fungal growth and enzyme secretion adapt to variable lignin composition. Notably, no significant reductions in growth were observed highlighting the ability of anaerobic fungi to degrade diverse feedstocks regardless of lignin composition. I have additionally included preliminary work intended to identify what epigenetic regulational strategies exist for anaerobic fungi, and how they relate to carbohydrate active enzyme expression. We hope to leverage this knowledge to engineer base enzyme cocktails that release significant portions of the fermentable sugars in untreated or mildly treated plant biomass as a means to make bioenergy technologies more efficient.</p>

Submitted by THIAGO YUKIO NITTA (thiago.nitta@unesp.br) on 2019-01-29T12:58:15Z No. of bitstreams: 1 TESE_Thiago Y. Nitta.pdf: 1430317 bytes, checksum: f0157f184021dc8ba21a719787b120cd (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2019-01-29T13:35:03Z (GMT) No. of bitstreams: 1 nitta_ty_dr_bot.pdf: 1430317 bytes, checksum: f0157f184021dc8ba21a719787b120cd (MD5) / Made available in DSpace on 2019-01-29T13:35:03Z (GMT). No. of bitstreams: 1 nitta_ty_dr_bot.pdf: 1430317 bytes, checksum: f0157f184021dc8ba21a719787b120cd (MD5) Previous issue date: 2019-01-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo do presente estudo foi avaliar os efeitos fisiológicos e metabólicos da inclusão dietética de matodextrina na dose de 1g/kg em equinos submetidos a dois testes padrão de exercícios progressivos realizados em esteira de alta velocidade, T1 e T2. Para tanto, foram utilizados oito equinos da raça Puro Sangue Árabe, distribuídos de forma que todos animais passaram por dois grupos, com intervalo entre eles de 7 dias: Grupo maltodextrina (GM n=8), que ingeriram 1g de maltodextrina + veículo, 45 min antes de cada teste e Grupo controle (GC n=8) que ingeriram veículo (1g/kg de farelo de trigo + 0,1g/kg de óleo de soja) 45 min antes de cada teste. O teste padrão de exercício progressivo (TPEP) consistiu em inclinação da esteira a +6%, com velocidade inicial de 1,8 m/s por 5min, a 4 m/s por 3min, a 6 m/s por 2 min, seguidos de fases a 7, 8 e 9 m/s por dois minutos cada, de modo que a manta da esteira foi parada quando os cavalos não conseguiram acompanhar a velocidade, mesmo sendo estimulados. Após o término do exercício o animal foi monitorado quanto a concentração de glicose plasmática e lactato sanguíneo, além dos valores da frequência cardíaca e índices de desempenho: distância percorrida, V200 e V4. Para análise estatística os os animais foram considerados como blocos, para diminuir o efeito da variabilidade devido ao desempenho de cada animal. A normalidade das variáveis-resposta foi confirmada utilizando o teste de Shapiro-Wilk e o teste de Tukey. Análise estatística através de modelos mistos demonstrou que não houve diferença estatística dos valores de glicose plasmática, lactato e índices de desempenho entre GM e GC e nem entre T1 e T2. Desta forma, o consumo voluntário de maltodextrina na dose de 1g/kg não influenciou na glicemia, lactatemia e nos índices de desempenho atlético nos dois testes padrão de exercício progressivo em um intervalo de seis horas entre eles e equinos submetidos a dois testes de exercício progressivo (TPEP) máximo, realizados em esteira ergométrica de alta velocidade, com intervalo de 6 horas não apresentam variação nos valores de glicose, lactato e índices de desempenho atlético. / The aim of this search was to evaluate the effect of the voluntary intake 1g/kg matodextrin in horses submitted to two standard tests of progressive exercises performed on a high speed treadmill. Used eight geldings Arabian horses, distributed in such a way that all animals underwent two treatments, with a 7-day interval between treatments: Group maltodextrin (GM n=8), which ingested 1g of maltodextrin + vehicle, 45 min before each test and Control group (GC n=8) who ingested vehicle (1g/kg of wheat bran + 0.1g / kg of soybean oil) 45 min before each test. The progressive exercise test (TPEP) consisted of inclination of the treadmill at + 6%, initial velocity of 1.8m / s for 5min, at 4m / s for 3min, at 6m / s for 2min, followed by phases at 7 , 8 and 9 m / s for two minutes each, so that the mat of the mat was stopped when the horses could not keep pace, even though they were stimulated. After completing the exercise, the animal was monitored for glycemia and lactatemia, as well as heart rate and performance indexes: distance traveled, V200 and V4. The statistical design for the assessment of blood lactate levels consisted of an experimental design, considering the animals as blocks, to decrease the variability due to the performance of each animal. The normality of the response variables was confirmed using graphical evaluations and the ShapiroWilk test. The Tukey test was used in the multiple comparisons to verify which means had significant differences at different moments. Statistical analysis using mixed models showed that there was no statistical difference in the values of plasma glucose, lactate and speed indexes. Thus, voluntary consumption of 1 g / kg maltodextrin did not ix influence glycemia, lactatemia and athletic performance indices in the two standard progressive exercise tests in a six-hour interval between them

Dentre a gama de estudos existentes acerca das respostas de plantas ao incremento de CO2 atmosférico associado às mudanças climáticas, são poucas as investigações que contemplam espécies amazônicas frente à relevância desse ecossistema, e se desconhecem estudos desse aspecto com palmeiras. O açaí (Euterpe oleracea Mart., Arecaceae) é uma espécie típica de planícies da Floresta Amazônica sujeitas ao regime anual de inundação e, além de ser extremamente tolerante à anóxia, apresenta elevado valor comercial e um potencial para a produção de energia a partir de biomassa, gerando uma exploração economicamente sustentável. Este trabalho buscou caracterizar o crescimento inicial de plântulas de açaí quanto à fotossíntese, crescimento e metabolismo de carboidratos e investigar as respostas desses parâmetros ao incremento de CO2 atmosférico (de 380ppm - ambiente para 760ppm - elevado) em duas escalas temporais: ao longo do desenvolvimento das plântulas (entre 105 e 195 dias após a germinação, período de desenvolvimento da segunda folha) e ao longo de 24 horas (aos 175 dias após a germinação). Foram analisadas medidas de altura, área foliar, acúmulo e alocação de biomassa, curvas de resposta da fotossíntese à luz, conteúdo de clorofila e concentração de carboidratos não-estruturais. Foi verificado que o período de estabelecimento das plântulas se estende até cerca de 150 dias após a germinação e se sobrepôs ao experimento com elevado CO2. A presença de outra via de entrada de carbono além da fotossíntese tamponou mas não inibiu os efeitos do aumento de CO2 atmosférico. Foram observadas ao longo do experimento reduções na área foliar, condutância estomática e respiração no escuro e aumentos na fotossíntese e eficiência de uso da água, além de aumento na concentração de carboidratos não-estruturais e na biomassa. Os dados obtidos após o esgotamento das reservas do endosperma não apresentaram sinais de que a planta aclimatará ao incremento de CO2, pois aos 90 dias de experimento foram observadas as maiores porcentagens de aumento na fotossíntese, 89%, e no teor de amido, 300%. Esses resultados indicam que essa espécie apresenta uma grande capacidade de aumento da força de dreno, o que diminui a sinalização por açúcares possibilitando a manutenção de uma resposta positiva ao incremento de CO2 atmosférico / Despite all knowledge available nowadays on plant responses to increasing atmospheric CO2, few are the studies that focus on Amazonian species in contrast to the biological relevance of this ecosystem, and no record has been found of Palm species analysis on this area of research. Açaí palm (Euterpe oleracea Mart., Arecaceae) is a typical species of Amazon plains subjected to annual periods of flooding. Apart from being extremely anoxia tolerant, this species presents high commercial value and a potential of energy production through biomass utilization, counting towards a sustainable economic exploitation. This study aimed to characterize the initial growth of açaí palm seedlings as for photosynthesis, growth and carbohydrate metabolism and to investigate these parameters\' responses to an increase on atmospheric CO2 (from 380ppm - ambient to 760ppm - elevated) on two temporal scales: throughout seedling development (from 105 to 195 days post germination, time span of second leaf development) and throughout 24 hours (at 175 days post germination). Analysis included: height, leaf area, biomass increase and allocation, light-response curves, chlorophyll content and non-structural carbohydrate concentration. It was observed that the seedling establishment period extends to 150 days post germination and overlapped the elevated CO2 experiment. The presence of another carbon entry pathway besides photosynthesis buffered the effects of elevated CO2, but did not inhibit them. Results observed were reductions on leaf area, stomatal conductance and dark respiration and increases on photosynthesis, water use efficiency, non-structural carbohydrate content and biomass. The results obtained after seed reserves were depleted did not seem to indicate that this species will present acclimation to elevated CO2, since the greater percentages of increase on photosynthesis (89%) and starch content (300%) occurred at the end of the experiment (90 days on elevated CO2). These results indicate that açaí palm seedlings have a large capacity of sink strenght increase, which reduces sugar signaling and maintains a positive response to the increase on atmospheric CO2

As galectinas, proteínas que compartilham características como afinidade por ?-galactosídeos e a presença de um domínio de reconhecimento ao carboidrato (CRD), tem como importante papel a decodificação de mensagens moleculares. As galectinas são encontradas em uma grande variedade de células e tecidos animais e estão envolvidas em diversos processos celulares relacionados a resposta imune e inflamatória. O grupo tandem-repeat das galectinas é formado por proteínas que apresentam dois CRDs distintos (CRD1 e CRD2) conectados por um peptídeo de ligação, ao qual pertencem as galectinas-4 e -12 humanas, alvos de nosso estudo. Durante o desenvolvimento do presente projeto foram utilizadas abordagens multidisciplinares através de técnicas biofísicas, cristalográficas, computacionais e imunoquímicas. Foi iniciado o estudo de caracterização estrutural da galectina-4 humana (hGal4) e seus domínios hGal4-CRD1 e hGal4-CRD2, de forma independente, através da produção heteróloga das proteínas e ensaios de estabilidade térmica e cristalização. As estruturas cristalográficas dos domínios foram determinadas a 1,48 e 2,46 Å de resolução, respectivamente e utilizadas para a construção de um modelo para a hGal4. Com base nos estudos de dinâmica molecular e estabilidade térmica foi possível propor um modelo de interação entre os CRDs através do peptídeo de ligação. Ensaios de dinâmica da hGal4 com LacNAc sugeriram uma diferença de plasticidade dos CRDs no reconhecimento do ligante. Foram também realizadas incessantes tentativas de desenvolver um protocolo de expressão heteróloga para as isoformas e domínios da galectina-12 humana (hGal12). Como resultado, observamos uma alta tendência à formação de corpos de inclusão e agregados resistentes a desnaturação, além da susceptibilidade ao ataque proteolítico. Os modelos estruturais dos domínios da hGal12 não permitiram identificar nenhuma característica que justificasse o comportamento das proteínas. Foram realizados estudos de localização celular em células HL-60 e foi possível identificar a presença da hGal12 na superfície das gotas de lipídio, organelas responsáveis pelo armazenamento de energia e o processo de catabolismo celular. A alta insolubilidade observada para as isoformas e domínios da hGal12, a sua localização em gotas de lipídeos, a divergência evolutiva da hGal12 quando comparada com outras galectinas do mesmo grupo e incluindo o fato de um dos CRDs não apresentar os requerimentos estruturais básicos para ligar ?-galactosídeos, nos leva a especular se essa proteína estaria, na verdade, sendo expressa como parte de um heterocomplexo proteico, que estabilizaria a estrutura da hGal12 e a endereçaria para as gotas de lipídeo. Em nossa hipótese, o domínio hGal12-CRD2 poderia ter evoluído para favorecer a interação com outros parceiros macromoleculares. Devido ao importante papel desempenhado pelas galectinas em inúmeros processos celulares, os resultados aqui obtidos representam uma importante contribuição no entendimento do papel que as galectinas hGal4 e hGal12 exercem nos diferentes eventos celulares, além de fornecem ferramentas experimentais para desenvolvimento de futuros estudos funcionais. / Galectins are proteins that share characteristics such as affinity for ?-galactosides and the presence of a carbohydrate recognition domain (CRD). They belong to a family of proteins that display the important role of decoding molecular messages. Galectins are found in a variety of cell types and are involved in several biological phenomena related to immune response and inflammation. The tandem-repeat group of galectins consists of proteins with two distinct CRDs (CRD1 and CRD2) connected by a peptide linker, in which belong galectins-4 and -12, targets of our study. During the development of the present project a multidisciplinary approach was used including the use of biophysical, crystallographic, computational and immunochemical techniques. The structural characterization of human galectin-4 (hGal4) and its hGal4-CRD1 and hGal4-CRD2 independent domains was initiated by their heterologous protein production, thermal stability and crystallization assays. The crystallographic structure of domains were determined at 1.48 e 2.46 Å resolution, respectively, and used for constructing a model for hGal4. Based on molecular dynamics and thermal stability studies we propose a model of interaction between CRDs mediated by linker peptide. Dynamics simulation of hGal4 with LacNAc suggested a difference in plasticity between CRDs and ligand recognition. In addition, several attempts have been made towards the development of a protocol for expression of isoforms and independent domains from human galectin-12 (hGal12). As result, we observed a high tendency to body inclusion formation and denaturing resistant aggregates, besides high susceptibility to proteolytic attack. Structural models for the hGal12 domains did not allow the identification of any feature to justify proteins behavior. Cell location studies in HL-60 cells were performed and hGal12 was found to be located on the surface of lipid droplets, organelles responsible for energy storage and catabolism. The high insolubility displayed by isoforms and domains from hGal12, together with its location on lipid droplets, the evolutionary divergence of hGal12 compared to tandem-repeat proteins, and the fact that hGal12-CRD2 does not present the structural requirements for ?-galactosides binding, suggest that hGal12 is, in fact, expressed as part of a protein complex that could both stabilize hGal12 structure and guide it to the lipid droplets. In our hypothesis, the hGal12-CRD2 could have evolved in order to favor the interaction with other macromolecular partners. Due to crucial roles displayed by galectins in innumerous biological process, we believe that our results represent an important contribution for the understanding of hGal4 e hGal12 proteins roles within the cell, besides providing experimental tools for the development of further functional studies.

O objetivo desse estudo foi investigar a associação da inflamação com a resistência periférica à insulina (RI) na vigência de dietas obesogênicas, suplementação com óleo de peixe (SOP) e ausência de TLR-4. Os animais receberam dieta balanceada (DB), DB e leite condensado (DB+LC), hiperlipídica (HFD) ou HFD+LC. DB+LC e HFD levaram ao aumento: no ganho de massa corpórea (MC) e expressão de F4/80 e leptina no tecido adiposo (TA) em relação à DB. Em relação à HFD, HFD+LC provocou: aumento no HOMA-IR (4x), nos conteúdos de IL-6, IL-10, TNF-a, IL-1b, ICAM-1, VCAM-1 e leptina no TA. Comparada à DB+LC, HF+LC aumentou: a MC, HOMA-IR e expressão de F4/80 e reduziu a expressão de leptina no TA. A SOP nos animais em DB reduziu a expressão de F4/80, TNF-a e TLR-4 no fígado. A SOP em HFD reduziu o MC, HOMA-IR e aumentou o conteúdo de adiponectina no TA. A deleção de TLR-4 elevou o MC em DB+OP e a gordura mesentérica no HFD+OP. Assim, o consumo elevado de gordura ou açúcar causou obesidade e a associação de ambos potencializou a obesidade e induziu RI e inflamação no TA. A suplementação com OP atenuou a inflamação associada à DB e a obesidade e RI na vigência de HFD, sendo esses efeitos dependentes de TLR-4. / The aim of this study was to investigate the association between inflammation and insulin resistance (IR) under obesogenic diets, fish oil supplementation (FOS) and TLR-4-/-. The mice were fed a balanced diet (BD), BD and condensed milk (BD+CM), high-fat diet (HFD) or high-fat diet and condensed milk (HFD+CM). The BD+CM and HFD increased: body weight gain (BWG), F4/80 and leptin in adipose tissue (AT). The HFD+CM compared to HFD increased: HOMA-IR, the contents of IL-6, IL-10, TNF-a, IL-1b, ICAM-1, VCAM-1 and leptin in AT. As compared to BD+CM, HFD+CM increased: BWG, HOMA-IR, F4/80 and reduced the leptin in AT. The FOS+BD reduced F4/80, TNF-a and TLR-4 in the liver. The FOS+HFD reduced BWG, HOMA-IR and increased the adiponectin in AT. TLR-4 deletion increased BWG in the FOS+BD and mesenteric fat in the FOS+HFD groups. The high consumption of fat or sugar induced obesity and the association of both potentiated this effect leading to IR and inflammation in adipose tissue and perivascular fat. FO supplementation attenuated the inflammation when associated with BD and the obesity and IR induced by HFD in a TLR-4 dependent-manner.

As enzimas exógenas são mecanismos de redução de custos e otimização da eficiência do uso de ingredientes aumentando a digestão e reduzindo a excreção de nutrientes ao ambiente. Desta forma, foram realizados dois experimentos para avaliar o efeito da adição de enzima exógena sobre o desempenho e qualidade dos ovos de poedeiras comerciais. O primeiro estudo teve como intuito avaliar os efeitos da dieta com nutrientes reduzidos e suplementados com fitase para poedeiras comerciais de 70 a 86 semanas de idade sobre as características de desempenho, qualidade do ovo e parâmetros econômicos. As poedeiras comerciais Novogen White ® (n = 256) foram distribuídas de acordo com um delineamento inteiramente casualizado em quatro tratamentos, com oito repetições de oito galinhas cada: controle positivo (CP): dieta convencional, não suplementada com fitase; dieta com níveis reduzidos de P (-0.12%), Ca (-0.10%) e ME (-14 kcal / kg) e suplementados com 300 FTU fitase / kg (CN300FTU); dieta com níveis reduzidos de P (-0,16%), Ca (-0,13%), ME (-18 kcal / kg), CP (-18%), aminoácidos sintéticos (-0,01%) e suplementados com 600 FTU fitase / kg (RN600FTU); e dieta com níveis reduzidos de P (-0.18% P), Ca (-0.15%), ME (-20 kcal / kg), CP (-20%), aminoácidos sintéticos (-0,01%) e suplementados com 900 FTU fitase / kg (CN900FTU). Durante o experimento foi coletado os dados desempenho, a qualidade dos ovos e os dados de análise econômica. As poedeiras alimentadas com a dieta CN300FTU apresentaram uma produção de ovos 2,68% maior que as alimentadas com a dieta CP. As poedeiras alimentadas com CP tiveram os ovos mais pesados (66,80 g). A massa de ovos produzida por poedeiras alimentadas com CP e CN300FTU foi estatisticamente similar. As características de qualidade dos ovos não foram influenciadas pelos tratamentos dietéticos, com exceção da espessura da casca do ovo, cujo maior valor foi obtido nas poedeiras alimentadas por CP. O custo da ração da dieta CN900FTU foi aproximadamente 9% menor do que o da dieta CP. Este estudo mostrou que as poedeiras alimentadas com uma dieta de nutrientes reduzidos e suplementados com fitase de 300FTU produziram o melhor desempenho e resultados econômicos. No segundo experimento, foi avaliado os efeitos dos níveis de energia metabolizável e suplementadas com complexo enzimático para poedeiras comerciais, de segundo ciclo, de 84 a 100 semanas de idade em seu desempenho, qualidade do ovo e parâmetros econômicos. As poedeiras comerciais Hy-line White ® (n = 224) foram distribuídas de acordo com um delineamento inteiramente casualizado em esquema fatorial 2 X 2, dois níveis de energia metabolizável (2.626, 2.776 Kcal de energia metabolizável) e com e sem suplementação de carboidrase (5g/t), totalizando quatro tratamentos com sete repetições. Houve interação entre o nível de energia e o complexo enzimático para produção e massa de ovo. Sendo, maior produtividade e massa de ovos foi para as poedeiras alimentadas com maior nível de energia e a suplementada com o complexo enzimático. Conclui-se que a suplementação de enzimas exógenas (fitase e carboidrase) para poedeiras comerciais nos dois experimentos apresentou efeitos satisfatórios para as características zootécnicas e econômicas. / The exogenous enzymes are mechanisms for cost reduction and optimization of the efficiency of the use of ingredients enhancing digestion and reducing the excretion of nutrients. In this way, two independent experiments were performed to evaluate the effect the addition of exogenous enzyme on egg production and quality characteristics of the egg of laying hens. The firty study, the objective of this study was to evaluate the effects of feeding reduced-nutrient diets supplemented with phytase to commercial laying hens from 70 to 86 weeks of age on their performance, egg quality, and economic parameters. Novogen White ® commercial laying hens (n=256) were distributed according to a completely randomized design into four treatments, with eight replicates of eight hens each: Positive control: conventional diet, not supplemented with phytase; diet with reduced levels of P (-0.12%), Ca (-0.10%), and ME (-14 kcal/kg), and supplemented with 300 FTU phytase/kg (RN300FTU); diet with reduced levels of P (-0.16%), Ca (-0.13%), ME (-18 kcal/kg), CP (-18%), synthetic amino acids (-0,01%), and supplemented with 600 FTU phytase/kg (RN600FTU); and diet with reduced levels of P (-0.18% P), Ca (-0.15%), ME (-20 kcal/kg), CP (-20%), synthetic amino acids (-0,01%), and supplemented with 900 FTU phytase/kg (RN900FTU). Performance, egg quality, and economic parameters data were collected during the experiment. The layers fed the RN300FTU diet presented 2.68% higher egg production than those fed the PC diet. PC-fed hens laid the heaviest eggs (66.80g). Egg mass produced by PC- and RN300FTU-fed hens were statistically similar. Egg quality traits were not influenced by the dietary treatments, except for eggshell thickness, which highest value was obtained in the PC-fed hens. The feeding cost of the RN900FTU diet was approximately 9% lower compared with that of the PC diet. This study showed that feeding layers with a reduced-nutrient diet supplemented with 300FTU phytase yielded the best performance and economic results. In the second experiment, study was to evaluate the effects of feeding levels of metabolizable energy diets supplemented with enzymatic complex to commercial laying hens, second cycle, from 84 to 100 weeks of age on their performance, egg quality, and economic parameters. Novogen White ® commercial laying hens (n=256) were distributed according to a completely randomized design into four treatments, with eight replicates of eight hens each: Positive control: conventional diet, not supplemented with phytase. Hy-line White ® commercial laying hens (n= 224) were distributed according to a completely randomized design into factorial arrangement 2 X 2, two levels of metabolizable energy (2,626, 2,776 Kcal of metabolizable energy) and with and without supplementation carbohydrase (0, 5 / t), totaling four treatments with seven repetitions. There was interaction between the energy level and the enzymatic complex for production and egg mass. Being, greater productivity and egg mass was for layers that had the highest energy level and the inclusion of the enzyme complex. It was concluded that the supplementation of exogenous enzymes (phytase and carbohydrase) for commercial laying hens in both experiments presented satisfactory effects for the zootechnical and economic characteristics.

Having access to mild and operationally simple techniques for attaining carbohydrate targets will be necessary to facilitate advancement in biological, medicinal, and pharmacological research. Even with the abundance of elegant reports for generating glycosidic linkages, the stereoselective construction of alpha- and beta-oligosaccharides and glycoconjugates is by no means trivial. In an era when expanded awareness of the impact we are having on the environment drives the state-of-the-art, synthetic chemists are tasked with developing cleaner and more efficient reactions for achieving their transformations. This movement imparts the value that prevention of waste is always superior to its treatment or cleanup. Chapter 1 of this thesis will highlight recent advancement, in this regard, by examining strategies that employ transition metal catalysis in the synthesis of oligosaccharides and glycoconjugates. These methods are mild and effective for constructing glycosidic bonds with reduced levels of waste through utilization of sub-stoichiometric amounts of transition metals to promote the glycosylation. The development of a general and practical method for the stereoselective synthesis of beta-O-aryl-glycosides that exploits the nature of a cationic palladium(II) catalyst, instead of a C(2)-ester directing group, to control the beta-selectivity is described in chapter 2. The beta-glycosylation protocol is highly diastereoselective and requires 2-3 mol % of Pd(CH3CN)4(BF4)2 to activate glycosyl trichloroacetimidate donors at room temperature. The method has been applied to D-glucose, D-galactose, and D-xylose donors with a non-directing group incorporated at the C(2)-position to provide the O-aryl glycosides with good to excellent beta-selectivity. In addition, its application is widespread to electron-donating, electron-withdrawing, and hindered phenols. The glycosylation is likely to proceed through a seven-member ring intermediate, wherein the palladium catalyst coordinates both the C(1)-trichloroacetimidate nitrogen and C(2)-ether oxygen, blocking the alpha-face. As a result, the phenol nucleophile preferentially approaches from the top face of the activated donor, leading to the formation of the beta-O-aryl glycoside. The area of sugar urea derivatives has received considerable attention in recent years because of the unique structural properties and activities that these compounds display. The urea-linkage at the anomeric center is a robust alternative to the naturally occurring O- and N-glycosidic linkages of oligosaccharides and glycoconjugates, and the natural products that have been identified to contain these structures show remarkable biological activity. While methods for installing the beta-urea-linkage at the anomeric center have been around for decades, the first synthesis of alpha-urea glycosides has been much more recent. In either case, the selective synthesis of glycosyl ureas can be quite challenging, and a mixture of alph- and beta-isomers will often result. Chapter 3 provides a comprehensive review of the synthetic approaches to alpha- and beta-urea glycosides and examines the structure and activity of the natural products, and their analogues, that have been identified to contain them. There are only a handful of reports for the construction of beta-urea glycosides, and even fewer that are able to attain the alpha-urea structures. Chapter 4 will cover two of these methods, where a transition metal catalyst is employed to facilitate the alpha-selective transformation. The 1st-generation process, covered in section 4.1, involves the cationic palladium(II)-catalyzed rearrangement of glycal trichloroacetimidate to alpha-glycal trichloroacetamide in its key step. The transformation is carried out with only 0.5 mol% Pd(CH3CN)4(BF4)2 catalyst and is both highly alpha-selective and tolerant to a diverse array of protecting groups. The glycal product of the rearrangement is functionalized to pyranoside, protected, and then converted to glycosyl urea in 3-steps. A diverse array of primary and hindered secondary nitrogen nucleophiles have been coupled with the alpha-acetamide products, generating alpha-urea glycosides with retention of stereochemical integrity at the anomeric center. This is the first synthesis of alpha-glycosyl urea to rely on the nature of the catalyst/ligand complex, rather than substrate, to control selectivity. This method, however, suffers from limitations in scope and a dependence on toxic osmium tetroxide to functionalize the glycal. In section 4.2, the development and mechanistic investigation of a 2nd-generation process, able to overcome the limitations of the glycal methodology to provide an efficient and highly stereoselective access to alpha-urea glycosides, is decribed. This two-step procedure begins with a highly selective nickel-catalyzed conversion of alpha-glycosyl trichloroacetimidate to alpha-trichloroacetamide. The alpha-selectivity in the reaction is controlled with a cationic nickel(II) catalyst, Ni(dppe)(OTf)2. Mechanistic studies have identified a coordination of the nickel catalyst with equatorial C2-ether group of the glycosyl trichloroacetimidate to be paramount for achieving an á-selective transformation. A cross-over experiment has indicated that the reaction does not proceed in an exclusively-intramolecular fashion. The alpha-trichloroacetamide products are directly converted into alpha-urea glycosides by reacting them with a variety of nucleophilic amines in presence of cesium carbonate. Only alpha-urea products are observed, as the reaction retains stereochemical integrity at the anomeric center during the urea-forming step.