It is increasingly important to identify the nature of the interfacial water in biology in order to explain how biological functions and systems work. It is not simply a matter of which biomolecules are present in a cell, but also of how these biomolecules interact with one another. This body of work uses neutron scattering techniques to explain the nature of the vibrational dynamics of water interacting with biomolecules and systems that mimic the biological molecular crowding environment of a cell. Recent work in science has seen the synthesis of periodic mesoporous organosilicas with organic groups attached. In the first paper in this thesis, the use of one of these materials is highlighted to look at confined water, equivalent to the water found in a crowded cellular environment. Here it is shown that the properties of the water within the pores and water molecules around the surface were shown to be different and then identified as interfacial and bulk water respectively. In order to develop the investigation of interfacial water with biological matter, it seemed appropriate to start with the most basic molecules, amino acids. The second paper presents a complete survey of the 20 biologically important amino acids using one of the world's highest resolution neutron scattering spectrometer (TOSCA at ISIS, Rutherford Appleton Laboratory). Computer simulation of the experimental work through molecular dynamics, allows many vibrational modes to be assigned for the first time and correlated with the broader vibrational peaks previously observed for proteins. Comparison of the dry states with the hydrated states of amino acids, gives some insight into the sites within the amino acid side chains where water molecules are likely to bind. For serine this is the hydroxyl group in the side chain. The third paper focuses on IINS data of serine in more detail and discusses several low energy vibrational modes that have been assigned and for the first time, shows how the presence of water molecules changes the dynamic behaviour of librational and torsional modes differently. The combination of these studies allows a clearer picture of how water in biology interacts with biomolecules and of the importance of water to our existence.

Pre-eclampsia (PE) is a serious disease affecting approximately 5% of pregnancies per annum. The disease etiology is complex but its origin lies in abnormal placental development and function. PE is associated with inflammation, increased nitrative stress and abnormal renewal of syncytiotrophoblast (STB), the transporting epithelium of the placenta. STB is renewed by cytotrophoblasts (CTBs) that proliferate, differentiate and fuse with STB and this is balanced by apoptosis. The amino acid taurine facilitates proliferation, differentiation and apoptosis in non-placental tissues. Taurine is also cytoprotective, protecting cells from damage by inflammatory cytokines. Taurine is transported from maternal blood into STB by the amino acid transporter TauT. In isolated STB membranes, TauT activity is inhibited by agents that nitrate tyrosine residues. This thesis tested the hypothesis that STB TauT activity is down-regulated in PE due to post-translational modification of TauT through tyrosine nitration which lowers intracellular taurine and contributes to altered STB renewal. Placentas were collected from normal pregnancy (NP) and PE (blood pressure >140/90mmHg after 20 weeks gestation in previously normotensive women plus proteinuria >300 mg/L in a 24-hour collection). STB TauT activity, measured as Na+-dependent uptake of 3H-taurine into placental villous fragments, was significantly lower in PE (n=24) compared to NP (n=44). Western blotting of membrane enriched homogenates showed that TauT protein expression (normalised to β-actin) was significantly higher in placentas from PE (n=8) compared to NP (n=9). The presence of nitrotyrosine residues (marker of nitrative stress) in placentas of women with PE and NP was assessed by immunohistochemistry (IHC). The intensity of STB nitrotyrosine staining was greater in PE placentas that had reduced TauT activity (n=8) than in NP (n=7). To determine the effect of nitrative stress on TauT activity and STB renewal, placental villous explants from NP were cultured (7 days; n=6) and treated with SIN-1 (1mM; days 5,6) to induce nitrative stress. STB nitrotyrosine (IHC) and TauT activity (3H-taurine uptake) was determined on day 7 and STB renewal was assessed by IHC for apoptosis (M30), proliferation (dual staining for Ki67 and the CTB marker E-cadherin) and STB integrity (cytokeratin 7). SIN-1 increased STB nitrotyrosine staining intensity compared to controls, confirming induction of nitrative stress. SIN-1 reduced STB TauT activity, increased apoptosis, reduced CTB proliferation and altered STB regeneration compared to control. To determine the effect of reducing intracellular taurine on STB renewal, villous explants were cultured for 7 days with 2.5mM β-alanine to competitively inhibit taurine uptake (n=6). At day 7, intracellular taurine, measured as the steady-state accumulation of 3H-taurine, was 15% of normal. STB turnover was assessed at day 7 as described above. β-alanine significantly increased apoptosis and altered STB regeneration compared to controls. Following statistical analysis all p <0.05.In conclusion, STB TauT activity was lower, and protein expression higher, in PE compared to NP. STB nitrotyrosine was elevated in PE and nitrative stress inhibited STB TauT activity and disrupted STB renewal in vitro. Reducing intracellular taurine also disrupted STB renewal in vitro. Overall the data support the hypothesis that post-translational modification of TauT by nitration inhibits TauT activity in PE. This reduces intracellular taurine which contributes to abnormal renewal of STB. Further work is needed (a) to confirm that TauT is nitrated in PE and that reduced STB TauT activity lowers intracellular taurine and reduces taurine delivery to the fetus and (b) to determine the mechanism/s by which taurine regulates CTB apoptosis and facilitates renewal of STB.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-08-15T19:37:52Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eloisa.pdf: 351801 bytes, checksum: 5d8bb006aa699c1f75f27ffc6a85479b (MD5) / Made available in DSpace on 2017-08-15T19:37:52Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Eloisa.pdf: 351801 bytes, checksum: 5d8bb006aa699c1f75f27ffc6a85479b (MD5) Previous issue date: 2017-05-23 / L-arginine has been used as a dietary supplement to minimize the side effects of chemotherapy, but it is not fully elucidated its actions and nor the ideal dose that can minimize the side effects. The objective of this study was to evaluate the effect of L-arginine supplementation on the formation of micronuclei in 70 Wistar rats after chemotherapy with 5-FU divided into 7 batches (10 male rats weighing 200 g/batch): LC water ad libitum; LArg2% and LArg4% mice fed commercial feed and 2% and 4% of L-arginine in water ad libitum, respectively; LC rats fed commercial feed, water ad libitum and a dose of 50 mg cyclophosphamide/kg; L5-FU rats fed commercial feed, water ad libitum and a dose of 200 mg of 5-FU/kg was applied; LArg2%+5-FU and LArg4%+5-FU rats fed commercial feed, 2% and 4% L-arginine were added in water ad libitum, respectively, and a dose of 200 mg of 5-FU/Kg. The mice were sacrificed 72 hours after the application of the chemotherapeutics to evaluate the formation of micronuclei. In the data analysis, one-way ANOVA was applied and followed by the Tukey test at 5%. Mice supplemented ad libitum with 2% and 4% L-arginine showed a reduction (P < 0.05) in the formation of micronuclei in bone marrow cells after chemotherapy, indicating that these supplements minimize the mutagenic effect of 5-FU. / A suplementação com L-arginina tem sido utilizada para minimizar os efeitos colaterais da quimioterapia, no entanto, ainda não está totalmente elucidado seus benefícios e nem determinado uma dose ideal de suplementação que pode minimizar esses efeitos. Objetivou avaliar o efeito da suplementação com L-arginina na formação de micronúcleos em 70 ratos Wistar após a aplicação da 5-FU. Estes ratos foram divididos em sete lotes (10 ratos/lote): Lc ratos alimentados com ração comercial e água ad libitum; LArg2% e LArg4% ratos alimentados com ração comercial e adicionou-se 2% e 4% de L-arginina na água ad libitum, respectivamente; Lciclo ratos alimentados com ração comercial, água ad libitum e aplicou-se uma dose de 50 mg de ciclofosfamida/Kg; L5-FU ratos alimentados com ração comercial, água ad libitum e aplicou-se uma dose de 200 mg de 5-FU/Kg; LArg2%+5-FU e LArg4%+5-FU ratos alimentados com ração comercial, adicionou-se 2% e 4% de L-arginina na água ad libitum, respectivamente, e aplicou-se uma dose de 200 mg de 5-FU/Kg. A contagem de micronúcleos nos eritrócitos policromáticos dos ratos foi realizada 72 horas após a aplicação dos quimioterápicos. Os resultados obtidos foram analisados pela ANOVA one-way seguido do teste de Tukey a 5%. Os ratos dos lotes LArg2%+5-FU e LArg4%+5-FU apresentaram redução (P < 0,05) na formação de micronúcleos nos eritrócitos policromáticos após a quimioterapia, indicando que estas suplementações com L-arginina minimizou o efeito mutagênico da 5-FU.

Submitted by Michele Mologni (mologni@unoeste.br) on 2018-11-27T17:31:18Z No. of bitstreams: 1 Carolini Rossetti Cervini.pdf: 405222 bytes, checksum: 83a6122ca8a8ad6a37bff5bf733a0373 (MD5) / Made available in DSpace on 2018-11-27T17:31:18Z (GMT). No. of bitstreams: 1 Carolini Rossetti Cervini.pdf: 405222 bytes, checksum: 83a6122ca8a8ad6a37bff5bf733a0373 (MD5) Previous issue date: 2017-09-27 / . / .

The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.

Main goal of classifying proteins into families is to understand structural, functional and evolutionary relationships between individual proteins, which are not easily deducible from available data. Since the structure and function of proteins are closely related, determination of function is mainly based on structural properties, that can be obtained relatively easily with current resources. Protein classification is also used in development of special medicines, in the diagnosis of clinical diseases or in personalized healthcare, which means a lot of investment in it. I created a new hierarchical tool for protein classification that achieves better results than some existing solutions. The implementation of the tool was preceded by acquaintance with the properties of proteins, examination of existing classification approaches, creation of an extensive data set, realizing experiments and selection of the final classifiers of the hierarchical tool.

La fluorooléfine (CF=CH), motif isostère et isoélectronique de la liaison amide, peut être utilisé comme mime efficace de la liaison peptidique. De plus, ce motif confère une meilleure résistance à la dégradation enzymatique comparé à la liaison peptidique. Cette thèse s’inscrit dans notre programme de développement de nouvelles méthodologies d’accès aux fluoropseudopeptides. Dans une première partie, nous appliquons diverses stratégies originales du laboratoire vers la synthèse d’un analogue du neuropeptide 26RFa. Dans une seconde partie est présentée une stratégie générale vers l’accès à des pseudopeptides possédant un motif proline, un acide aminé extrêmement important. Ainsi, la synthèse asymétrique d’analogues fluorés de dipeptide incluant l’unité proline (AA-[(Z) ou (E)CF=C]-Pro), de conformation cisoïde ou transoïde, a été développée. Enfin, nous avons étendu cette méthodologie à la synthèse d’un analogue de l’Enalapril®, molécule biologiquement active. / The Fluoroolefin moiety (CF=CH) can be used as an effective peptide bond mimic due to isoelectronic and isosteric properties. Moreover, this moiety provides better resistance to enzymatic degradation compared to native peptide bond. This thesis is part of our program aiming at developing new methodologies towards fluoropseudopeptides. In a first part, we apply various innovative strategies from the laboratory to the synthesis of an analog of neuropeptide 26RFa. In the second part is presented an overall strategy towards fluorinated pseudopeptide including a proline residue, an amino acid extremely important. Thus, the asymmetric synthesis of fluorinated dipeptide analogues AA-[(Z) or (E) CF=C]-Pro, under cisoid or transoid conformation, has been developed. Finally, we extend this methodology to the synthesis of an analogue of biologically active Enalapril®.

The objective of the experiments conducted for this dissertation was to determine the standardized ileal digestibility (SID) of amino acids (AA) in a variety of feed ingredients for broiler chickens and pigs. The effects of casein in experimental diets on the SID of AA in corn distillers’ dried grains with solubles (DDGS) fed to pigs were evaluated. The SID of AA in feed ingredients, which include full-fat soybean (FFSB), two soybean meals (SBM), peanut flour (PNF), full-fat canola seeds (FFCS), canola meal (CM), canola expellers (CE), hydrolyzed feather meal (HFM), flash dried poultry protein (FDPP), poultry meal (PM), and meat and bone meal (MBM), were compared in broiler chickens and pigs. One of the studies determined the ileal digestibility of AA in casein by regression analysis and investigated the effects of 60 g/kg casein in experimental diets on the SID of AA in DDGS. The ileal digestibility of AA in casein were close to 100%, ranging from 95.5% (SE = 9.10) for Cys to 103.1% (SE = 4.40) for Arg. In addition, the SID of Lys and Phe in DDGS determined by pigs fed the diet containing DDGS and casein were greater (<i>P</i> < 0.05) than the values determined by pigs fed the diet containing DDGS without casein. Based on the results of this experiment, two additional experiments were conducted to determine the effects of graded concentrations of casein from 55 to 165 g/kg in experimental diets on the SID of AA in DDGS and to determine the effects of dietary DDGS concentrations (i.e., 155.6 or 466.8 g/kg) and addition of casein in experimental diets on the SID of AA in DDGS. The SID of indispensable AA, except for Arg and Lys, linearly decreased (<i>P</i> < 0.05) as the concentration of casein in experimental diets increased. Moreover, pigs fed the diets containing 155.6 g/kg DDGS had less (<i>P</i> < 0.05) SID of indispensable AA, except for Trp, in DDGS than those fed the diets containing 466.8 g/kg DDGS regardless of the addition of casein in experimental diets. Therefore, it may be concluded that the addition of casein improves the SID of AA in DDGS, but reduced DDGS concentration in experimental diets decreases the SID of AA in DDGS. In one pair of experiments conducted to compare the SID of AA in FFSB, SBM containing 430 g/kg crude protein, SBM containing 470 g/kg crude protein, and PNF between broiler chickens and pigs, the SID of AA, except for Trp, Ala, and Glu, in test ingredients for pigs were greater (<i>P</i> < 0.05) than the values for broiler chickens. In addition, in both broiler chickens and pigs, the SID of Ile, Leu, and Val in FFSB were less (<i>P</i> < 0.05) than in the other test ingredients. In another pair of experiments conducted to compare the SID of AA in FFCS, CM, and CE between broiler chickens and pigs, interactions (<i>P</i> < 0.05) between experimental diets and species were observed in the SID of AA, except for Lys, Gly, Pro, and Ser. The SID of AA in FFCS for broiler chickens were greater (<i>P</i> < 0.05) than pigs; however, there was no difference in the SID of AA in CM or CE between broiler chickens and pigs. The objective of a third pair of experiments was to compare the SID of AA in HFM, FDPP, PM, and MBM fed to broiler chickens and pigs. There were interactions (<i>P</i> < 0.05) between experimental diets and species in the SID of His, Thr, Trp, and Val. In broiler chickens, the SID of His, Thr, and Trp in FDPP and PM were greater (<i>P</i> < 0.05) than in HFM but were less (<i>P</i> < 0.05) than MBM; however, difference in SID of His, Thr, and Trp among FDPP, PM, and MBM was not observed in pigs. Based on the results of three pairs of studies, it was revealed that differences in SID of AA in common feed ingredients for both broiler chickens and pigs were affected by species. Therefore, it may be concluded that the effects of feed ingredient-specific factors on the SID of AA are different between broiler chickens and pigs.

Amino acid phenylalanine plays a key role in numerous biological processes and is also involved in amyloid fibril diseases. The aim of the thesis is to deepen our understanding of its behavior and partitioning at interfaces, and to investigate its clustering. Classical atomistic molecular dynamics simulations were performed for phenylalanine and three other aromatic molecules which chemical structure is derived from it - phenylglycine, phenylacetic acid and tyrosine. Molecules are simulated at both water-air and at water-DPPC-air interfaces. Phenylalanine, phenylglycine and phenylacetic acid demonstrate surface activity at the water-air interface, whereas tyrosine is not surface active. All molecules interact with the lipid monolayer at the water-DPPC-air interface but only phenylalanine penetrates deep into the monolayer. Formation of transient clusters is observed in the interfacial regions, mostly for phenylalanine. Powered by TCPDF (www.tcpdf.org)

Metabolic cross-feeding is an important process that can broadly shape microbial communities. Comparative genomic analysis of >6000 sequenced bacteria from diverse environments provides evidence to suggesting that amino acid biosynthesis has been broadly optimized to reduce individual metabolic burden in favor of enhanced cross-feeding to support synergistic growth across the biosphere. Still, little is known about specific cross-feeding principles that drive the formation and maintenance of individuals within a mixed population. Here, we devised a series of synthetic syntrophic communities to probe the complex interactions underlying metabolic exchange of amino acids. We experimentally analyzed multi-member, multi-dimensional communities of Escherichia coli of increasing sophistication to assess the outcomes of synergistic cross-feeding. We find that biosynthetically costly amino acids including methionine, lysine, isoleucine, arginine and aromatics, tend to promote stronger cooperative interactions than amino acids that are cheaper to produce. Furthermore, cells that share common intermediates along branching pathways yielded more synergistic growth, but exhibited many instances of both positive and negative epistasis when these interactions scaled to higher-dimensions. This system enabled the identification of synergistic pairings and optimal expression levels of amino acid exporters of arginine, threonine and aromatics towards drastic improvements of ecosystem productivity. Tradeoffs identified in these mutualistic systems between secretion, relative abundance and absolute community productivity have implication in the evolution of cooperative behaviors. Long-term evolution of these synthetic communities highlight transporter over-expression, amino acid pool redistribution, and perturbations to nitrogen regulation as strategies to circumvent imposed metabolic dependencies. To address this potentially problematic genomic plasticity, a genetically reassigned organism is leveraged to investigate synthetic metabolic dependencies showing improved biocontainment and potential for microbial consortia control. These results improve our basic understanding of microbial syntrophy while also highlighting the utility and limitations of current approaches to modeling and controlling the dynamic complexities of microbial ecosystems. This work sets a foundation for future endeavors in microbial ecology and evolution, and presents a platform to develop better and more robust engineered synthetic communities for industrial biotechnology.