<p>The delivery of therapeutic peptides and proteins is often challenged by a short circulation half-life, necessitating frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. The covalent conjugation of therapeutic peptides and proteins, and more recently oligonucleotide-based drugs, with the “stealth” polymer poly(ethylene glycol) (PEG), termed PEGylation, is one of the most commonly used approaches to increase the in vivo half-life and reduce the immunogenicity of these therapeutic biomolecules. However, after several decades of research and clinical use, the limitations of PEGylation have begun to emerge.</p><p>Conventional methods for synthesizing peptide/protein-polymer conjugates have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of control over site and stoichiometry of conjugation, which results in heterogeneous products with significantly compromised biological activity. Additionally, anti-PEG antibodies have been induced in patients treated with PEGylated drugs and have been shown to correlate with rapid clearance of these drugs. High levels of pre-existing anti-PEG antibodies have also been found in individuals naïve to PEGylated agents, which are associated with serious first-exposure allergic reactions.</p><p>To address the synthetic limitations of PEGylation, a general approach for the high-yield synthesis of site-specific (C-terminal) and stoichiometric (1:1) peptide/protein-polymer conjugates, named sortase-catalyzed polymer conjugation, was developed. Demonstrating proof-of-concept of the approach with green fluorescent protein (GFP) as a model protein, sortase A from Staphylococcus aureus was used to site-specifically attach an initiator solely at the C-terminus of GFP, followed by in situ growth of the PEG-based brush polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) from the protein macroinitiator by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeded with high specificity and near-complete (~ 95%) product conversion. Subsequent in situ ATRP in aqueous buffer produced 1:1 stoichiometric conjugates with > 90% yield, tunable MW, low dispersity, and no denaturation of the protein. The extraordinarily high yield compares favorably to order of magnitude losses typically seen in conventional PEGylation processes.</p><p>Next, the therapeutic potential of POEGMAlation, or the conjugation of POEGMA to a peptide or protein, was demonstrated by implementing the developed sortase-catalyzed polymer conjugation strategy with exendin-4 (exendin), a therapeutic peptide for treating type 2 diabetes, to synthesize exendin-C-POEGMA conjugates with a wide and tunable range of molecular weights (MWs) and low dispersity. A single subcutaneous injection of exendin-C-POEGMA conjugates lowered blood glucose for up to 120 h in a diabetic mouse model. Most intriguingly, we showed that appending PEG as oligomeric side-chains on the conjugated POEGMA and tuning the side-chain length completely eliminated the reactivity of exendin-C-POEGMA conjugates toward patient-derived anti-PEG antibodies without compromising in vivo efficacy. Clinically, the lack of anti-PEG antigenicity of POEGMA conjugates is expected to completely eliminate serious first-exposure allergic reactions and the accelerated blood clearance of POEGMA-drug conjugates due to pre-existing anti-PEG antibodies in patients.</p><p>Collectively, these results establish POEGMAlation as a next-generation PEGylation technology that is highly useful for improving the pharmacological performance of therapeutic biomolecules while providing a timely solution to the increasing levels of pre-existing anti-PEG antibodies in patients that are seriously hindering the safety and efficacy of traditional PEGylated drugs.</p> / Dissertation

The purpose of this study was to identify and compare preferences and perceptions of orthodontists and general dentists when restoring peg-shaped lateral incisors. The investigation sought to summarize these preferences with regard to treatment planning, tooth preparation and interdisciplinary communication. A pair of mailed and electronic surveys was distributed to 1,500 general dentists and orthodontists, respectively. The results indicated that general dentists perceived that general dentists held the primary decision-making responsibility, while orthodontists disagreed (P<0.0001). Orthodontists prioritized the treatment goals of Class I canine relationship and overbite/overjet more significantly than general dentists, whom valued tooth proportions more highly (P<0.0001). General dentists reported receiving significantly less input than orthodontists report seeking (P<0.0001).The consensus of both groups showed that the tooth should be positioned centered mesiodistally and guided by the gingival margins incisogingivally. Both groups agree that orthodontists must improve communication to improve treatment results.

Submitted by Jadson Francisco de Jesus SILVA (jadson@uefs.br) on 2018-01-26T21:53:28Z No. of bitstreams: 1 disserta??o Eliza 14 de dezembro final.pdf: 1828093 bytes, checksum: 7d102752271132d8745e82e9533c13b3 (MD5) / Made available in DSpace on 2018-01-26T21:53:28Z (GMT). No. of bitstreams: 1 disserta??o Eliza 14 de dezembro final.pdf: 1828093 bytes, checksum: 7d102752271132d8745e82e9533c13b3 (MD5) Previous issue date: 2017-07-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The stress situation in Caatinga is aggravated by climate changes, from which are predicted higher temperatures; water scarcity and higher soil salinization. Studies evaluating differences in seeds tolerance to abiotic stress among samples or genotypes of the same species can constitute physiologic tools and significant and complementary information for the selection of the most adapted group to environmental pressures. This work aimed to compare the seed vulnerability of different samples of Erythrina velutina (Fabaceae) to abiotic stress; evaluate que physiologic quality of seeds and determine the tolerance limits to saline, hydric and thermic stress. The assays were conducted at EMBRAPA semiarido Seeds laboratory and at the Germination laboratory of State University of Feira de Santana. In addition, the assays were entirely randomly mounted, four repetitions of 10 seeds for the vigor test and 50 seeds for the stress test. In the initial vigor, the seeds of all samples showed 100% of radicle emission at the first count. The seeds samples were affected by the addition of sodium chloride in the solution, exhibiting critical limit at 36dSm-1 for some samples. The radicular protrusion was affected with the decrease of the osmotic potential of the solution, which was caused by PEG 6000, limiting the germination in -0,9 MPa. The seed germination of E. velutina occurs at broad limits of temperature, from 5 to 40?C. Generally, the stress imposed by sodium chloride and PEG did not compromise the viability of the seed germinal power as the seeds of the samples showed high rate of germination when the ideal conditions were reestablished, excepting Cai?ara 2011. The great temperature of E. velutina germination occurs between 25 to 30?C. / A situa??o de estresse na caatinga tende a se agravar pelas mudan?as no clima onde se prev? maiores temperaturas, escassez de ?gua e maior saliniza??o do solo. Estudos que avaliem as diferen?as de toler?ncia de sementes aos estresses abi?ticos entre acessos ou gen?tipos da mesma esp?cie podem constituir ferramentas fisiol?gicas importantes e complementares para a sele??o de grupos de plantas mais adaptadas ?s press?es ambientais. Esse trabalho teve como objetivos comparar a vulnerabilidade de sementes de diferentes acessos de Erythrina velutina (Fabaceae) ? estresses abi?ticos; avaliar a qualidade fisiol?gica das sementes; e determinar os limites de toler?ncia aos estresses salino, h?drico e t?rmico. Os ensaios foram conduzidos nos laborat?rios de sementes da Embrapa Semi?rido (Petrolina-PE) e no de Germina??o da UEFS (Feira de Santana ?BA). Os ensaios foram montados em delineamento inteiramente casualizado, quatro repeti??es de 10 sementes para o teste de vigor e de 50 sementes para os estresses. Em rela??o ao vigor inicial as sementes de todos os acessos apresentaram 100% de emiss?o de rad?cula na primeira contagem. As sementes dos acessos foram afetadas pela adi??o de cloreto de s?dio na solu??o apresentando limite cr?tico de germina??o aos 36 dS.m-1 para alguns. A protrus?o radicular foi afetada tamb?m com a diminui??o do potencial osm?tico da solu??o causada pelo PEG 6000 tendo limite de germina??o em -0,9 MPa. A germina??o de sementes de E. velutina ocorreu sob amplo limites de temperatura, 5 a 40 ?C. De modo geral os estresses impostos pelo cloreto de s?dio e PEG n?o comprometeram a viabilidade do poder germinativo, visto que as sementes dos acessos apresentaram alta porcentagem de germina??o quando as condi??es ideais foram reestabelecidas, exceto as de Cai?ara 2011. A temperatura ?tima de germina??o de E. velutina ocorreu na faixa de 25 a 30 ?C.

<p>Istraživanja su sprovedena na jedanaest genotipova &scaron;ećerne repe (Beta vulgaris ssp. vulgaris, L., označeni brojevima 1-11) koji su u poljskim uslovima ispoljili razlike u opservacionom testu nivoa tolerantnosti prema su&scaron;i (visoko, srednje i nisko tolerantni). U prvom delu eksperimenta biljke su gajene u polukontrolisanim uslovima, u stakleniku u supstratu koji je bio me&scaron;avina zemlje i peska. Biljke su svakodnevno zalivane tokom 90 dana, nakon čega je izazvan vodni deficit prestankom zalivanja, dok je kod kontrolnih biljaka zalivanje nastavljeno. Pet dana po prestanku zalivanja analizirani su parametri koji bi trebalo da ukažu na genotipske razlike u smislu tolerantnosti prema nedostatku vode. Utvrđen je sadržaj vode/suve materije u zemlji&scaron;tu i u biljnim tkivima (koren stablo i list). Lisna povr&scaron;ina i koncentracija pigmenata hloroplasta su ustanovljeni kod kontrolnih biljaka s ciljem utvrđivanja genotipskih specifičnosti. Osim toga, mereni su parametri fluorescencije hlorofila (F0, Fm, Fv, Fv/Fm i t1/2) kako bi se utvrdio efekat stresa izazvanog nedostatkom vode na inhibiciju transporta elektrona kroz PSII, i koncentracija slobodnog prolina, amino kiseline koja se nakuplja u uslovima stresa. U drugom delu eksperimenta ovi genotipovi su testirani u kontrolisanim, in vitro uslovima gajenja. Aksilarni izdanci su gajeni na podlozi za mikropropagaciju sa 0 (kontrola), 3 i 5% polietilen glikola (PEG 6000) pune četiri nedelje, a potom su vr&scaron;ene analize. Utvrđena je sveža masa izdanaka, sadržaj suve materije i koncentracija slobodnog prolina. U trećem delu eksperimenta upoređene su razlike u ekspresiji 13 kandidat-gena, koji su povezani sa reakcijom biljaka na uslove stresa, posebno su&scaron;e, u listovima biljaka gajenih u polukontrolisanim uslovima.<br />Biljke izložene stresu u polukontrolisanim uslovima gajenja su u proseku imale oko tri lista manje, za četiri procenta veći udeo suve materije i sedmostruko veći sadržaj prolina. Koncentracija prolina u listovima je bila vi&scaron;a u uslovima nedostatka vode u polukontrolisanim uslovima kod svih genotipova, a posebno kod nekih iz slabo tolerantne (2, 6) i visoko tolerantne (4) grupe.<br />U in vitro uslovima usled tretmanom PEG-om smanjila se ukupna suva masa i vi&scaron;e nego prepolovio broj aksilarnih izdanaka. PEG u koncentraciji od 3% je doveo do<br />povećanja ukupne sveže mase, a koncentracija prolina se povećala se porastom koncentracije PEG. Koncentracija prolina u uslovima stresa je kod oba eksperimenta bila značajno povećana u odnosu na kontrolu i to u in vitro uslovima &scaron;est puta, a u polukontrolisanim uslovima u stakleniku &scaron;esnaest puta u odnosu na odgovarajuće kontrole. Povećanje sadržaja prolina u biljkama gajenim u polukontrolisanim uslovima je bilo tri puta veće u odnosu na eksperiment sa PEG-om.<br />Koncentracija slobodnog prolina, kao jedan od potencijalnih parametara - pokazatelja tolerantnosti genotipova &scaron;ećerne repe prema su&scaron;i, je adekvatniji od ukupne suve mase. Rezultati su pokazali da je test u in vitro uslovima (posebno tretman 3% PEG-om) efikasiji za ocenu tolerantnosti prema su&scaron;i od eksperimenta u polukontrolisanim uslovima. Grupisanje genotipova prema nivou tolerisanja nedostatka vode na osnovu koncentracije prolina ustanovljene u ekperimentu in vitro dalo je isti rezultat kao i opservacioni test u poljskim uslovima.<br />Utvrđene su promene u ekspresiji kandidat-gena u uslovima su&scaron;e u odnosu na kontrolu, a ustanovljene su i razlike između genotipova. Jedan od analiziranih kandidat-gena može da posluži za dalji razvoj markera.<br />Ovi rezultati mogu da se primene u procesu oplemenjivanja &scaron;ećerne repe koje je usmereno na povećanje tolerantnosti prema ovom abiotičkom činiocu.</p>

In a 2011 paper by Beeler and Hoilman, the game of peg solitaire is generalized to arbitrary boards. These boards are treated as graphs in the combinatorial sense. An open problem from that paper is to determine the minimum number of edges necessary for a graph with a fixed number of vertices to be solvable. This thesis provides new bounds on this number. It also provides necessary and sufficient conditions for two families of graphs to be solvable, along with criticality results, and the maximum number of pegs that can be left in each of the two graph families.

A retrospective chart review was conducted on pediatric patients at the Intermountain Cystic Fibrosis Center who had a percutaneous endoscopic gastrostomy (PEG) placed between 1993 and 1999. Height velocity improved significantly in the group of patients with a PEG placed; however, pulmonary function declined more significantly over time. Questionnaires regarding attitude toward PEG placement were sent to patients enrolled in accredited cystic fibrosis centers in the mountain west region and to their parents. The overall response rate was 54.25% for the PEG questionnaire and 24% for the non-PEG questionnaire. Ninety-six percent of the patients with a PEG reported that weight was a problem at time of placement, and 91% reported weight gain after PEG ill placement. Sixty-four percent of the patients with a PEG reported that they would have a PEG placed if they made the decision again. Of the patients without a PEG, 60.7% thought a PEG looked bad, and 59.2% would be embarrassed to have a PEG. Forty-nine percent of patients without a PEG expressed a lack of knowledge of the pros and cons of PEG placement and 35.4% had no opinion about their knowledge of PEGs. PEG placement can be beneficial in improving nutritional status. Optimal time for PEG placement may be earlier rather than after pulmonary function has declined. People with a PEG have felt positive toward placement, and those without a PEG seem to lack knowledge about the pros and cons of PEG placement.

Dans le but de réduire l'utilisation des solvants organiques dans les réactions et dans la purification, nous avons envisagé de coupler la synthèse supportée sur PEGs dans l'eau aux microréacteurs en flux continu et à l'ultrafiltration comme technique de purification. Dans un premier temps nous avons synthétisé des 3,4-dihydropyrimidin-2-ones (DHPMs) en utilisant comme étape clef la réaction multicomposant de Biginelli avec un aldéhyde supporté sur PEG. Dans ce but, nous avons trouvé que la liaison du PEG au 4-carboxybenzaldehyde, décrite dans le CH2Cl2, a lieu dans le PEG fondu (70 °C) en absence de solvant. La synthèse des DMPMs a ensuite été réalisée dans l'eau et dans le PEG fondu, à partir de l'aldéhyde supporté, et les produits finaux ont été isolés après saponification. Cette synthèse a ensuite été optimisée en microréacteurs en flux continu dans l'eau et la purification des adduits supportés a été réalisée par ultrafiltration dans l'eau.La stratégie décrite ci-dessus a été appliquée à des synthèses multi-étapes, la réaction de Baylis-Hillmann étant l'étape clef pour la préparation de tétrahydro-1,3-oxazine, tétrazoles, 1,4-oxazepin-3-ones et tétrahydro-1,3-pyrimidin-4-ones, composés dotés d'une activité biologique potentielle. Les synthèses de tétrahydro-1,3-oxazines et d'un tétrazole ont d'abord été optimisées par lots avec les purifications réalisées par ultrafiltration dans l'eau, puis adaptées en microréacteurs en flux continu.Tous les adduits supportés sur PEG ont été caractérisés par RMN et MALDI-Tof, et des nouvelles méthodes d'analyse RMN ont été mises au point afin de réduire l'intensité des signaux dus au PEG et de faciliter l'étude des molécules supportées.En conclusion nous avons développé la synthèse supportée, en milieux éco-compatibles de DHPMs, tétrahydro-1,3-oxazines et d'un tétrazole, par lots et en microréacteurs en flux continu, en utilisant les réactions de Biginelli et Baylis-Hillmann comme étapes clefs. Toutes les purifications ont été réalisées par ultrafiltration dans l'eau, afin de réduire l'utilisation de solvants organiques.

<p>This thesis describes syntheses and characterization of star-like amphiphilic block copolymers consisting of poly(ethylene glycol) (PEG) as the hydrophilic block,polycarbonate as the hydrophobic block and an amine-containing dendrimer as the core molecule. The macromolecules were synthesized by either a convergent or adivergent approach that includes tandem click reactions and ring opening polymerizations (ROP) of methyl trimethyl carbonates (MTC) with differentfunctionalities. The ROP of MTC monomers was performed using organocatalysts that allow mild reaction condition and reasonable molecular weight distribution(PDI~1.3). These synthetic approaches provide the resultant polymers with three different conformations, which are; mikto-arm type, comb-block with short PEGbrushes, and linear block with long PEG chain. The star-like polymers that were synthesized were all water soluble and most of them formed nano aggregates inwater. Different morphologies were observed in AFM study depending on the polymer conformation. Interestingly, some of them had indications pointing towards alower critical solution temperature.</p>

Adsorption of proteins at interfaces has an important role in biotechnological and pharmaceutical applications. Indeed, several undesirable processes are related to protein adsorption, as for example: fouling of contact lenses, clotting on blood contacting devices, triggering inammation around articial organs, diminished circulation time of therapeutic proteins and drug bearing liposomes. Neutral water soluble polymers, such as poly(ethylene glycol) (PEG), are used to repress protein adsorption: by coating the surface with a polymer brush, a "cushion" is created between the protein and the surface, that can reduce, or even completely repress the adsorption. Understanding the mechanism that inhibits the adsorption at interfaces is an active field of research, and could lead to relevant improvements in biomaterials performances and design. A clear understanding of the mechanism of protein adsorption onto polymer brushes is still missing. The first models describing the interactions of a polymer brush with adsorbing particles predicted two adsorption modes: primary adsorption at the grafting surface, and secondary adsorption at the outer edge of the brush (occurring for large cylindrical proteins). Primary adsorption can be repressed by increasing the grafting density of the brush, and secondary adsorption by increasing its thickness, in agreement with the experiments reported in the literature. But experimental evidences (a maximum in the adsorbed amount observed for long brushes) suggested then the existence of a third mode: ternary adsorption within the brush itself, due to attractive interactions between the protein and the brush. Standard techniques can in general only probe the total adsorbed amount. The aim of this work was to separate primary and ternary adsorption isotherms, by using neutron reectivity and deuterated proteins. As neutrons interact differently with hydrogen and deuterium atoms, the contrast between the hydrogenated brush and the deuterated protein is high enough to separate the two contributions. We studied the adsorption of deuterated myoglobin on PEG brushes with different degrees of polymerisation (N = 56, 146 and 770), and as a function of the area per grafted chain. The contribution of primary and ternary adsorption was separated for the different systems, and the adsorbed amount was extracted and the adsorption isotherms compared to the theoretical predictions. The ability to distinguish between the different adsorption modes, and the quantification of their relative contribution to the overall amount of adsorbed proteins, represents a major advance in optimising surface properties. In particular, the occurrence of ternary adsorption onto PEG brushes affects their status as tool for repressing protein adsorption. L’adsorption de protéines aux interfaces a un rôle important pour certaines applications pharmaceutiques ou biotechnologiques. En effet, plusieurs processus indésirables sont liés à l’adsorption de protéines, par exemple l’encrassement de lentilles de contact, la coagulation dans des appareils contenant du sang, l’inflammation d’organes artificiels ou encore la diminution du temps de circulation dans le corps de protéines ou liposomes thérapeutiques. Certains polymères, tels que le polyéthylène glycol (PEG), sont utilisés pour réprimer l’adsorption de protéines : en greffant une brosse de PEG sur la surface, une couche est créée entre la protéine et celle-ci qui diminue, voire même réprime complètement l’adsorption. Comprendre le mécanisme qui entrave l’adsorption aux interfaces est un sujet de recherche actif, qui pourrait mener à des améliorations significatives dans la conception de biomatériaux. À ce jour, la compréhension du mécanisme d’adsorption de protéines sur des brosses de polymère n’est pas claire. Les premiers modèles décrivant les interactions entre brosses de polymères et particules adsorbantes prédisaient deux modes d’adsorption : l’adsorption primaire sur la surface de greffage, et l’adsorption secondaire à l’extérieur de la brosse (pour les grandes protéines cylindriques uniquement). L’adsorption primaire peut-être réprimée en augmentant la densité de greffage de la brosse, et l’adsorption secondaire en augmentant son épaisseur, en accord avec les expériences reportées dans la littérature. Mais d’autres évidences expérimentales (un maximum dans la quantité adsorbée observé pour les brosses longues) ont ensuite suggéré l’existence d’un troisième mode : l’adsorption ternaire à l’intérieur même de la brosse, due aux interactions attractives entre la protéine et la brosse. Les techniques standards peuvent en général mesurer la quantité adsorbée totale. Le but de ce travail était de séparer les isothermes d’adsorption primaire et ternaire, en utilisant la réflectivité de neutrons et des protéines deutérées. Comme les neutrons interagissent différemment avec les atomes d’hydrogène ou de deutérium, le contraste entre la brosse hydrogénée et la protéine deutérée est ainsi suffisant pour séparer les deux contributions. Nous avons étudié l’adsorption de myoglobine deutérée sur des brosses de PEG avec différents degrés de polymérisation (N = 56, 146 and 770), en fonction de l’aire par chaîne Σ. La contribution des adsorptions primaire et ternaire put être séparée pour les différents systèmes, et les quantités adsorbées extraites pour finalement comparer les isothermes d’adsorption aux prédictions théoriques. La possibilité de distinguer les différents modes d’adsorption, et la quantification de leur contribution relative à la quantité totale de protéines adsorbées représente une avancée majeure dans l’optimisation des propriétés des surfaces. L’adsorption ternaire dans les brosses de PEG en particulier remet en question leur utilisation pour réprimer l’adsorption de protéines.

En 1998, le traitement de la co-infection VHC rarement discuté avant l'ère des HAART, compte tenu d'une réponse médiocre à la monothérapie par IFNα et d'un pronostic de vie lié au VIH estimé en moyenne à 10 ans, fût reconsidéré. C'est ainsi que débuta en 2000, l'essai RIBAVIC HC02, essai randomisé et multicentrique comparant l'association de la ribavirine 800 mg/j à l'Interféron 3 MUI x3/semaine ou au PEG-α-2b Interféron 1,5 μg/kg/semaine pendant 48 semaines. Une cohorte des patients inclus dans l'essai RIBAVIC (cohorte RIBAVIC EP10) débuta en 2001 pour évaluer le devenir à long terme de ces patients. L'essai RIBAVIC et la cohorte RIBAVIC ont apporté les enseignements suivants : - la cinétique de la charge virale VHC peut différer selon la nature du traitement antirétroviral. - la prévalence et les facteurs de risque de la stéatose sont similaires à ceux observés dans la population mono-infectée VHC - le taux de réponse virologique soutenue est inférieur chez les patients co-infectés (27%) comparé aux patients mono-infectés VHC (50%) - le taux de non réponse virologique (diminution de la charge virale VHC inférieure à 2 log à S12) sous traitement par pegIFN plus ribavirine est plus élevé (33%) comparé aux patients monoinfectés VHC (14%). L'interaction entre la ribavirine et l'abacavir pourrait être un facteur de risque. - l'indétectabilité de l'ARN VHC dès S4 est prédictive de la réponse à long terme (valeur prédictive positive 97%) et la décroissance de la charge virale VHC est significativement plus lente chez les patients rechuteurs comparée aux patients répondeurs long terme à S2 et à S4 - Au cours du traitement anti-VHC :1- le risque d'anémie est élevé et majoré par la coprescription de zidovudine et de ribavirine ; 2- l'amaigrissement est fréquent et sévère et peut être révélateur d'une toxicité mitochondriale ; 3- le risque bactérien n'est pas lié au taux des polynucléaires neutrophiles mais à la fibrose hépatique ; 4- le risque de toxicité mitochondriale, d'aggravation de la fibrose et de décompensation hépatique est majoré par l'interaction entre la didanosine et la ribavirine - une réponse virologique soutenue est associée à un bénéfice histologique et clinique.