Kliniska användandet av antibiotika är under ett allt större hot då bakterier har utvecklat resistens mot många klasser av antibiotika som används i sjukvården. Resistensgener hos bakterier finns kromosomalt eller burna av plasmider som kan förvärvas från andra bakterier. Spridning av resistens mot β-laktamantibiotika är oroande hos Enterobacteriaceae där inte många antibiotikaalternativ finns. Några plasmidburna β-laktamaser med ett utökat spektrum (ESBL) har blivit vanligare. Syftet med studien var jämförelse och utvärdering av tre kommersiella kromogena medier (CHROMagar™ C3GR, Chromatic™ ESBL+AmpC och CHROMagar™ mSuperCARBA™) selektiva för ESBL-producenter och jämföra dessa mot nuvarande metod (selektivt medium för gramnegativa bakterier och antibiotikadiskar). Kända stammar (11 st) med ESBLA / ESBLM tillväxte på C3GR och ESBL+AmpC men tendens till starkare tillväxt fanns på ESBL+AmpC. mSuperCARBA™ detekterade samtliga (14 st) karbapenemresistenta stammar. Vid jämförelse av 85 kliniska prov som analyserades med nuvarande metod för detektion av ESBL-producerande bakterier, och kromogena medier detekterades alla nio positiva för ESBLA på C3GR och ESBL+AmpC. Två ESBLM-producenter detekterades med säkerhet på ESBL+AmpC men ej på C3GR. Bland de negativa proverna gav 11växt på C3GR och sju på ESBL+AmpC, dessa innehöll bakterier som hade minskad cefalosporin-känslighet men ej klassificerades som ESBL-producerande. mSuperCARBA™ detekterade ESBLCARBA-producenter bättre än nuvarande metod som missade vissa. Slutsats från studien blev att ESBL+AmpC gav bättre tillväxt och selekterade bort fler arter och isolat som bar på minskad känslighet mot cefalosporiner men ej klassificerades som ESBL-producerande. Användning av medierna ESBL+AmpC och mSuperCARBA i kombination som ersättning för nuvarande metod innebär ökad känslighet och förenklad avläsning. / The clinical use of antibiotics is becoming less successful as bacteria have developed resistance to many antibiotics used in health care. Resistance genes in bacteria are chromosomal or carried by plasmids that can be acquired from other bacteria. The resistance to β-lactam antibiotics is particularly worrying in Enterobacteriaceae where there are few options available for treatment. Some plasmid-encoded β-lactamases with extended spectrum (ESBLs) have become common. The aim of the study was to compare and evaluate three commercial chromogenic media (CHROMagar™ C3GR, Chromatic™ ESBL+AmpC and CHROMagar™ mSuperCARBA™) selective for ESBL-producing bacteria and comparing them to the current method using selective medium for gram negative bacteria and antibiotic disks. All known ESBL-producing strains tested (11) with ESBLA or ESBLM grew on C3GR and ESBL+AmpC but there was a tendency towards stronger growth on ESBL+AmpC. mSuperCARBA™ detected all tested (14) carbapenem-resistant strains. In an analysis of 85 clinical samples, the chromogenic media detected all positive ESBLA producers, both C3GR and ESBL+AmpC. Two positive ESBLM producerswere detected by ESBL+AmpC with certainty but not on C3GR. Among the negative samples 11 gave growth on C3GR and seven on ESBL+AmpC. These bacteria had a slightly decreased cephalosporin sensitivity but were not classified as ESBL producers. mSuperCARBA™ detected ESBLCARBA producers better than the current method. The conclusion was that ESBL+AmpC gave better growth and was more selective against species and isolates carrying reduced susceptibility to cephalosporines, without being ESBL producing. ESBL+AmpC and mSuperCARBA™ could be used together for detecting the ESBL producing bacteria to increase sensibility.

Γενετικές παραλλαγές σε γονιδιακούς τόπους που βρίσκονται εντός (cis) αλλά και εκτός (trans) του συμπλέγματος των ανθρώπινων β-σφαιρινικών γονιδίων έχει δειχθεί πως συσχετίζονται σημαντικά με τα επίπεδα της εμβρυϊκής αιμοσφαιρίνης (HbF). Ένα από τα γονίδια αυτά είναι το γονίδιο MAP3K5 (ή ASK1), το οποίο είναι μέλος της οδού της MAP κινάσης, ενεργοποιείται από διάφορες παθολογικές καταστάσεις και από προ-φλεγμονώδεις κυτοκίνες, συμβάλλοντας στην κυτταρική απόπτωση. Πρόσφατα αποτελέσματα από το εργαστήριό μας έδειξαν ότι η παρουσία του σπάνιου αλληλομόρφου C σε μονονουκλεοτιδικούς πολυμορφισμούς (SNP) του συγκεκριμένου γονιδίου, και συγκεκριμένα στους rs9376230 και rs9483947, συσχετίζεται με μειωμένα επίπεδα της εμβρυϊκής αιμοσφαιρίνης (HbF) (Tafrali et al., 2013). Για το λόγο αυτό, υποθέσαμε ότι οι πολυμορφισμοί αυτοί συνδέονται με πιθανές αλλαγές σε ρυθμιστικές περιοχές του γονιδίου ΜΑΡ3Κ5 οι οποίες τροποποιούν τα επίπεδα έκφρασής του. Έτσι, ερευνήσαμε τον υποκινητή του γονιδίου ΜΑΡ3Κ5 ως προς τη συχνότητα παρουσίας τεσσάρων ή πέντε αντιγράφων της μικροδορυφορικής αλληλουχίας 5’ – GCGCG – 3’ (θέση -51 έως -27 από τη θέση έναρξης της μεταγραφής). Με την προσέγγιση αυτή θελήσαμε να εξακριβώσουμε εάν οι μικροδορυφορικές αυτές αλληλουχίες συνδέονται με τους μονονουκλεοτιδικούς πολυμορφισμούς και, κατά συνέπεια, με τα επίπεδα της HbF σε ασθενείς με ενδιάμεση ή μείζονα β-μεσογειακή αναιμία και σε μη-θαλασσαιμικά άτομα. Για τον σκοπό αυτό, χρησιμοποιήθηκαν 11 ασθενείς με ενδιάμεση β-μεσογειακή αναιμία, 15 ασθενείς με μείζονα β-μεσογειακή αναιμία και 60 μη-θαλασσαιμικά άτομα ελληνικής καταγωγής. Ο προσδιορισμός του γονότυπου πραγματοποιήθηκε με εκλεκτική ενίσχυση του τμήματος του υποκινητή του γονιδίου ΜΑΡ3Κ5, μεταξύ των θέσεων -273 έως και +79, και στη συνέχεια, είτε με αλληλούχιση σε αυτοματοποιημένο αναλυτή ή με ανάλυση ετεροδιμερών. Η ανάλυση έδειξε ότι υπάρχει σχεδόν απόλυτη σύνδεση των σπάνιων αλληλομόρφων C τόσο για τον rs9376230 όσο και για τον rs9483947 με τη μικροδορυφορική αλληλουχία των πέντε επαναλήψεων, σχηματίζοντας έναν απλότυπο. O συγκεκριμένος απλότυπος συσχετίζεται με χαμηλά επίπεδα της HbF και το φαινότυπο της βαριάς β-μεσογειακής αναιμίας, αφού σε ασθενείς με μείζονα β-μεσογειακή αναιμία εντοπίζεται με συχνότητα 60% και αποκλίνει στατιστικώς σημαντικά από τις αντίστοιχες συχνότητες σε ασθενείς με ενδιάμεση β-μεσογειακή αναιμία (p=0,04) και σε μη-θαλασσαιμικά άτομα (p=0,003). Τα αποτελέσματα αυτά δείχνουν ότι οι πολυμορφισμοί rs9376230 και rs9483947 συνδέονται με τη μικροδορυφορική αλληλουχία του υποκινητή, η οποία έχει πιθανό λειτουργικό ρόλο στα επίπεδα έκφρασης του γονιδίου ΜΑΡ3Κ5. / Genetic variations in loci that are in cis or trans of the human beta-globin gene cluster are shown to correlate significantly with the levels of fetal hemoglobin (HbF). One of these genes is MAP3K5 (or ASK1), a member of the MAPK pathway, which is activated by various stresses and pro-inflammatory cytokines that contribute to cellular apoptosis. Recent results from our laboratory have shown that the presence of the rare C allele in two intronic SNPs of MAP3K5, namely rs9376230 and rs9483947, is associated with reduced levels of fetal hemoglobin (HbF) (Tafrali et al., 2013). For this reason, we assumed that these polymorphisms may be associated with changes in the regulatory regions of MAP3K5 which could modify its expression levels. Thus, we investigated the frequency of a microsatellite marker at the proximal promoter region of MAP3K5, regarding the presence of four or five repeats of a 5-bp short tandem repeat (STR), am y 5’ – GCGCG – 3’ (positio -51 to -27 from transcription start site). With this approach, we wanted to ascertain whether this STR is associated with the previously studied SNPs (Tafrali et al., 2013) and consequently with th v s o bF i β-thalassemia intermediate or major patients compared to non-thalassemic individuals. For this purpose, we analyzed the DNA samples from 11 β-tha ass mia i t rm ia a 15 β-thalassemia major patients, as well as 60 non-thalassemic controls of Western Greek origin. Genotyping was performed by selectively amplifying a MAP3K5 gene promoter fragment between positions -273 to +79, followed by either sequencing or heteroduplex analysis. The analysis showed that there is an almost perfect correlation of the rare C allele for rs9376230 and the rare C allele for rs9483947 with the five STR repeats, forming a haplotype associated with low levels of HbF and with the phenotype of severe b-thalassemia. This haplotype is detected with a r qu cy o 60% i β-thalassemia major patients and it statistica y sig i ica t y viat s rom th corr spo i g r qu ci s i β-thalassemia intermedia patients (p = 0.04) and in non-thalassemic individuals (p = 0.003). These results indicate that the polymorphisms rs9376230 and rs9483947 are in linkage with the promoter STR polymorphism, which may have a functional role in MAP3K5 gene expression.

Rutinosides are very common glycosidic aroma precursors. The glycosidic moiety influences wine aroma, flavour and taste of juices, so its cleavage has many consequences. These interesting insights led us to a diglycosidase - the extracellular β-rutinosidase from Aspergillus niger. The purified β- rutinosidase was partly analyzed by MALDI-TOF/TOF. The insert encoding for β-rutinosidase was ligated into the expression vector pPICZα A. Pichia pastoris KM71H was used as an expression system. It was find out, that β rutinosidase gene consists of a 1137 bp, encoding protein with 379 amino acids. The enzyme was determined to have relative molecule mass 60 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were found to be 3,0 and 50 řC, respectively. p-Nitrophenyl-β-rutinoside was used as a substrate Powered by TCPDF (www.tcpdf.org)

Des études montrent que l’accumulation de lipides dans l’hypothalamus serait responsable de l’installation d’une lipotoxicité centrale : phénomène qui pourrait jouer un rôle dans l’apparition d’une insulino-résistance périphérique et du diabète de type II en dérégulant le contrôle nerveux de l’homéostasie glucidique. Il est connu que l'accumulation des céramides est impliquée dans le développement d’une lipotoxicité des tissus périphériques. L’objectif de cette étude est de déterminer le rôle du métabolisme des céramides au niveau hypothalamique dans l’installation d’une insulino-résistance centrale et d'en étudier les mécanismes impliqués. Nous avons également déterminé le rôle du métabolisme des céramides hypothalamiques dans la dérégulation de l’homéostasie glucidique induite par l’obésité.L’installation d'une insulino-résistance centrale est étudiée à l'aide d'approches in vitro, en utilisant des cellules hypothalamiques de souris GT1-7 traitées avec du palmitate pendant 24h. L'action de l’insuline est mesurée par la quantification d’Akt phosphorylée (western blot). Les céramides sont quantifiées par lipidomique, l'expression d’ARNm des gènes codant pour les enzymes de la voie de synthèse de novo des céramides par qRT-PCR. Des rats Zucker obèses sont perfusés avec la myriocine (inhibiteur de la synthèse de novo des céramides) en ICV pendant 21 jours. Des tests de sensibilité à l'insuline et de tolérance au glucose sont réalisés. A la fin du traitement, ils reçoivent une injection ICV d'insuline, la sensibilité à l’insuline ainsi que les taux de céramides sont quantifiés dans l’hypothalamus. Les îlots de Langerhans sont isolés pour des tests de sécrétion d'insuline.Nous avons mis en évidence une insulino-résistance dans la lignée hypothalamique GT1-7 traitées avec le palmitate qui s’accompagne d’une accumulation de céramides. En présence de myriocine, les céramides ne sont plus accumulés et le l’insulino-résistance induite par le palmitate est contre-carrée. En utilisant un inhibiteur de la PKCζ et un adénovirus codant pour un dominant-négatif de la PKCζ, nous avons montré que le palmitate n'est plus capable d'induire une insulino-résistance et ce malgré la présence d'une accumulation de céramides. Chez le rat Zucker obèse, nous avons mis en évidence une accumulation de céramides hypothalamiques qui est contre-carrée par la myriocine. Ceci est associé avec une amélioration de la sensibilité à l’insuline dans l’hypothalamus. De façon, intéressante, ces animaux améliorent leur tolérance au glucose qui est associée à une augmentation du tonus parasympathique conduisant à une augmentation de la sécrétion d’insuline. Les îlots de Langerhans isolés à partir de ces rats présentent une capacité sécrétoire augmentée lors du traitement avec la myriocine.Au final, notre étude révèle que la lipotoxicité hypothalamique est associée à une accumulation de céramides dans cette structure, responsable de l’installation d’une insulino-résistance. Ces résultats mettent également en évidence le rôle clé du métabolisme des céramides au niveau de l’hypothalamus dans la dérégulation du contrôle nerveux de l’homéostasie glucidique induit par l’obésité / Studies show that hypothalamic lipid accumulation is responsible for the development of central lipotoxicity, a phenomenon that could play a role in the installation of peripheral insulin resistance and type II diabetes by deregulating the nervous control of glucose homeostasis. It is known that the accumulation of ceramides is involved in the development of lipotoxicity of peripheral tissues. The objective of this study is to determine the role of the hypothalamic ceramide metabolism on the installation of a central insulin resistance and to study the mechanisms involved on this phenomenon. We also determined the role of hypothalamic ceramide metabolism in the deregulation of obesity-induced glucose homeostasis.The installation of a central insulin resistance is studied using in vitro approaches using hypothalamic GT1-7 mouse cells treated with palmitate for 24 hours. The action of insulin is measured by the quantification of phosphorylated Akt (western blot). The ceramides are quantified by lipidomic assay, mRNA expression of genes encoding enzymes of de novo synthesis pathway of ceramides by qRT-PCR. Obese Zucker rats were perfused with myriocin (an inhibitor of de novo synthesis of ceramides) in ICV for 21 days. Insulin sensitivity and glucose tolerance tests are performed. At the end of treatment, they receive an ICV injection of insulin, insulin sensitivity and ceramide levels are quantified in the hypothalamus. Islets of Langerhans are isolated for insulin secretion tests.We have demonstrated that palmitate is able to induce insulin resistance in the hypothalamic GT1-7, which is accompanied by an accumulation of ceramides. In the presence of myriocin, ceramides are no longer accumulated and the insulin resistance induced by palmitate is counteract. Using an inhibitor of PKCζ and an adenovirus encoding a dominant-negative of PKCζ, we have shown that palmitate is no longer able to induce insulin resistance despite the presence of an accumulation of ceramides. In the obese Zucker rat, we have demonstrated an accumulation of hypothalamic ceramides which is counteract by myriocin. This is associated with an improvement in insulin sensitivity in the hypothalamus. Interestingly, these animals improve their glucose tolerance which is associated with an increase in parasympathetic tone leading to an increase in insulin secretion. Islets of Langerhans isolated from these rats have increased secretory capacity when treated with myriocin.In conclusion, our study reveals that hypothalamic lipotoxicity is associated with an accumulation of ceramides in this structure, responsible for the installation of insulin resistance. These results also highlight the key role of ceramide metabolism at the hypothalamus level in the deregulation of nervous control of obesity-induced carbohydrate homeostasis

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-02-16T15:54:37Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) INFLUÊNCIA DA RADIAÇÃO GAMA NA COMPOSIÇÃO QUÍMICA DO TOMATE (Solanum Lycopersicum).pdf: 1401969 bytes, checksum: feb3a80a7b1738da16023772afc5c60f (MD5) / Made available in DSpace on 2017-02-16T15:54:37Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) INFLUÊNCIA DA RADIAÇÃO GAMA NA COMPOSIÇÃO QUÍMICA DO TOMATE (Solanum Lycopersicum).pdf: 1401969 bytes, checksum: feb3a80a7b1738da16023772afc5c60f (MD5) Previous issue date: 2016-07-29 / A técnica de conservação de alimentos por meio da radiação gama tem-se mostrado eficiente para o aumento do tempo de prateleira de diversos alimentos de origem vegetal. Dentre os vegetais com grandes índices de perda pós–colheita encontrasse o tomate (Lycopersicumesculentum), sendo este a segunda cultura agrícola, de maior difusão no mundo para consumo in natura ou processado. Este alimento tem sido amplamente estudado, uma vez que contêm diversos antioxidantes, como carotenóides, vitamina C, além de tocoferóis e flavonóides que contribui para diversas funções no organismo. Este trabalho tem por objetivo analisar os efeitos da radiação gama nos teores de licopeno, β-caroteno, vitamina C, pH, acidez titulável, coliformes e Samonellas, em tomates comercializados em feiras livres de Recife–PE. Sendo os tomates divididos em três grupos, um controle (não irradiado) e dois irradiados a uma dose de 0,5 kGy e 1,0 kGy a partir de uma fonte de 60Co, com taxa de dose de 2,629 kGy/h. Dentre as observações constatadas pode-se verificar que a radiação ionizante provocou uma diminuição nos teores de licopeno e β- caroteno. Constatou-se uma redução nos teores de vitamina C para as amostras irradiadas, sendo que a dose de 1,0 kGy apresentou maior redução. A análise estatística comprovou que a radiação gama nos tomates estudados permitiu um aumento no tempo de prateleira, evidenciando ser está uma técnica efetiva para conservação no pós-colheita. / The food storage technique by means of gamma radiation has proved effective in increasing the shelf life of many foods of plant origin. Among the vegetables with large post-harvest loss ratios found tomatoes (Lycopersicumesculentum), which is the second crop, the most widespread in the world for fresh consumption or processed. This food has been widely studied since they contain many antioxidants such as carotenoids, vitamin C, and tocopherol, and flavonoids that contributes to various functions in the body. This work aims to analyze the effects of gamma radiation on lycopene content, β-carotene, vitamin C, pH, titratable acidity, coliforms and Samonellas in tomatoes sold in street markets of Recife-PE. As the tomatoes divided into three groups, a control (non-irradiated) and two irradiated at a dose of 0.5 kGy 1.0 kGy from a 60Co source, dose rate of 2,629 kGy / hr. Among the noted observations it can be seen that ionizing radiation caused a decrease in levels of lycopene and β- carotene. It was observed a reduction in vitamin C content for samples irradiated, and the dose of 1.0 kGy showed a reduction. Statistical analysis has shown that the gamma radiation tomatoes studied led to an increase in shelf life, thus demonstrating that it is an effective technique for conservation in postharvest.

>Magister Scientiae - MSc / The complete degradation of recalcitrant lignocellulose biomass into value-added products requires the efficient and synergistic action of lignocellulose degrading enzymes. This has resulted in a need for the discovery of new hydrolytic enzymes which are more effective than commonly used ones. β-xylosidases and β-glucosidases are key glycoside hydrolases (GHs) that catalyse the final hydrolytic steps of xylan and cellulose degradation, essential for the complete degradation of lignocellulose. Functional-based metagenomics has been employed successfully for the identification and discovery of novel GH genes from a metagenome library. Therefore, this approach was used in this study to increase the chances of discovering novel glycoside hydrolase genes from a horse manure metagenomic DNA library constructed in a previous study. Three fosmid clones P55E4, P81G1, and P89A4 exhibiting β-xylosidase activity were found to encode putative glycosyl hydrolases designated XylP55, XylP81, and BglP89. Amino acid sequence analysis revealed that XylP55, XylP81, and BglP89 are members of the GH43, GH39, and GH3 glycoside hydrolase families, respectively. Phylogenetic analysis of XylP81 and BglP89 indicated that these showed relatively low sequence similarities to other homologues in the respective GH families. The enzymes were expressed and purified, and only XylP81 and BglP89 were biochemically characterized. XylP81 (~58 kDa) and BglP89 (~84 kDa) both showed optimum activity at pH 6 and 50℃ and retained 100% residual activity at 55℃ after 1-hour indicating that they are moderately thermostable. XylP81 had high specific activity against 4-nitrophenyl-β-D-xylopyranoside (pNPX; 122 U/mg) with a KM value of 5.3 mM, kcat/KM of 20.3 s-1mM-1, and it showed enzyme activity against α-L-arabinofuranosidase, β-galactosidase, and β-glucosidase activity. BglP89 had a high specific activity for 4-nitrophenyl-β-D-glucopyranoside (pNPG; 133.5 U/mg) with a KM value of 8.4 mM, kcat/KM of 22 s-1mM-1 and also showed α-L-arabinofuranosidase, β-galactosidase, β-glucosidase, and low β-xylosidase activity. BglP89 also showed low hydrolytic activity on cellobiose, β-glucan, and lichenan indicating that it is a broad specificity β-glucosidase. XylP81 retained ~40% activity in the presence of 3 M xylose whilst BglP89 showed considerable glucose tolerance at 150 mM glucose and retained ~46% residual activity. This study reveals two metagenomic derived enzymes (β-xylosidase and β-glucosidase) showing characteristics that could make them potential candidates for lignocellulose biomass degradation in biotechnological and industrial applications.

The effects of supplementation of the leucine metabolite β-hydroxy-β- methylbutyrate (HMB) were examined in a resistance training study. Thirty- nine men and 36 women between the ages of 20-40 y were randomized to either a placebo (P) supplemented or HMB supplemented (3.0 g HMB/d) group in two gender cohorts. All subjects trained three times per week for 4 wk. In the HMB group, plasma creatine phosphokinase levels tended to be suppressed compared to the placebo group following the 4 wk of resistance training (HMB:174.4 ± 26.8 to 173.5 ± 17.0 U/L; P:155.0 ± 20.8 to 195.2 ± 23.5 U/L). There were no significant differences in strength gains based on prior training status or gender with HMB supplementation. The HMB group had a greater increase in upper body strength than the placebo group (HMB:7.5 ± 0.6 kg; P:5.2 ± 0.6 kg; P = 0.008). The HMB groups increased fat-free weight by 1.4 ± 0.2 kg and decreased percent fat by 1.1% ± 0.2% while the placebo groups increased fat-free weight by 0.9 ± 0.2 kg and decreased percent fat by 0.5% ± 0.2% (fat-free weight P = 0.08, percent fat P = 0.08, HMB compared to placebo). In summary, this is the first short-term study to investigate the roles of gender and training status on the effects of HMB supplementation on strength and body composition. This study showed, regardless of gender or training status, HMB may increase upper body strength and minimize muscle damage when combined with an exercise program.

The use of 4-acetoxy-2,2-dimethylbutanoyl protecting group for the C2-hydroxyl allows the selective formation of β-glycosides without producing α-glycosides. This very bulky protecting group can be removed under mild conditions.

Indiana University-Purdue University Indianapolis (IUPUI) / A hallmark of diabetes is the loss of physical or functional Beta (β) cell mass. Maladaptive intrinsic β cell responses to islet inflammatory stress may exacerbate diabetes development, suggesting that β cells themselves may not be innocent bystanders in diabetes development. MicroRNAs (miRNAs), small RNAs that repress mRNA translation, serve as important regulators of β cell development and function. β cell microRNA 21 (miR-21) is increased in models of diabetes and I have identified Hypoxia Inducible Factor 1 Subunit Alpha (Hif1a) as a regulator of β cell miR-21. However, β cell effects of miR-21, remain poorly defined. To define the effects of miR-21, an in silico analysis of predictive targets of miR-21 identified multiple targets associated with maintenance of β cell identity, including the SMAD Family Member 2 (Smad2) mRNAs in the Transforming Growth Factor Beta 2 (Tgfb2) pathway. Based on this, I hypothesized that β cell miR-21 induces dysfunction via loss of β cell identity. To test this, I developed a tetracycline-on system of miR-21 induction in clonal β cells and human islets, as well as novel transgenic zebrafish and mouse models of inducible β cell specific miR-21 overexpression. β cell miR-21 induction increased aldehyde dehydrogenase (aldh1a3), but reduced expression of transcription factors associated with β cell identity, and glucose stimulated insulin secretion (GSIS), consistent with β cell dedifferentiation and dysfunction. Predicted targets Tgfb2 and Smad2 were reduced by miR-21 overexpression and confirmed to directly bind miR-21 using streptavidin-biotin pulldown. In vivo models of β cell miR-21 induction exhibited hyperglycemia, increased glucagon expression, and decreased insulin expression. These findings implicate miR-21- mediated reduction of mRNAs regulating β cell identity as a contributor to β cell dedifferentiation and dysfunction during islet inflammatory stress. / 2022-05-19

In the study of structurally restricted cyclic β-amino acids and peptides, methanopyrrolidine-5-carboxylic acids (MetPyr-5-acids), or 5-syn-carboxy-2-azabicyclo[2.1.1] hexanes, and derivatives were investigated. MetPyr-5-acids are a series of highly conformationally constrained β-proline derivatives, which belong to a novel category of β-amino acids utilized as building blocks for the synthesis of β-peptides. These β-peptides lack the backbone hydrogen bonds necessary for folding in the usual manner. Substituents and functional groups in this ring system were envisioned to impact the folding properties and functionalities of the corresponding β-peptides. In the present study, the analogues of MetPyr-5-acids with C6- substitutions were prepared, and the folding properties of their peptides were explored. To introduce different functionalities at C6 in MetPyr-5-acids, 6-syn-hydroxymethyl substituted derivatives were synthesized and were used as key intermediates. In the synthesis of this core structure, the major steps in their preparation included the Michael addition of benzyloxymethyl allyl amine to 3-butynone, followed by UV light irradiation of the diene to afford 5-acetyl-6-benzyloxymethyl-2-azabicyclo[2.1.1]hexane. Haloform (Br2/NaOH) oxidation of the acetyl group leads to the 6-substituted MetPyr-5-acid. Resolution of the racemate was achieved either by resolving (±)-6-syn-benzyloxymethyl-MetPyr-5-acid via a classical crystallization resolution method using (S)-(-)-α-methylbenzylamine, or by chiral preparative HPLC separation of (±)-6-syn-benzyloxymethyl-MetPyr-5-acid methyl ester. The absolute stereochemistry was confirmed by X-ray crystallography of a derivative. Novel analogs with a range of functionalities incorporated at the C6 position in MetPyr-5-acid were synthesized from 6-syn-hydroxymethyl-MetPyr-5-acid methyl ester, and include hydrophilic groups such as hydroxyl, amino, methyl ether, and hydrophobic groups, such as substituted phenyl groups and triazole. From the protected C6-substituted analogs of MetPyr-5-acids, peptide oligomers of C6-benzyloxymethyl-2,4-methanopyrrolidine-b-amino acid were prepared up to the length of octomer in high yields. This series of oligomers were characterized by circular dichroism (CD) and indicated enhanced order of folding uniformity for the tetramer and up, with increasing ordered folding for longer oligomers. The octomer exhibited minimal solvent effects, and was stable with increasing temperature up to 80 °C. Analysis by NMR of the iso-butyric amide capped monomer indicated a mixture of cis/trans conformation favoring the cis conformation. This was slightly different from the C6 unsubstituted iso-butyric amide derivative, which favored the trans conformation. For the dipeptide, the C6-benzyloxymethyl substitution increased the percentage of cis conformation of the dipeptide amide bond, but the major peptide had the trans conformation. This demonstrated that C6 substitutions could shift the cis/trans equilibrium towards the cis conformation. Longer oligomers showed ordered secondary folding structure as demonstrated by the increase in ellipticity per amino acid unit, but was too complicated to be determined by NMR analysis. Both the CD patterns and molecular model calculation predicted that the longer oligomers (tetramer and above) favor the trans conformation. This preference was driven by the backbone dipole effect. II. SYNTHESIS OF 2,2-DISUBSTITUTED PYRROLIDINE-3-CARBOXYLIC ACIDS Due to the perceived steric influence of 2,2-disubstitution in the pyrrolidine-3-carboxylic acid, it is believed that the adjacent amide/peptide bonds should result in a trans amide bond conformation. Because of the difficulty in introducing disubstitution at the hindered C2 position, the synthesis of such derivatives has not been successful. For this reason a new method was introduced to prepare novel derivatives, at the N- and C- termini of protected 2,2-dimethyl pyrrolidine-3-carboxylic acid, i.e., benzyloxycarbonyl protected 2,2-dimethylpyrrolidine-3-carboxylate. This procedure included the Michael addition of 2-nitropropane to dimethyl fumarate, followed by ring closure of the amino ester derived from reduction of the nitro ester providing the pyrrolidinone. Reduction of the pyrrolidinone to the pyrrolidine with borane finished 2,2-dimethylpyrrolidine-3-carboxylate in moderate overall yield. A preliminary set of two amides, iso-butyric amide and 3,5-dichlorobenzamide of this 2,2-dimethylpyrrolidine-3-carboxylate, were also prepared. NMR analysis of this pyrrolidine derivative suggested the amide bonds adopted the trans conformation. It was concluded that steric bulk of the 2,2-disubstitution favorably influenced the trans amide conformation. This demonstrated that trans amide conformation control of a β-proline amide was possible. / Chemistry