Master of Science / Food Science Institute, Agriculture / Katherine A. Tilley / Michael Tilley / The wheat tortilla is a chemically leavened circular light colored flat bread. Desirable characteristics for good quality tortilla include large diameter, softness, flexibility and long shelf stability. Important components influencing quality are wheat flour properties, which have not been optimized for tortilla industrial production thus far. The studies presented here investigated the effects of high molecular weight glutenin subunits (HMW-GS) on tortilla quality. Two approaches were employed: biotypes derived from Centurk and OK102 cultivars expressing defined HMW-GS compositions and transgenic wheat lines over-expressing HMW-GS 10. Analysis of protein expression and protein extractability were conducted to characterize wheat flours and suitable assays carried out to determine the respective dough properties. Tortillas were prepared by the hot-press method and quality parameters were measured at days 0, 2, 4, 7 and 14. Tortillas derived from Centurk biotypes possessing HMW-GS 2*, 7+9, 2+12, 2*, 7+8, 5+10 and 2*, 7+9, 5+10 exhibited superior texture profiles over time, but smaller diameters than the biotype 2*, 7+8, 2+12. Tortillas containing HMW-GS 7+9 and 2+12 revealed a texture profile similar to tortillas containing 5+10. Tortillas from the OK biotype 2*, 7+9, 3+12 exhibited larger diameter and texture profiles equivalent to tortillas containing 5+10. Therefore, this biotype showed the best quality within this cultivar. Tortillas derived from transgenic flours over-expressing HMW-GS 10 exhibited an undesirable rough appearance with decreased diameter, greater thickness, lower rollability scores, lower stretchability and greater rupture force over time. Over-expression of HMW-GS 10 in a wheat line containing 1RS-translocation did not promote the same deleterious effects in tortilla quality as it did in transgenic lines without 1RS translocation.

Staphylococcus aureus is a highly problematic human pathogen due to its ability to cause devastating infections, paired with a capacity to withstand the action of a large fraction of available antibiotics. Both pathogenicity and antibiotic resistance are encoded by numerous genomic elements, though the expression of these factors is energetically costly and not always beneficial for cellular survival. Therefore, S. aureus has developed sophisticated regulatory networks to integrate a multitude of signals, enabling it to navigate the delicate balance between its pathogenic lifestyle and baseline needs for cellular energy homeostasis. It is thus imperative to study S. aureus behavior and its underlying regulatory circuits not as isolated entities, but rather holistically as part of an optimized, highly interconnected system. To do so, we must seek to achieve a comprehensive understanding of all encoded regulators, that is, not only historically well defined elements like transcription factors, two-component systems and σ factors, but also the lesser studied ’non-classical’ regulators like small regulatory RNAs and regulatory subunits of RNA-dependent RNA polymerase (RNAP). To this end, we describe here the identification of numerous, previously unidentified sRNAs and their incorporation into a new standardized cataloging and annotation system. The identification and annotation procedures are based on a number of RNAseq experiments performed in three different S. aureus backgrounds (MRSA252, NCTC 8325, and USA300). We then apply RNAseq to evaluate the expression patterns of these elements when grown in human serum, thus probing for possible connections between sRNAs and S. aureus pathogenicity. In addition, we characterize the role of two small RNAP subunits, δ and ω, for S. aureus RNAP function. δ is of particular interest, as it is unique to Gram-positive bacteria; deletion of the subunit results in a loss of transcriptional stringency and decreased expression of numerous virulence determinants. These alterations are accompanied by impaired survival of the δ mutant in whole human blood, increased phagocytosis by human leukocytes, and decreased survival in a murine model septicemia when compared to its parental strain. In contrast, there is no indication of direct and gene-specific transcriptional functions for ω. Rather, we describe a role for ω in the structural integrity of the RNAP complex, where its loss leads to a structural disturbance in the RNAP complex that causes altered affinities for (alternative) σ factors and the δ subunit. Overall, the findings presented here contribute to a better understanding of the intricate regulatory systems that guide the lifestyle of an organism that presents an immense burden to patients and our health care system alike.

Morphine is a clinically very important drug from the opioid group that is used for treatment of severe pain because of its strong analgetic effect. Opioid receptors mediating the morphine effect interact with the Gi/o class of trimeric G-proteins. Opioid receptors also occur in heart tissue and morphine can thus potentially exercise its effect on the function of this organ. The major aim of this project was to pursue consequences of long-term treatment with morphine on expression and distribution of selected heterotrimeric G-protein subunits in the rat heart. Potential cardioprotective effects of this drug have also been studied. Laboratory rats of the Wistar strain were treated with morphine (1 mg/kg/day or 10 mg/kg/day) for 10 or 28 days. The control group was treated with saline solution. Prolonged treatment with morphine did not cause any effects on Gs, Gi, Gz, Gq/11, G subunits, but the expression of Go rather decreased. The results of subsequent experiments showed that prolonged administration of high doses of morphine may reduce the area affected by infarction and reduced the frequency of ventricle arrhythmias depending on dose and duration of morphine administration. Key words: morphine, myocardium, opioid receptor, G-protein subunits, infarction.

Signal transduction by G-proteins (a heterotrimer membrane protein composed of an α, β, and γ subunit) requires that the α-subunit undergoes a transition from a GDP-bound inactive state to an activated GTP-bound state. The exchange of GDP for GTP leads to a conformational change in the α-subunit that results in the loss of affinity for the βγ subunits. We predicted that appropriate genetic manipulation of key regions of the α-subunit could result in the induction of the active conformation that would mimic at least in part the activated GTP-bound state. We have demonstrated that the substitution of the 38 amino acid residue carboxyl termimus of Gαs with the last 36 amino acid residues of Gαi2 resulted in a chimeric Gα-subunit (C4) that exhibits a constitutively active Gαs-like activity. Similarly, the substitution of the amino terminal 61 amino acid residues of Gαs with the first 54 residues of Gαi2 also resulted in a chimeric Gα-subunit that is persistently active (Gs like). We have also generated point mutations in the Gαs subunit that are comparable to the activating mutations in the ras protein. Our results suggest that point mutations in the signature sequence of the A (Val 49) and C (Thr 225) homologous regions that are implicated in regulating the GTPase activity of the molecule also resulted in the activation of the subunit. The present study has identified four key regions of the α-subunit that are critical for the activity and regulation of the Gs protein.

<p>Ribosome assembly is a complex process, facilitated by more than 20 protein factors in bacteria. GTPases and ATPases represent the energy driving force of these factors. In my research as a PhD student, I studied the function of two GTPases, YjeQ and RbgA, involved in the assembly of the small and the large ribosomal subunits, respectively.</p> <p>We isolated and characterized <em>in-vivo</em> assembled immature small (30S) and large (50S) subunits using a perturbation in the genes coding for these proteins. We observed that both subunits contained an incomplete ribosomal protein content, mainly lacking late-binding r-proteins. Additionally, we observed distortions in the functional core of the immature ribosomal subunit, particularly in the mRNA decoding center of the 30S subunit, the peptidyltransferase center of the 50S subunit, and tRNA binding sites.</p> <p>Additionally, we have determined that the YjeQ protein interacts with the 30S subunit through its N-terminal OB-fold domain, and C-terminal Zn-finger motif. The binding site of YjeQ on the 30S subunit prevents the interaction with tRNAs, translation factors, and the 50S subunit.</p> <p>Finally, we uncovered a novel functional interplay between RbgA and the ribosomal protein L16 during late stages of ribosomal assembly. We proposed that recruitment of L16 to the assembling 50S subunit would induce a conformational rearrangement that would ultimately promote the GTP-dependent release of RbgA.</p> <p>The function of the assembly factors associated with the process of <em>in-vivo</em> ribosome assembly is not known, and thus a framework on how ribosomes are built is still elusive. I believe the research presented in this thesis provides novel insights into the role of YjeQ and RbgA in the assembly of ribosomes</p> / Doctor of Philosophy (PhD)

La protéine kinase caséine kinase 2 (CK2) est une sérine/thréonine kinase hautement pléiotrope dont la liste des substrats est supérieure à 500 protéines, lesquelles sont impliquées dans un large éventail de fonctions cellulaires. Les sous-unités catalytiques de CK2 (alpha et/ou alpha') sont constitutivement actives soit seules soit en combinaison avec les sous-unités régulatrices béta pour former une protéine hétérotétramérique (holoenzyme). Une troisième isoforme de la sous-unité catalytique, désignée CK2α'', a été découverte plus récemment et peu d'informations sont actuellement disponibles. L'activité hautement constitutive de CK2 est suspectée de contribuer au phénomène de néoplasie. Une stratégie de conception d'inhibiteurs tétracycliques ciblant le site ATP de la CK2 a permis l'élaboration de trois séries de composés comportant le motif indéno[1,2-b]indole. Un procédé multi-étapes de synthèse a permis de fonctionnaliser précisément le cycle D du noyau indéno[1,2-b]indole et de générer une première chimiothèque de molécules originales. Toutes les molécules finales ont été testées sur la protéine kinase humaine CK2 (Muenster) et certaines ont présentées des CI50 de l'ordre du submicromolaire. L'analyse des Relations Structure-Activité (SAR) et la construction d'un modèle 3D-QSAR (Duesseldorf) a contribué à affiner le choix des substituants introduits sur le châssis moléculaire développé. Les indéno[1,2-b]indoles fonctionnalisés les plus prometteurs ont été également testés sur d'autres cibles biologiques comme la phosphatase CDC25A (Metz) et la kinase DYRK1B (Saarbruecken). Des études de modélisation moléculaire (Duesseldorf) utilisant les données cristallographiques disponibles de l'enzyme ont permis d'analyser les interactions ligand-protéine. Les inhibiteurs les plus puissants in vitro ont été testés sur quatre lignées cellulaires normales afin d'établir leur profil cytotoxique (Centre de Recherche en Cancérologie de Lyon) / Synthesis and evaluation of indéno[1,2-b]indole derivatives as potential inhibitors of human protein kinase CK2 Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase highly pleiotropic listed substrates it is greater than 500 proteins, which are involved in a wide range of cellular functions. The catalytic subunits of CK2 (α and/or α') are constitutively active either alone or in combination with the regulatory subunits to form a hetero- beta protein holoenzyme). A third isoform of the catalytic subunit, designated CK2 α', was discovered more recently and little information is currently available. The high constitutive activity of CK2 is suspected of contributing to the phenomenal of neoplasia. A design strategy tetracyclic inhibitors targeting the ATP site of CK2 resulted in the development of three series of compounds containing the motif indeno[1,2-b]indole. A multi-step synthesis process has specifically functionalize the D ring of the core indeno[1,2-b]indole and generate a first combinatorial library of original molecules. All final compounds were tested on human protein kinase CK2 (Muenster), and some have reported IC50 of the order of sub-micromolar. Analysis of Structure-Activity Relationships (SAR) and the construction of a 3D-QSAR model (Duesseldorf) helped to refine the choice of substituents introduced into the moleculair frame developed. The indeno[1,2-b]indole the most promising functionalized indoles were also tested on other biological targets such as phosphatase CDC25 A (Metz) and kinase DYRK1B (Saarbruecken). Of molecular modeling studies (Duesseldorf) using the crystallographic data of the enzyme were used to analyze protein-ligand interactions. The most potent in vitro inhibitor were tested on four normal cell lines to determine their cytotoxic profile (Cancer Research Center of Lyon)

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. / Bizkaia:Talent and the European Union's Seventh Framework Program (Marie Curie Actions; COFUND; to S.C., A.S., T.K.); Marie Curie Actions Career Integration Grant (PCIG14-GA-2013-632072 to P.F.); Ministerio de Economía Y Competitividad (CTQ2014-55907-R to P.F., S.C.); FIRB Futuro in Ricerca from the Italian Ministero dell'Istruzione, dell'Universitá e della Ricerca (RBFR130VS5_001 to A.F.); Peruvian Programa Nacional de Innovación para la Competitividad y Productividad (382-PNICP-PIBA-2014 (to P.M. and A.F.)). Funding for open access charge: Institutional funding. / Revisión por pares

Foi levado a efeito estudo sobre formas múltiplas de aldolase de músculo de coelho. A enzima foi purificada a pH 7,5 por eluição com substrato a partir de coluna de fosfocelulose. A enzima foi ainda cristalizada por diálise dessas preparações contra solução saturada de sulfato de amônio. Formas múltiplas de aldolase foram obtidas por fracionamento a diferentes pI por eletrofocalização em gradiente de Ampholine na faixa de pH entre 7,0 a 10,0. Nessas condições foram separados cinco híbridos resultantes da associação ao acaso das sub-unidades &#945; e &#946;, os quais foram analisados em estado de dissociação a partir de proteínas carboximetiladas e separadas por eletroforese em gel de poliacrilamida na presença de uréia 8M. Foi também estudado o aparecimento de sub-unidade &#945; e &#946; em músculo de coelhos de idades que variavam de 1 a 240 dias, verificando-se que em coelhos de 1 dia existia apenas a proteína &#945;4, surgindo sub-unidades &#946; já em animais de 10 dias. / Not available.

A hemoglobina extracelular gigante do anelídeo Glossoscolex paulistus (HbGp) é homológa à hemoglobina da Lumbricus terrestris (HbLt). Baseado nos estudos de MALDI-TOF-MS foi determinada a massa molecular (MM) das subunidades da HbGp. Entretanto, ainda não era possível propor o valor exato da MM para a HbGp íntegra, pois a estequiometria deste oligômero ainda não era totalmente clara. Este trabalho objetiva avaliar a massa molecular do oligômero em dois estados de oxidação: oxi- e cianometa-HbGp, bem como avaliar a estabilidade desta proteína, ou seja, a dissociação e desnaturação em função do pH em meio ácido. O estudo por ultracentrifugação analítica permitiu uma avaliação independente da massa molecular da HbGp. Valores de MM de 3600 &plusmn; 100 e 3700 &plusmn; 100 kDa foram obtidos para a oxi- e cianometa-HbGp, respectivamente. Estes valores está de acordo com a massa esperada, assumindo a estequiometria proposta por Vinogradov para a HbLt. Os dados de ultracentrifugação para as amostras do monômero d puro mostraram um coeficiente de sedimentação de 1,95 &plusmn; 0,04 S para ambos os valores de pH 7,0 e 10,0. Além disso, as distribuições c (s) do monômero d puro indicaram que uma pequena contribuição (5%) de dímero de monômeros, d2, com valores de s020,w de 3,2 S estava presente em solução. Para a oxi-HbGp íntegra no pH 10,0 nenhuma contribuição em 58 - 59 S foi observada, sugerindo completa dissociação oligomérica. As distribuições c (s) mostraram dois picos adicionais em relação ao monômero puro: um pico em 4,2 - 4,4 S, que está associado ao trímero, abc; e um segundo pico em 5,8 - 6,0 S, que poderia ser associado ao tetrâmero, abcd. A adição de &beta;-mercaptoetanol leva ao desaparecimento do pico em 4,2 S, consistente com a redução das pontes dissulfeto do trímero abc e produção dos monômeros a, b e c. Cerca de 19 % da forma cianometa-HbGp íntegra coexiste em equilíbrio com as subunidades dissociadas. Finalmente, estudos em meio ácido mostravam que na faixa de pH 5,0 - 7,0 as três formas de oxidação de HbGp apresentaram alta estabilidade oligomérica. Abaixo de pH 5,0 os dados de fluorescência mostrava que a estabilidade diminui na sequência cianometa > oxi > meta. Assim a estabilidade das formas oxi-, meta- e cianometa-HbGp foi avaliada, ficando evidenciada a maior estabilidade da forma cianometa-HbGp. / The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is homologous to Lumbricus terrestris (HbLt). Based on MALDI-TOF-MS the molecular masses (MM) of HbGp subunits were determined. However, the exact value of the MM for the HbGp oligomer is not known. This study has as a main goal to evaluate the molecular weight of the oligomer in two oxidation states: oxy- and cyanomet-HbGp. Also the stability of the protein dissociation and denaturation as a function of pH was monitored. The present analytical ultracentrifugation study allowed us to assess the molecular mass of the whole oligomer giving valores of MM of 3600 &plusmn; 100 and 3700 &plusmn; 100 kDa for the oxy- and cyanomet-HbGp, respectively. These values are in agreement with the expected mass based on Vinogradov model for HbLt. Data were obtained of so20, w for the pure monomer d as 1.95 &plusmn; 0.04 S for both pH values 7.0 and 10.0. C(s) distributions for pure monomer indicated that a small contribution of dimer of monomers (5%), d2, was also present with so20, w of 3.2 S in solution. For the whole oxy- HbGp at pH 10.0 no contribution at 58 - 59 S was observed, suggesting complete oligomeric dissociation. C(s) distribution showed two additional peaks as compared to pure monomer: a peak at 4.2 - 4.5 S, probably due to the trimer, abc; a second peak at 5.8 - 6.0 S, that could be associated to the tetramer, abcd. Addition of beta-mercaptoethanol leads to the disappearance of the peak at 4.2 S, consistent with the reduction of the trimer abc disulfide bridges and production of monomers a, b and c. It may be noted that about 19% of cyanomet-HbGp, undissociated, coexist in equilibrium with the isolated subunits. Finally, studies in acidic pH values show that in the pH range 5.0-7.0 the oligomeric stability for the three oxidation forms of HbGp is quite high. Below pH 5.0, fluorescence emission data suggest that the stability is reduced in the following order: cyanomet > oxy > met-HbGp. Thus the stability of the oxy-, meta- and cyanomet-HbGp forms was evaluated evidenced making it clear the higher stability of the cyanomet-HbGp.

Introdução: Os receptores ionotrópicos do glutamato, como o N-metil-D-Aspartato (NMDA), estão envolvidos em desordens psiquiátricas. NMDARs são complexos heteroméricos que incorporam tres diferentes subunidades: NR1, NR2 e NR3. Objetivos: quantificar os níveis plasmáticos das subunidades NR1 e NR2 NMDAR em pacientes em primeiro episódio psicótico (PEP), em comparação com os irmãos e controles saudáveis. Métodos: Este é um estudo transversal de PEP na região de Ribeirão Preto, Brasil, sendo o grupo controle composto por indivíduos saudáveis, pareados por idade, sexo e mesma área de abrangência dos casos. Foram coletados 5 mL de amostra de sangue próxima a data de diagnóstico de PEP. A quantificação plasmática das subunidades NR1 e NR2 foi realizada por ELISA. Os dados foram analisados por ANOVA (significante se p<0,05) e curva ROC. Resultados: Foram incluídos 166 pacientes em PEP (idade: x = 30,34 ± 12,2 anos; 64% homens), destes 84 com diagnóstico de psicose não afetiva, 51 com transtorno bipolar e 31 com transtorno depressivo. Foram tambem incluídos 76 irmãos e 166 controles saudáveis. Os níveis plasmáticos das subunidades NR1 e NR2 foram significativamente menores em pacientes com transtornos psicóticos (NR1: x = 71,0 ± 100,3 pg/mL, NR2: x = 2,5 ± 2 ng/ml), transtorno bipolar (NR1: x = 185,7 ± 319,5 pg/ml; NR2: x = 2,1 ± 2,2 ng/ml), transtorno depressivo (NR1: x = 83,2 ±185,0 pg/ml; NR2: x = 2,1± 2,1 ng/ml) em comparação com os irmãos (NR1: x = 140,6 ± 193,8 pg/ml; NR2: = 6,2 ± 1,5 ng/ml) e voluntários saudáveis (NR1: x = 146,7 ± 361,1 pg/ml; NR2: x = 4,8 ± 2,2 ng/ml) [NR1 e NR2, p < 0,001]. Indivíduos com valores plasmáticos de NR2 inferiores a 3,648 ng/mL apresentam um risco 14,72 vezes maior de estar doente (PEP) de quem não possui o NR2 abaixo deste valor. Conclusões: Este é o primeiro estudo relatando a quantificação e a redução das concentrações plasmaticas das subunidades NR1 e NR2 em transtornos psiquiátricos graves quando comparados aos irmãos e controles, podendo a subunidade NR2 ser um candidato a biomarcador plasmático em pacientes com PEP. / Background: Ionotropic glutamate receptors, such as N-Methyl-D-Aspartate (NMDA), are involved in pathophysiology of several psychiatric disorders. NMDARs are described as heteromeric complexes incorporating distincts subunits within a repertoire of three types: NR1, NR2 and NR3. Aim: to quantify the NR1 and NR2 subunits NMDAR plasma levels in patients with first episode psychosis (FEP), compared with siblings and healthy controls. Methods: This is a cross-sectional study of FEP conducted in Ribeirão Preto, Brazil. The control group were composed by healthy subjects matched for age, sex and same coverage area of cases. 5 ml of blood sample were collected next to the date of FEP diagnosis. NR1 and NR2 subunits plasmatic quantification was performed by ELISA. Data were analyzed by ANOVA (significant at p < 0.05) and ROC curve. Results: FEP sample comprised 166 patients (age: x = 30.34 ± 12.2 years; 64% men), of these 84 with a diagnosis of psychotic disorder, 51 with bipolar disorder and 31 with depressive disorder. It was also included 76 siblings and 166 healthy controls. NR1 and NR2 subunits plasma levels were significantly lower in patients with psychotic disorders (NR1: x = 71.0 ± 100.3 pg / ml, NR2: x = 2.5 ± 2 ng/ml), bipolar disorder (NR1: x = 185.7 ± 319.5 pg/mL; NR2: x = 2.1 ± 2.2 ng/ml), depressive disorders (NR1: x = 83.2 ± 185.0 pg/mL; NR2: x = 2.1 ± 2.1 ng/ml) compared with siblings (NR1: x = 140.6 ± 193.8 pg/mL; NR2: x = 6.2 ± 1.5 ng/ml) and healthy volunteers (NR1: x = 146.7 ± 361.1 pg / mL; NR2: x = 4.8 ± 2.2 ng/ml) [NR1 and NR2, p < 0.001]. Interestingly, individuals with NR2 plasma values less than 3.648 ng/ml present 14.72 times a higher risk to be in FEP than other patients. Conclusions: This is the first study reporting the measurement and reduction of NR1 and NR2 subunits plasma concentrations in severe psychiatric disorders when compared to siblings and controls. And highlighting that NR2 subunit can be a candidate for plasma biomarker in patients with FEP.