Tesis: "Proteins alternatives"

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Dehouck, Yves. "Développement de potentiels statistiques pour l'étude in silico de protéines et analyse de structurations alternatives". Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211040.

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Cette thèse se place dans le cadre de l'étude in silico, c'est-à-dire assistée par ordinateur, des liens qui unissent la séquence d'une protéine à la (ou aux) structure(s) tri-dimensionnelle(s) qu'elle adopte. Le décryptage de ces liens présente de nombreuses applications dans divers domaines et constitue sans doute l'une des problématiques les plus fascinantes de la recherche en biologie moléculaire.

Le premier aspect de notre travail concerne le développement de potentiels statistiques dérivés de bases de données de protéines dont les structures sont connues. Ces potentiels présentent plusieurs avantages: ils peuvent être aisément adaptés à des représentations structurales simplifiées, et permettent de définir un nombre limité de fonctions énergétiques qui incarnent l'ensemble complexe d'interactions gouvernant la structure et la stabilité des protéines, et qui incluent également certaines contributions entropiques. Cependant, leur signification physique reste assez nébuleuse, car l'impact des diverses hypothèses nécessaires à leur dérivation est loin d'être clairement établi. Nous nous sommes attachés à l'étude de certaines limitations des ces potentiels: leur dépendance en la taille des protéines incluses dans la base de données, la non-additivité des termes de potentiels, et l'importance souvent négligée de l'environnement protéique spécifique ressenti par chaque résidu. Nous avons ainsi mis en évidence que l'influence de la taille des protéines de la base de données sur les potentiels de distance entre résidus est spécifique à chaque paire d'acides aminés, peut être relativement importante, et résulte essentiellement de la répartition inhomogène des résidus hydrophobes et hydrophiles entre le coeur et la surface des protéines. Ces résultats ont guidé la mise au point de fonctions correctives qui permettent de tenir compte de cette influence lors de la dérivation des potentiels. Par ailleurs, la définition d'une procédure générale de dérivation de potentiels et de termes de couplage a rendu possible la création d'une fonction énergétique qui tient compte simultanément de plusieurs descripteurs de séquence et de structure (la nature des résidus, leurs conformations, leurs accessibilités au solvant, ainsi que les distances qui les séparent dans l'espace et le long de la séquence). Cette fonction énergétique présente des performances nettement améliorées par rapport aux potentiels originaux, et par rapport à d'autres potentiels décrits dans la littérature.

Le deuxième aspect de notre travail concerne l'application de programmes basés sur des potentiels statistiques à l'étude de protéines qui adoptent des structures alternatives. La permutation de domaines est un phénomène qui affecte diverses protéines et qui implique la génération d'un oligomère suite à l'échange de fragments structuraux entre monomères identiques. Nos résultats suggèrent que la présence de "faiblesses structurales", c'est-à-dire de régions qui ne sont pas optimales vis-à-vis de la stabilité de la structure native ou qui présentent une préférence marquée pour une conformation non-native en absence d'interactions tertiaires, est intimement liée aux mécanismes de permutation. Nous avons également mis en évidence l'importance des interactions de type cation-{pi}, qui sont fréquemment observées dans certaines zones clés de la permutation. Finalement, nous avons sélectionné un ensemble de mutations susceptibles de modifier sensiblement la propension de diverses protéines à permuter. L'étude expérimentale de ces mutations devrait permettre de valider, ou de raffiner, les hypothèses que nous avons proposées quant au rôle joué par les faiblesses structurales et les interactions de type cation-{pi}. Nous avons également analysé une autre protéine soumise à d'importants réarrangements conformationnels: l'{alpha}1-antitrypsine. Dans le cas de cette protéine, les modifications structurales sont indispensables à l'exécution de l'activité biologique normale, mais peuvent sous certaines conditions mener à la formation de polymères insolubles et au développement de maladies. Afin de contribuer à une meilleure compréhension des mécanismes responsables de la polymérisation, nous avons cherché à concevoir rationnellement des protéines mutantes qui présentent une propension à polymériser contrôlée. Des tests expérimentaux ont été réalisés par le groupe australien du Professeur S.P. Bottomley, et ont permis de valider nos prédictions de manière assez remarquable.

The work presented in this thesis concerns the computational study of the relationships between the sequence of a protein and its three-dimensional structure(s). The unravelling of these relationships has many applications in different domains and is probably one of the most fascinating issues in molecular biology.

The first part of our work is devoted to the development of statistical potentials derived from databases of known protein structures. These potentials allow to define a limited number of energetic functions embodying the complex ensemble of interactions that rule protein folding and stability (including some entropic contributions), and can be easily adapted to simplified representations of protein structures. However, their physical meaning remains unclear since several hypotheses and approximations are necessary, whose impact is far from clearly understood. We studied some of the limitations of these potentials: their dependence on the size of the proteins included in the database, the non-additivity of the different potential terms, and the importance of the specific environment of each residue. Our results show that residue-based distance potentials are affected by the size of the database proteins, and that this effect can be quite strong, is residue-specific, and seems to result mostly from the inhomogeneous partition of hydrophobic and hydrophilic residues between the surface and the core of proteins. On the basis of these observations, we defined a set of corrective functions in order to take protein size into account while deriving the potentials. On the other hand, we developed a general procedure of derivation of potentials and coupling terms and consequently created an energetic function describing the correlations between several sequence and structure descriptors (the nature of each residue, the conformation of its main chain, its solvent accessibility, and the distances that separate it from other residues, in space and along the sequence). This energetic function presents a strongly improved predictive power, in comparison with the original potentials and with other potentials described in the literature.

The second part describes the application of different programs, based on statistical potentials, to the study of proteins that adopt alternative structures. Domain swapping involves the exchange of a structural element between identical proteins, and leads to the generation of an oligomeric unit. We showed that the presence of “structural weaknesses”, regions that are not optimal with respect to the folding mechanisms or to the stability of the native structure, seems to be intimately linked with the swapping mechanisms. In addition, cation-{pi} interactions were frequently detected in some key locations and might also play an important role. Finally, we designed a set of mutations that are likely to affect the swapping propensities of different proteins. The experimental study of these mutations should allow to validate, or refine, our hypotheses concerning the importance of structural weaknesses and cation-{pi} interactions. We also analysed another protein that undergoes large conformational changes: {alpha}1-antitrypsin. In this case, the structural modifications are necessary to the proper execution of the biological activity. However, under certain circumstances, they lead to the formation of insoluble polymers and the development of diseases. With the aim of reaching a better understanding of the mechanisms that are responsible for this polymerisation, we tried to design mutant proteins that display a controlled polymerisation propensity. An experimental study of these mutants was conducted by the group of Prof. S.P. Bottomley, and remarkably confirmed our predictions.

Doctorat en sciences appliquées
info:eu-repo/semantics/nonPublished

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Guellouz, Asma. "Création par évolution dirigée de protéines artificielles en alternatives aux anticorps". Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00767675.

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Les travaux décrits dans ce mémoire ont pour objectif d'une part le développementd'une nouvelle famille de protéines artificielles et d'autre part la création de nouveaux sitesde fixation spécifiques dans ces protéines. L'objectif général était de développer une approchegénérale permettant d'obtenir rapidement des protéines reconnaissant toute macromoléculecible choisie. On peut voir ces protéines artificielles spécifiques comme des sortes d'anticorpsartificiels pour leur spécificité et leur affinité mais dont les propriétés physiques : stabilité,solubilité, efficacité d'expression, insensibilité à l'agrégation sont nettement plus favorablesque celles des anticorps et de leur dérivés.Le premier chapitre, présente la conception et la construction d'une bibliothèque deprotéines artificielles dite de première génération où les protéines sont formées par larépétition d'un motif idéalisé à partir d'une famille de motifs naturels appelés HEAT repeats.Toutes les protéines de la bibliothèque, dénommées αRep, sont conçues pour avoir la mêmearchitecture générale mais diffèrent les unes des autres par le nombre de motifs et par laséquence dans certaines positions rendues variables au sein de chaque motif. Cette banquenous a permis de valider l'architecture αRep choisie : Les protéines s'expriment sous formesoluble, sont très stables et adoptent la structure secondaire et tertiaire attendue quel que soitla séquence des positions hypervariables. Le second chapitre présente alors les approchessuivies pour l'amélioration de la qualité et de la diversité de la bibliothèque et a conduit à laconstruction d'une bibliothèque d'αRep de deuxième génération. Cette dernière bibliothèque(2 .1) repose sur le même schéma général mais contient une diversité ayant été optimiséelors de la conception puis améliorée expérimentalement par une procédure dite deFiltration/shuffling. Cette bibliothèque très diverse (1.7*109 clones indépendants) a été alorsexploitée pour y rechercher, par des méthodes d'exposition sur phages, de nouvelles αRepreconnaissant des protéines cibles préalablement choisies. L'ensemble des résultats montretrès clairement que des αRep reconnaissant spécifiquement, avec une affinité élevée, desprotéines cibles choisies arbitrairement peuvent être effectivement obtenues. Les structurestridimensionnelles de plusieurs complexes formés entre les αRep et leur cible a été résoluepermet de comprendre la nature et l'organisation précise de ces capacités de reconnaissancemoléculaire nouvellement créées.

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Bysell, Lisa. "Vegetariska alternativ till kött i svenska livsmedelsbutiker : En fallstudie om utbud, hinder och drivkrafter". Thesis, KTH, Industriell ekologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-196683.

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A study of European households found that Food and drink causes a fourth of the total various environmental impacts. Globally does the food sector account for 22 % of all the greenhouse gas emissions. The climate impact from different foods do however vary significantly, and meat products does generally cause higher greenhouse gas emissions than plant-based foods. A reduction of the meat consumption is suggested by many researchers as one of the most important measures in order to move towards a more sustainable food consumption and production. The grocery retailers have an important role as gatekeepers between the consumer and the products, and the aim of this thesis is to study a transition towards a larger range and increased sales of plant-based protein alternatives to meat from the retailers’ perspective. It does so by attempting to identify drivers and barriers from their point of view. Data was collected through interviews with representatives for the largest retailer groups (n=3) and interviews with store managers (n=8) as well as by examining the product range in the stores (n=10). Grocery stores were located in two different cities; the district Södermalm in central Stockholm and Östersund, a mid-sized town in the Northern parts of Sweden. The results from the interviews showed an increase in the range as well as the sales of vegetarian products at all the participating retailers and stores, and all the interviewees believed that this will continue to increase even further in the future. Flexitarians and young people stands out as the main groups of consumers who buy these products and thus lies behind this increase. What drives the consumers has been identified as an increased awareness, an improved product range and media attention. From the retailers’ perspective it is also now considered a competitive advantage to offer an attractive range of these products. Price has been identified as one of the main barriers for a future expansion of the sales these vegetarian alternatives, but several of the participants also believed that a lot of people are not at all interested in lowering their meat consumption which would be a central obstacle to overcome to get a wide spread in society as a whole. Even if the products have improved recently they may still not be attractive enough to be considered as an alternative for all consumers. The findings also indicate that there is a relatively small difference between the two participating cities, with the main exception that the stores at Södermalm in general offers more perishable vegetarian alternatives than the stores in Östersund does. The most significant differences, regarding product range, was found within ICA (Sweden’s largest food retailer) where the stores are privately owned and the store managers can choose assortment which differs from the other two large retailers Axfood and Coop where it is centrally controlled. The representatives from the retailer groups had rather different views on their ownrole in the development of the vegetarian alternatives but also on what future measures they believed are needed for a future development and impact. This has consequences for the consumers as the range differs to a large degree, to some extent between the different retailer groups, but mainly within the largest one (ICA). The results from this study cannot be generalised, but may provide new insights to a perspective that seems to not have been studied before and might constitute a point of departure for future research.
Mat och dryck står för ungefär en fjärdedel av vår miljö- och klimatpåverkan och globalt står livsmedelssektorn för 22 % av alla växthusgasutsläpp. Klimatpåverkan varierar dock kraftigt mellan olika typer av livsmedel och generellt sett är utsläppen från kött flera gånger större än för vegetabiliska livsmedel. Att minska köttkonsumtionen och övergå till mer vegetabiliska proteiner anses vara en av de viktigaste åtgärderna för att nå en mer hållbar matkonsumtion. Som länken mellan varan och konsumenten har dagligvaruhandeln en viktig roll för att förutsättningarna ska finnas på plats. Den här studien syftar till att undersöka handelns syn på utvecklingen av en ökad´andel vegetariska alternativ till kött, genom att belysa drivkrafter, hinder samt hur utbudet ser ut. Data samlades in genom intervjuer med centrala representanter för ICA, Coop och Axfood (n=3) samt genom intervjuer med butikschefer (n=8) och sortimentsundersökningar i butik (n=10) i en stor respektive mellanstor svensk stad (på Södermalm i Stockholm samt i Östersund). Resultaten visar att försäljningen och utbudet av vegetariska produkter har de senaste åren ökat kraftigt hos alla medverkande butiker och bolag, och utvecklingen tros även fortsätta i framtiden. Det är främst flexitarianer och unga som är drivande för utvecklingen, och en ökad medvetenhet (miljö, hälsa och djurvälfärd) samt att utbudet blivit bättre pekas ut som bidragande orsaker. För butikerna och bolagen själva är det även en konkurrensfördel att ha ett bra vegetariskt utbud. Ett av de främsta hindren som kommit fram för den fortsatta utvecklingen är att det är många som inte är intresserade av att minska sin köttkonsumtion, att produkterna inte är tillräckligt attraktiva och att de kan vara dyra. Resultaten visar också att skillnaden i utbud mellan städerna är relativt liten, med undantaget att det är en mindre andel färska alternativ i Östersund än på Södermalm. De största skillnaderna i hur många vegetariska alternativ som kunden har att välja på (oberoende av stad) finns inom ICA som drivs av privata handlare och där butikerna i större grad kan påverka sitt sortiment, tillskillnad från Axfood och Coop där sortimenten centralstyrs. De centrala representanter som medverkat har olika syn på sin egen roll i utvecklingen samt vilka åtgärder som behövs för en fortsatt utveckling. För konsumenten finns det en relativt stor skillnad i bredden på det vegetariska utbudet beroende på vilken butik de väljer att gå till, vilket verkar vara en konsekvens av vilken nivå sortimentet bestäms på. Resultaten från studien går inte attgeneraliseras, men de kan ge nya insikter i ett perspektiv som inte verkar studerats tidigare och vara en utgångspunkt för framtida studier.

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Wiedmer, Stefanie, Alexander Erdbeer, Beate Volke, Stephanie Randel, Franz Kapplusch, Sacha Hanig y Michael Kurth. "Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa)". Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-231860.

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The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

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Wiedmer, Stefanie, Alexander Erdbeer, Beate Volke, Stephanie Randel, Franz Kapplusch, Sacha Hanig y Michael Kurth. "Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa)". EDP Sciences, 2017. https://tud.qucosa.de/id/qucosa%3A30707.

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The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

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Lindström, Mikael. "Functional characterization of the alternative reading frame protein p14ARF /". Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-917-x.

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Knave, Axel. "Production and characterization of alternative scaffold proteins for medical applications". Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-278838.

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Antibodies, as forerunners in the field of biological drugs, are originally an organism’s answer to the invasion of different pathogens. Today, antibodies are a common treatment for many chronic diseases such as the immune-mediated inflammatory diseases rheumatoid arthritis or psoriasis. It is suspected that the cytokines interleukin 17a (IL17a) and interleukin 17c (IL17c) are involved in those diseases and are commonly treated with antibodies that inhibit the cytokines. Even though antibodies have been a huge success as biological drugs they also have downsides when it comes to their production, size and stability. In quest of finding alternatives to antibodies in diagnostics and therapy, a novel class of biologics has been developed. So-called alternative scaffold proteins are small polypeptide chains that can be engineered to show affinity towards different biomarkers. ABD-Derived Affinity ProTeins or ADAPTs are one example of these alternative scaffolds that can be modified to bind a biomarker as target and keep their affinity to Human Serum Albumin (HSA) at the same time, making them bispecific. In this project, twenty-four previously selected ADAPT binder candidates that have shown good prospects towards IL17a and IL17c in previous experiments were cloned, produced, purified and characterized to determine if they show potential as tools in diagnostics or therapy of autoimmune diseases. The proteins were produced in E. coli, purified by affinity chromatography and characterized using Surface Plasmon Resonance (SPR), Circular Dichroism (CD) and Size Exclusion Chromatography (SEC). All candidates were successfully cloned into E. coli and out of these, 10 could be produced and 5 showed affinity towards their target using SPR. Examination by SEC and CD showed that the protein variants did not seem to be structurally stable and hints of impurities in the samples could be detected. This and a low yield could be further confirmed via SDS-PAGE. In conclusion, binders were produced that could theoretically be promising candidates as tools in diagnostics or therapy of chronic diseases were IL17a and/or IL17c are important. Nevertheless, in order to support these claims further investigations and developments are necessary.
Antikroppar, som föregångare inom området biologiska läkemedel, är ursprungligen en organisms svar på invasionen av olika patogen. Idag är antikroppar en vanlig behandling för många kroniska sjukdomar, såsom de immunmedierade inflammatoriska sjukdomarna reumatoid artrit eller psoriasis. Cytokinerna interleukin 17a (IL17a) och interleukin 17c (IL17c) tros vara involverade i dessa sjukdomar och behandlas vanligtvis med antikroppar som hämmar cytokinerna. Trots att antikroppar har varit en stor framgång som biologiska läkemedel har de också nackdelar när det gäller deras produktion, storlek och stabilitet. För att hitta alternativ till antikroppar inom diagnostik och terapi har en ny klass av biologiska läkemedel utvecklats. Så kallade alternative scaffold proteins är små polypeptidkedjor som kan manipuleras för att visa affinitet gentemot olika biomarkörer. ABD-Derived Affinity ProTeins eller ADAPTs är ett exempel på dessa alternative scaffolds som kan modifieras för att binda en biomarkör som mål utan att påverka affiniteten till Humant Serum Albumin (HSA), vilket gör dem bispecifika. I detta projekt klonades, producerades, renades och karakteriserades tjugofyra tidigare utvalda ADAPT-bindarkandidater som har visat goda förutsättningar gentemot IL17a och IL17c i tidigare experiment. Proteinerna producerades i E. coli, renades genom affinitetskromatografi och karakteriserades med användning av Surface Plasmon Resonance (SPR), Circular Dichroism (CD) och Size Exclusion Chromatography (SEC). Alla kandidater klonades framgångsrikt i E. coli och av dessa kunde 10 produceras. Fem bindare visade affinitet till deras mål med SPR. Undersökning med SEC och CD visade dock att proteinvarianterna inte var strukturellt stabila och antydan till föroreningar kunde detekteras i proverna. Detta och ett lågt utbyte kunde ytterligare bekräftas via SDS-PAGE. Sammanfattningsvis kunde bindare producerades och dessa kan teoretiskt vara lovande kandidater till diagnostik eller terapi av kroniska sjukdomar där IL17a och/eller IL17c är viktiga. För att stödja dessa påståenden krävs dock ytterligare experiment och utveckling av bindarna.

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Matlin, Arianne Jane. "Regulation of α-actinin alternative splicing by polypyrimidine tract binding protein and CUG binding proteins". Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614724.

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Gasparini, Isabella. "Alternativ splicing och hur den förhåller sig till växters alternativa splicing". Thesis, Linköping University, Linköping University, Department of Physics, Chemistry and Biology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-57190.

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Alternativ splicing är en process som ger upphov till att olika mRNA-sekvenser bildas från en enda gen, vilket bidrar till en ökad proteindiversitet hos organismen. Olika mRNA-sekvenser kan uppstå eftersom att det förekommer olika varianter av alternativ splicing som även kan kombineras på flera olika sätt: cassette exon (inkludering/exkludering av exon), intron retention (intronet behålls), alternative 5´splice-site choice (olika 5´ splice sites kan väljas) och slutligen alternative 3´ splice-site choice (andra 3´ splice sites kan väljas). För att alternativ splicing ska äga rum i olika pre-mRNA måste den regleras av cis-reglerande element. De cis-reglerande elementen utgörs av fyra grupper: exonic splicing enhancers (ESE), exonic splicing silencers (ESS), intronic splicing enhancers (ISE) samt intronic splicing silencers (ISS). Som namnen förtäljer finns de antingen i exoner eller introner, där de interagerar med transagerande faktorer, SR-proteiner (aktiverare) eller hnRNPs (hämmare). Alternativ splicing förekommer både i djur och i växter. Hos Homo sapiens genomgår över 74 % av de 25,000 gener som finns hos organismen, alternativ splicing. Däremot i växten Arabidopsis thaliana, genomgår endast 22 %, av den totala mängden på cirka 26,000 gener, alternativ splicing. Eftersom att processen bidrar till en ökad proteindiversitet, kommer det medföra att olika processer i organismerna påverkas, exempelvis celltillväxt, celldöd samt utvecklingen av olika sjukdomar, såsom Parkinson och cystisk fibros. Många studier har gjorts som bekräftar dess betydelse för organismerna men på grund av processens komplexitet är det fortfarande ett ämne som ständigt måste utforskas.

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Barbosa, Keila Abadia. "Avalia??o nutricional do farelo de crambe para codornas de corte". UFVJM, 2016. http://acervo.ufvjm.edu.br/jspui/handle/1/1288.

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Objetivou-se avaliar a energia metaboliz?vel aparente (EMA), a energia metaboliz?vel aparente corrigida (EMAn) e o coeficiente de metaboliza??o aparente da energia bruta (CMAEB) do farelo de crambe e sua inclus?o em ra??es para codornas de corte sobre o desempenho, caracter?sticas de carca?a e an?lise de rentabilidade econ?mica. O primeiro experimento foi realizado para a determina??o da EMA, da EMAn e o CMAEB pelo m?todo de coleta total de excretas. Para isso utilizou-se 315 codornas de corte machos, com 42 dias de idade, durante 10 dias, distribu?das em tr?s tratamentos, sendo T1: ra??o refer?ncia (RR); T2: 80% RR + 20% de farelo de crambe e T3: 70% RR + 30% de farelo de crambe, com sete repeti??es de 15 aves por unidade experimental. Para o segundo experimento foram utilizadas 390 codornas, Coturnix coturnix, da linhagem LF1, machos e f?meas, distribu?das em delineamento inteiramente casualizado, com cinco tratamentos e seis repeti??es de 13 aves por unidade experimental. As fases experimentais foram divididas em inicial (8 a 21 dias) e de crescimento (22 a 35 dias de idade). Os n?veis de substitui??o de parte da prote?na da ra??o pela prote?na do farelo de crambe foram: 0, 3, 6, 9 e 12%. Para o primeiro experimento os valores obtidos de EMA, EMAn e CMAEB foram de 2445,58 kcal; 2197,29 kcal e 51,97%, respectivamente, para a ra??o teste T2. Para ra??o teste T3, obteve-se para EMA, EMAn e CMAEB os valores de 1772,18 kcal; 1592,25 kcal e 37,66%, respectivamente. Para o segundo experimento observou-se que n?o houve diferen?as significativas pela substitui??o de parte da prote?na bruta da ra??o pela prote?na do farelo de crambe sobre o desempenho das codornas nas fases inicial e de crescimento. Recomenda-se a substitui??o de parte da prote?na da ra??o pela prote?na do farelo de crambe at? o n?vel de 12%, por n?o influenciar negativamente no desempenho das codornas de corte. Pela an?lise de rentabilidade o n?vel de 6% de substitui??o de parte da prote?na da ra??o pela prote?na do farelo de crambe apresentou o melhor resultado.
Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Zootecnia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016.
This study aimed to evaluate the apparent metabolizable energy (AME), the corrected apparent metabolizable energy (AMEn) and apparent metabolization coefficient of gross energy (AMCGE) of crambe meal and their inclusion in diets for meat quails on performance, carcass features, and economic profitability analysis. The first experiment was conducted to determine the AME, the AMEn and AMCGE by the total excreta collection method. Three hundred and fifteen male meat quails with 42 age days, during 10 days, distributed in three treatments, T1: basal diet (BD); T2: 80% BD + 20% crambe meal and T3: 70% BD + 30% crambe meal, with seven replicates and fifteen birds each. For the second experiment, three hundred and ninety quails, Coturnix coturnix, the LF1 strain, male and female, were distributed in a completely randomized design, with five treatments and six repetitions with thirteen birds each. Experimental phases were divided into initial phase (8 to 21 days) and growing phase (22 to 35 days of age). The replacement levels of feed protein by crambe meal protein were 0, 3, 6, 9 and 12%. For the first experiment AME, AMEn and AMCGE values were 2445.58 kcal, 2197.29 kcal and 51.97%, respectively, to feed test T2. To feed test T3, was obtained for AME, AMEn and AMCGE values of 1772.18 kcal, 1592.25 kcal and 37.66%, respectively. For the second experiment there were no significant differences by dietary crude protein replacing part by crambe meal protein on the quails performance in the initial and growing phases. It is recommended to replace part of the feed protein at level 12% of crambe meal, not influencing negatively the meat quails performance. For the profitability analysis the replacement level of 6% crambe meal have better results.

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11

Dickson, Alexa Megan. "Alternative RNA processing and strategies to modulate splicing". Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/6057.

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Thesis (Ph. D.)--University of Missouri-Columbia, 2008.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.

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Birzele, Fabian. "Alternative Splicing and Protein Structure Evolution". Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-96027.

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Marini, Wanda. "Comparing mutant p53 and a wild-type p53 isoform, p47 : rationale for the selection of mutant p53 in tumours". Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116033.

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One of the major unresolved questions in cancer biology is why the majority of tumour cells express mutant p53 proteins. p53 is considered the prototype tumour suppressor protein, whose inactivation is the most frequent single genetic event in human cancer (Bourdon et al., 2005). Genetically-engineered p53-null knockout mice acquire multiple tumours very early on in life and human Li-Fraumeni families who carry germline mutations in p53 are highly cancer-prone (reviewed in Vousden and Lane, 2007). p53 mutant proteins have been found to acquire novel functions that promote cancer cell proliferation and survival, yet exactly why mutant p53s acquire oncogenic activity is still poorly understood. Mutant p53 has also been found to complex with wildtype p53, thus acting in a dominant negative way. However, this inhibition is incomplete since many cancers with mutant p53 alleles also have a loss of the second wild-type p53 allele and thus only express the mutant p53 (Baker et al., 1989). An N-terminal truncated p53 isoform, p47, arising from alternative splicing of the p53 gene (Ghosh et al., 2004) or by alternative initiation sites for translation (Yin et al. , 2002), has been described. Alternative splicing was found to be universal in all human multi-exon genes (Wang et al., 2008) and therefore determining the role of the p47 isoform with respect to the p53 gene is essential. Evidence in this study suggests that mutant p53 (p53RI75H) has a similar structure and function as p47, including the ability to complex with and impair both p53 and p73. Therefore, in addition to expressing a tumour suppressor protein, the p53 gene can also express an onco-protein (p47). This study therefore argues that tumours select for mutant p53 because it has gained the ability to function like p47, a wild-type p53 isoform.

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14

Fischetti, Francesca. "Meat analogues: le nuove alternative ai prodotti carnei". Master's thesis, Alma Mater Studiorum - Università di Bologna, 2022.

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Il presente elaborato affronta la tematica dei prodotti analoghi della carne, che già a partire da qualche anno si stanno affacciando sul mercato internazionale, con una domanda e offerta continuamente in espansione. Lo scopo dello studio è stato quello di delineare un quadro generale di tali prodotti da un punto di vista delle sue caratteristiche intrinseche e analizzandone le prospettive future di mercato, effettuando un’indagine sul consumatore, riguardante la percezione, la propensione all’acquisto e i motivi di tale scelta. La tesi si distingue in una prima sezione di tipo compilativo, basata su una ricerca bibliografica attraverso l’utilizzo di parole chiave. La seconda sezione dell’elaborato è di tipo sperimentale, basata sulla stesura e distribuzione di un questionario con l’obiettivo di ricavare informazioni sulle conoscenze e percezioni di potenziali consumatori all’acquisto di prodotti analoghi della carne. Nel dettaglio, il primo capitolo affronta il contesto in cui i prodotti surrogati della carne si trovano; si evidenziano i principali riferimenti legislativi in Italia e nel contesto della Comunità europea sui novel food, e sulla denominazione commerciale dei prodotti analoghi di origine vegetale. Il secondo capitolo affronta il processo tecnologico a cui vanno incontro le proteine per ottenere le medesime caratteristiche (fisiche e sensoriali) dei prodotti carnei. Il terzo capitolo contiene un’analisi della formulazione, con particolare riferimento alle fonti proteiche di origine vegetale e alla classe degli additivi. Inoltre, delinea in parte l’aspetto nutrizionale. Il quarto, ed ultimo, capitolo si apre delineando le prospettive di mercato future, e mettendo in evidenzia gli attori principali sul mercato che hanno optato per lanciare le proprie linee di analoghi della carne di origine vegetale e i canali distributivi in Italia. È seguita la descrizione del questionario posto agli intervistati, con relativi risultati e discussione.

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15

Finkler, Joana Karin. "Farinha de penas em dietas para Tilápia do Nilo". Universidade Estadual do Oeste do Paraná, 2013. http://tede.unioeste.br:8080/tede/handle/tede/1529.

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Current analysis evaluates the inclusion effect of feather meal in diets with and without aminoacids supplementation on the performance, centesimal composition, hematological parameters and feeding costs of Nile tilapia fingerlings, Oreochromis niloticus. One thousand and four hundred Nile tilapia fingerlings, initial average weight 4.98±0.08 g, distributed in 28 small cages of 1m³, with 50 fish each, were used. Three levels (8, 16 and 24%) of hydrolyzed feather meal (FM) inclusion and two groups, one with and the other without synthetic aminoacids supplementation (AAs), were evaluated during 65 days. Control treatment with neither feather meal nor aminoacids supplementation was also employed, totalizing seven treatments and four replications. Productive performance analysis including total length (TL), weight gain (WG), apparent food conversion (AFC), survival (SU), condition factor (CF) and protein efficiency rate (PER) was performed at the end of the experiment, coupled to body parameters such as visceral fat index (VFI) and hepatosomatic index (HIS). Blood samples were collected for erythrocytes counting and determination of hemoglobin, hematocrit, average corpuscular volume (ACV) and average corpuscular hemoglobin concentration (ACHC). The entire fish was used for the analysis of centesimal composition, namely, moisture (MO), protein (P), ether extract (EE) and mineral matter (MM)). Diet costs per kilo of WG were also assessed. Treatments did not influence (p>0.05) SU, HIS, CF, MO and MM and best results for FM and WG were those of treatments with 8% FM with or without AAs supplementation. AFC was higher and PER lower in treatments with 24% FM without supplementation. P rate was lower in treatment with inclusion of 24% FM without supplementation when compared to control, whereas EE had higher levels of inclusion when compared to control. Hematologic parameters were kept within the species´s normal variation level. Treatment with 8% FM with supplementation presented lower feeding costs. Results show that feather meal may be used in diets for Nile tilapia up to 8% inclusion, with or without supplementation of AAs, without any liability in performance, chemical composition and higidity. In fact, supplementation at the above inclusion level with AAs is economically more viable
Esse estudo teve por objetivo avaliar o efeito da inclusão de farinha de pena em dietas com e sem suplmentação de aminoácidos sobre o desempenho, composição centesimal, parâmetros hematológicos e custos de alimentação de alevinos de tilápia do Nilo Oreochromis niloticus. Para isso, foram utilizados 1.400 alevinos de tilápia do Nilo com peso inicial médio de 4,98 ± 0,08 g distribuídos em 28 hapas de 1 m³ com 50 peixes cada. Durante 65 dias foram avaliados três níveis de inclusão de farinha de pena hidrolisada (FP) (8, 16 e 24%) e dois grupos, um com e outro sem suplementação de aminoácidos sintéticos (AAs). Além disso, foi utilizado um tratamento controle, cuja dieta não continha nem farinha de pena e nem suplementação de aminoácidos, totalizando sete tratamentos com quatro repetições. Ao final do experimento foram realizadas análises de desempenho produtivo (comprimento total (CT), ganho de peso (GP), conversão alimentar aparente (CAA), sobrevivência (SO), fator de condição (FC) e taxa de eficiência proteica (TEP) e dos parâmetros corporais, (índice de gordura visceral (IGV) e índice hepatossomático (IHS)). As mostras de sangue foram coletadas para contagem de eritrócitos e determinação de hemoglobina, hematócrito, volume corpuscular médio (VCM) e concentração de hemoglobina corpuscular média (CHCM). Foram utilizados peixes inteiros para a análise da composição centesimal (umidade (UM), proteína (PB), extrato etéreo (EE) e matéria mineral (MM)). Também foi avaliado o custo das dietas por quilo de GP. Os tratamentos não influenciaram (p>0,05) na SO, IHS, FC, UM e MM. Os melhores resultados de PF e GP foram dos tratamentos com 8% de FP com ou sem suplemetação de AAs. A CA foi maior e a TEP menor no tratamento com 24% de FP sem suplementação. A PB foi menor no tratamento com inclusão de 24% FP sem suplementação em relação ao controle, e o EE aumentou com níveis mais altos de inclusão em relação ao controle. Os parâmetros hematológicos mativeram-se dentro da faixa de variação normal para a espécie. O tratamento com 8% de FP com suplementação apresentou o menor custo de alimentação. Conclui-se que a farinha de penas pode ser utilizada em dietas para tilápia do Nilo em até 8% de inclusão, com ou sem suplementação de AAs, sem prejuízos no desempenho, composição química e higidez, sendo que a suplementação deste nível de inclusão com AAs é economicamente mais viável

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16

Chick, Joel. "Proteomic analysis of liver membranes through an alternative shotgun methodology". Phd thesis, Australia : Macquarie University, 2009. http://hdl.handle.net/1959.14/42528.

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Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009.
Bibliography: p. 200-212.
Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References.
The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments.
Mode of access: World Wide Web.
609 p. ill. (some col.)

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17

Coelho, Vitor Lima. "Análise funcional de eventos de Splicing alternativo". Laboratório Nacional de Computação Científica, 2015. https://tede.lncc.br/handle/tede/212.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Splicing Alternativo (SA) é um mecanismo pós-transcricional que produz mais de um produto de gene, através da combinação de diferente éxons do lócus genômico, gerando grande parcela da variedade do proteoma dos eucariotos. Ao longo da última década, como seu papel regulatório tornou-se mais e mais evidente, SA tornou-se um fator chave para criação de complexidade de diferentes organismos dado um repertório constante de genes através das gerações. Através da inserção, exclusão ou substituição de partes da sequência do transcrito, SA pode obviamente também ter um impacto nos domínios funcionais de proteínas. Apesar de algumas tentativas que tem sido principalmente prejudicadas por questões técnicas causada pela redundância nas sequências transcritos alternativos, os efeitos em larga escala do SA em nível funcional tem sido pouco estudados até os dias de hoje. Este projeto descreve o desenvolvimento de uma ferramenta computacional chamada ASTAFUNK - Alternative Splicing Trancriptional Analyses with FUNctional Knowledge - um programa stand-alone automatizado e eficiente para estudar como a diversidade de determinado transcriptoma é traduzido em variação funcional, baseado em um padrão de anotação de transcriptomas (em GTF, Gene Transfer Format) e perfis de domínios (no formato do Pfam). Resumidamente, ASTAFUNK traduz as regiões que sofreram splicing alternativo de open read frames em sequências de aminoácidos, que subsequentemente são alinhadas com o profile Hidden Markov Model da base de dados do Pfam, empregando programação dinâmica padrão (algoritmo de Viterbi) com alguns refinamentos técnicos (isto é, abordagem branch-and-bound). Em contraste com ferramentas convencionais de predição de domínios (por exemplo, HMMER), o algoritmo de ASTAFUNK foi projetado para evitar escaneamentos redundantes de sequências em transcriptomas com alto grau de SA. Neste trabalho, aspectos teóricos e práticos da abordagem do ASTAFUNK são avaliados, e a eficiente implementação em JAVA é disponível livremente na internet sob BSD 3-clause open source license.
Alternative splicing (AS) is a mechanism that produces more than one gene product at the transcriptional level, by combining different exons of a gene, generating a major part of the proteome diversity in eukaryotes. Over the last decade, as the regulatory role of splicing has become more and more evident, AS turned a key factor in creating different organism complexity given a rather constant repertoire of genes across generations. By inserting, deleting or substituting part of the transcript sequence, AS can obviously also have an impact on functional protein domains. Despite some attempts that have mainly been hampered by technical issues caused by the redundancy in alternative transcript sequences, the large-scale effects of AS on the functional level has been poorly studied so far. This project describes the development of a computational tool called ASTAFUNK - Alternative Splicing Trancriptional Analyses with FUNctional Knowledge - an automated and efficient stand-alone program to study how diversity of a custom transcriptome translates into functional variation, based on standard transcriptome annotations (in GTF, Gene Transfer Format) and domain profiles (in Pfam format). In a nutshell, \afunk{} translates the alternatively spliced parts of open reading frames on the fly into amino acid sequences, which subsequently are aligned with the profile Hidden Markov Models from Pfam employing standard dynamic programming (Viterbi's algorithm) with some technical refinements (i.e., a branch-and-bound approach). In contrast to conventional domain prediction tools (e.g., the HMMER aligner), the ASTAFUNK algorithm has been designed to avoid redundant sequence scans in AS-enriched transcriptomes. In this work, theoretical and practical aspects of the ASTAFUNK approach are evaluated, and the efficient JAVA implementation is made freely available over the internet under the BSD 3-clause open source license.

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18

Resch, Alissa Marie. "Alternative splicing protein impact and genome evolution /". Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1472152071&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.

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Bremang, Michael Anthony. "The mouse protein repertoire : studies on alternative splicing, function and quality". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610626.

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Stark, Jeremy M. "SR proteins can function during alternative splicing to mediate exon/exon associations /". Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5020.

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ARAÚJO, Sandra Alves de. "Potencial anti-helmíntico de extratos proteicos de Leucaena leucocephala (Linn.) (Fabaceae) e Spigelia anthelmia (Linn.) (Loganiaceae) contra Haemonchus contortus (Rudolphi, 1803)". Universidade Federal do Maranhão, 2017. http://tedebc.ufma.br:8080/jspui/handle/tede/1787.

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Haemonchus contortus (RUDOLPHI, 1803) is a species of gastrointestinal nematode of great importance due to the damages caused in livestock. Natural products have been studied as an alternative to the use of commercial anthelmintics, responsible for the development of resistance in parasites. This study has as main objective to verify the anthelmintic activity of protein extracts of Leucaena leucocephala (Linn.) and Spigelia anthelmia (Linn.) against the nematode H. contortus. The present work was divided in two chapters. In chapter 1, L. leucocephala seeds were ground and the resulting flour, homogenized in 100 mM sodium phosphate buffer, pH 7. The suspension obtained was centrifuged (15,000 x g at 4 ° C for 30 min). After centrifugation, the obtained supernatant was denominated: Total extract (TE), cotyledon extract (CE) and shell extract (SE). The protein content, proteolytic, protease inhibitory, chitinolytic and anthelmintic activity of L. leucocephala extracts on H. contortus were verified. For each treatment, the effective concentration (EC) of the samples on the nematode was calculated. The extracts TE and CE presented a high protein content, besides having proteolytic, chitinolytic and protease inhibitory activity and inhibiting egg hatching (EC50 0.33 and 0.48 mg mL -1, respectively). However, L. leucocephala samples did not inhibit the larvae of H. contortus. It is concluded that proteins are correlated with the action of L. leucocephala on H. contortus. In Chapter 2, the parts of S. anthelmia were separated into leaves, roots and stem and, washed with distilled water, lyophilized and subsequently crushed. Subsequently the proteins were solubilized in 100 mM sodium phosphate buffer, pH 7. The suspension was centrifuged (15,000 x g at 4 ° C for 30 min) and the supernatant collected for analysis. From the ammonium sulfate precipitation (0-90%) of S. anthelmia extracts and membrane dialysis with 14 kDa molecular exclusion, protein fractions were obtained and denominated: Leaf protein fraction (LPF), stem protein fraction (SPF), root protein fraction (RPF). The samples were submitted to analysis by high performance liquid chromatography for the detection of compounds of secondary metabolism and mass spectrometry to identify possible bioactive proteins present. The protein content, proteolytic, protease inhibitory, chitinolytic, haemagglutinating and anthelmintic action of S. anthelmia protein fractions on H. contortus were verified. For each treatment, the effective concentration (EC) of the samples on the nematode was calculated. Fractions of S. anthelmia had an inhibitory effect on egg hatching, and this effect was more pronounced by LPF (EC50 0.17 mg mL -1). In addition, greater inhibition of larvae by LPF and RPF (EC50 0.27 and 0.25 mg mL -1, respectively) was observed. The protein fractions of root, stem and leaf of S. anthelmia were effective on the inhibition of larval migration (EC50 0.11, 0.14 and 0.21 mg mL -1, respectively). No secondary metabolite compounds were detected in the S. anthelmia fractions, while several proteins with anthelmintic potential were identified by mass spectrometry. It is concluded that proteins are correlated with the action of S. anthelmia on H. contortus, having potential for the development of anthelmintic products. Thus, the bioactive proteins present in L. leucocephala and S. anthelmia have promising pharmacological properties for the control of the H. contortus nematode.
Haemonchus contortus (RUDOLPHI, 1803) é uma espécie de nematoide gastrintestinal de grande importância devido aos prejuízos que causa na pecuária. Produtos naturais vêm sendo estudados como alternativa ao uso de anti-helmínticos comerciais, responsáveis pelo desenvolvimento de resistência nos parasitos. Este estudo tem como principal objetivo verificar a atividade anti-helmíntica de extratos proteicos de Leucaena leucocephala (Linn.) e Spigelia anthelmia (Linn.) contra o nematoide H. contortus. O presente trabalho foi dividido em dois capítulos. No capítulo 1, sementes de L. leucocephala foram trituradas e a farinha resultante, homogeneizada em tampão fosfato de sódio 100 mM, pH 7. A suspenção obtida foi centrifugada (15.000 x g a 4 ºC por 30 min). Após centrifugação, o sobrenadante obtido foi denominado: Extrato total (TE), extrato de cotilédone (CE) e extrato de casca (SE). O teor de proteínas, atividade proteolítica, inibitória de protease, quitinolítica e ação anti-helmíntica dos extratos de L. leucocephala sobre H. contortus foram verificados. Para cada tratamento foi calculada a concentração efetiva (EC) das amostras sobre o nematoide. Os extratos TE e CE apresentaram elevado teor proteico, além de possuírem atividade proteolítica, quitinolítica e inibitória de protease e inibirem a eclosão dos ovos (EC50 0,33 e 0,48 mg mL-1, respectivamente). No entanto, amostras de L. leucocephala não inibiram o desembainhamento larvar de H. contortus. Conclui-se que proteínas estão correlacionadas com a ação de L. leucocephala sobre H. contortus. No capítulo 2, as partes de S. anthelmia foram separadas em folhas, raízes e caule e, lavadas com água destilada, liofilizadas e subsequentemente trituradas. Posteriormente as proteínas foram solubilizadas em tampão fosfato de sódio 100 mM, pH 7. A suspensão foi centrifugada (15.000 x g a 4 ºC por 30 min) e o sobrenadante recolhido para análises. A partir da precipitação com sulfato de amônio (0-90%) dos extratos de S. anthelmia e diálise em membrana com exclusão molecular de 14 kDa, frações proteicas foram obtidas e denominadas: Fração proteica de folha (LPF), fração proteica de caule (SPF), fração proteica de raiz (RPF). As amostras foram submetidas a análise por cromatografia líquida de alta eficiência para detecção de compostos do metabolismo secundário e, espectrometria de massas para identificação de possíveis proteínas bioativas presentes. O teor de proteínas, atividade proteolítica, inibitória de protease, quitinolítica, hemaglutinante e ação anti-helmíntica das frações proteicas de S. anthelmia sobre H. contortus foram verificados. Para cada tratamento foi calculada a concentração efetiva (EC) das amostras sobre o nematoide. As frações de S. anthelmia apresentaram efeito inibitório sobre a eclosão dos ovos, sendo este efeito mais pronunciado pela LPF (EC50 0,17 mg mL-1). Além disso, foi verificada maior inibição do desembainhamento larvar por LPF e RPF (EC50 0,27 e 0,25 mg mL-1, respectivamente). As frações proteicas de raiz, caule e folha de S. anthelmia foram efetivas sobre a inibição da migração larvar (EC50 0,11; 0,14 e 0,21 mg mL-1, respectivamente). Não foram detectados compostos do metabolismo secundário nas frações de S. anthelmia, enquanto diversas proteínas com potencial anti-helmíntico foram identificadas por espectrometria de massas. Conclui-se que proteínas estão correlacionadas com a ação de S. anthelmia sobre H. contortus, tendo potenciais para o desenvolvimento de produtos anti-helmínticos. Assim, as proteínas bioativas presentes em L. leucocephala e S. anthelmia possuem propriedades farmacológicas promissoras para o controle do nematoide H. contortus.

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22

Bouchnak, Imen. "Biogénèse du chloroplaste : Voies d'import alternatives". Thesis, Université Grenoble Alpes (ComUE), 2018. http://www.theses.fr/2018GREAV074.

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Le chloroplaste est un composant majeur de la cellule végétale. Cet organite est le fruit d’une endosymbiose, survenue entre une cellule eucaryote et une cyanobactérie. Ainsi, 95% des gènes codant pour les protéines plastidiales ont été transférés vers le génome nucléaire au cours de l’évolution. En conséquence, la plupart des protéines chloroplastiques sont aujourd’hui codées par le noyau, synthétisées dans le cytosol sous forme de précurseurs dotés d’une une extension N-terminale clivable (le "peptide de transit") et ensuite importées sans les chloroplastes via le système TOC/TIC (Translocons localisés au niveau des membranes externe et interne de l'enveloppe des chloroplastes). Jusqu'à récemment, toutes les protéines destinées aux compartiments chloroplastiques internes étaient censées posséder une séquence d’adressage N-terminale clivable et engager la machinerie d’import général TOC/TIC. Cependant, des études récentes reposant sur des approches protéomiques ont révélé l’existence de plusieurs protéines chloroplastiques dépourvues de la séquence additionnelle clivable. La première évidence de telles protéines dites non canoniques a été fournie par notre équipe, étudiant le protéome de l’enveloppe du chloroplaste d’Arabidopsis, qui a conduit à l’identification d’une protéine quinone oxidoréductase homologue nommée « ceQORH ». Bien que dépourvues de peptide de transit clivable, il s’est avéré que ces protéines sont capables de rejoindre les compartiments chloroplastiques internes. D’autre part, il a été également montré que l’import de ces protéines dans le chloroplaste n’est pas médiée par la machinerie de translocation générale TOC/TIC. De plus, il s’est avéré que ces protéines ont la particularité d’être multilocalisées dans les cellules de différents tissus de la feuille. Cependant, les mécanismes moléculaires qui contrôlent la localisation sub-cellulaire de telles protéines chloroplastiques non canoniques demeurent encore inconnus. Pour mieux caractériser fonctionnellement les composantes des systèmes d’import alternatifs de protéines chloroplastiques non canoniques, nous avons adopté une approche directe qui reposait sur des techniques biochimiques combinant le crosslink chimique, la purification par affinité et la spectrométrie de masse. Cette stratégie nous a permis d’identifier un partenaire, impliqué dans le contrôle de l’adressage de la protéine ceQORH dans le chloroplaste. Alternativement, nous avons réalisé une bio-analyse du protéome de l’enveloppe du chloroplaste et qui nous a permis de revisiter la composition du protéome de l’enveloppe du chloroplaste. Afin d’expliquer la localisation sub-cellulaire variable de la protéine ceQORH, les membres de l’équipe ont émis l’hypothèse d’une interaction probable de cette protéine avec un partenaire cytosolique. Dans la dernière partie de cette étude, nous avons validé l’interaction, in planta, entre ceQORH et son partenaire par une approche génétique qui portait sur l’analyse de l’impact de l’absence de ce dernier sur la régulation de la localisation sub-cellulaire de la protéine ceQORH
Chloroplasts are a major component of plant cells. Their origin traces back to a cyanobacterial ancestor that was engulfed by an ancient eukaryotic cell and eventually integrated as an organelle during evolution. As a result, more than 95% of the ancestral cyanobacterial genes were transferred to the host cell nucleus. Proteins encoded by these relocated genes need to return to internal chloroplast compartments. This import is mainly achieved by the general TOC/TIC machinery located at the chloroplast surface. Until recently, all proteins destined to chloroplast were believed to possess an N-terminal and cleavable chloroplast targeting peptide, and to engage the TOC/TIC machinery. However, recent studies have revealed the existence of several non-canonical preproteins, lacking cleavable transit peptides. The first evidence for such ‘non-canonical’ chloroplast proteins was provided by our team studying the Arabidopsis chloroplast envelope proteome, leading to the identification of a quinone oxidoreductase homologue termed « ceQORH ». Furthermore, a few such proteins were demonstrated to use alternative targeting pathways, independent of the TOC/TIC machinery. To better characterize components of such alternative targeting machineries, a targeted study combining affinity purification and mass spectrometry aiming to identify alternative receptors at the chloroplast surface has been performed. This study allowed us to identify new “partner” involved in the control of chloroplast targeting of ceQORH protein. Alternatively, we also revisited the chloroplast envelope proteome composition and initiated a gene candidate approach. In addition, some non-canonical proteins are shared by plastids and other cell compartments. However, molecular mechanisms controlling subcellular localization of these non-canonical plastid proteins remain unknown. In order to explain the variable subcellular localization of ceQORH protein, our team hypothesized a probable interaction of ceQORH with a cytosolic partner. In the last part of this study, we validated the interaction between ceQORH and its partner in planta by a genetic approach analyzing the impact of the absence of the cytosolic partner on the regulation of the sub-cellular localization of ceQORH protein

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23

Carratalá, Tomás Jose Vicente. "Development and characterization of protein nanoformulations as alternative therapeutics to reduce antibiotic usage". Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/673321.

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L’aparició de farmacoresistència bacteriana als antibiòtics convencionals és una situació d’alarma mundial. Aquest escenari ha obligat a implementar mesures com la millora de les pràctiques d’higiene o l’administració controlada d’antibiòtics per reduir el seu ús en totes les àrees en les que s’utilitzen comunament, inclosa la medicina humana i animal i la indústria animal de producció d’aliments . Totes aquestes mesures estan destinades a disminuir l’aparició i propagació de la resistència als antibiòtics entre els bacteris, però quan es tracta de combatre als bacteris que ja presenten una o múltiples resistències, es necessiten amb urgència alternatives als antibiòtics. En aquest context, s’han proposat diferents estratègies, entre les quals s’inclouen l’ús de citoquines i pèptids antimicrobians (AMPs). Les citoquines són petites proteïnes reguladores intercel·lulars que tenen un paper central en l’inici, manteniment i regulació de la resposta immune innata. Concretament, l’IFN-γ té un paper fonamental en la promoció de la immunitat protectora contra les infeccions. D’altra banda, els AMPs, com GWH1, són generalment petits pèptids catiònics de naturalesa amfipàtica que tenen activitats antibacterianes, antifúngiques i antivirals d’ampli espectre, la capacitat de modular la resposta immune de l’hoste i una possibilitat reduïda d’induir resistència bacteriana. Malgrat el seu potencial com a agents antiinfecciosos, les citoquines i els AMPs estan subjectes a diversos desavantatges que s’han d’abordar abans de la seva possible aplicació biomèdica. En particular, la baixa estabilitat és un inconvenient comú associat a aquest tipus de compostos proteics. En aquest sentit, s’han aplicat diferents estratègies per superar aquesta limitació i millorar l’eficàcia d’aquests fàrmacs després de la seva administració. La majoria d’aquestes estratègies consisteixen en la vehicularización d’aquestes proteïnes o pèptids en estructures superiors, proporcionant un entorn protector i permetent la possibilitat d’alliberar-los en localitzacions concretes. La formació d’aquestes estructures pot ser modulada a través d’un disseny racional sobre el gen de la proteïna recombinant, com, per exemple, la incorporació de certs pèptids en l’estructura de la proteïna per generar components individuals amb capacitat autoensamblant (nanopartícules solubles), o per obtenir proteïnes propenses a l’agregació (cossos d’inclusió -IBs-). Encara que aparentment diferents, tots aquests complexos s’originen a partir d’interaccions proteiques impulsades per factors termodinàmics i cinètics similars que finalment condueixen a la formació de diferents formats de proteïnes; arranjaments estructurals superiors que involucren a través d’interaccions dirigides o no dirigides, la convergència de formes proteiques aïllades. En aquesta tesi s’ha caracteritzat i avaluat la nanoformulación d’aquestes molècules (citocines i AMPs) en diferents formats proteics incloent IBs i nanopartícules solubles autoensamblants amb l’objectiu de desenvolupar alternatives terapèutiques als antibiòtics. A més, s’ha llançat una mica de llum sobre les forces que governen el procés d’agregació i com es pot modular per promoure la formació d’aquests IBs.
La aparición de farmacorresistencia bacteriana a los antibióticos convencionales es una situación de alarma mundial. Este escenario ha obligado a implementar medidas como la mejora de las prácticas de higiene o la administración controlada de antibióticos para reducir su uso en todas las áreas en las que se utilizan comúnmente, incluida la medicina humana y animal y la industria animal de producción de alimentos. Todas estas medidas están destinadas a disminuir la aparición y propagación de la resistencia a los antibióticos entre las bacterias, pero cuando se trata de combatir a las bacterias que ya presentan una o múltiples resistencias, se necesitan con urgencia alternativas a los antibióticos. En este contexto, se han propuesto diferentes estrategias, entre las que se incluyen el uso de citoquinas y péptidos antimicrobianos (AMPs). Las citoquinas son pequeñas proteínas reguladoras intercelulares que desempeñan un papel central en el inicio, mantenimiento y regulación de la respuesta inmune innata. Concretamente, el IFN-γ tiene un papel fundamental en la promoción de la inmunidad protectora contra las infecciones. Por otro lado, los AMPs, como GWH1, son generalmente pequeños péptidos catiónicos de naturaleza anfipática que tienen actividades antibacterianas, antifúngicas y antivirales de amplio espectro, la capacidad de modular la respuesta inmune del huésped y una posibilidad reducida de inducir resistencia bacteriana. A pesar de su potencial como agentes antiinfecciosos, las citoquinas y los AMPs están sujetos a varias desventajas que deben abordarse antes de su posible aplicación biomédica. En particular, la baja estabilidad es un inconveniente común asociado a este tipo de compuestos proteicos. En este sentido, se han aplicado diferentes estrategias para superar esta limitación y mejorar la eficacia de estos fármacos tras su administración. La mayoría de estas estrategias consisten en la vehicularización de estas proteínas o péptidos en estructuras superiores, proporcionando un entorno protector y permitiendo la posibilidad de liberarlos en localizaciones concretas. La formación de estas estructuras puede ser modulada a través de un diseño racional sobre el gen de la proteína recombinante, como, por ejemplo, la incorporación de ciertos péptidos en la estructura de la proteína para generar componentes individuales con capacidad autoensamblante (nanopartículas solubles), o para obtener proteínas propensas a la agregación (cuerpos de inclusión -IBs-). Aunque aparentemente diferentes, todos estos complejos se originan a partir de interacciones proteicas impulsadas por factores termodinámicos y cinéticos similares que finalmente conducen a la formación de diferentes formatos de proteínas; arreglos estructurales superiores que involucran a través de interacciones dirigidas o no dirigidas, la convergencia de formas proteicas aisladas. En esta tesis se ha caracterizado y evaluado la nanoformulación de estas moléculas (citoquinas y AMPs) en diferentes formatos proteicos incluyendo IBs y nanopartículas solubles autoensamblantes con el objetivo de desarrollar alternativas terapéuticas a los antibióticos. Además, se ha arrojado algo de luz sobre las fuerzas que gobiernan el proceso de agregación y cómo se puede modular para promover la formación de dichos IBs.
The emergence of bacterial drug resistance to conventional antibiotics is a global alarming situation. This worrying scenario has forced the implementation of measures such us improved hygiene practices or antibiotic stewardship to reduce antimicrobial usage in all areas in which these therapeutics are commonly used, including human and animal medicine and food-producing animal industry. All these measures are intended to diminish the appearance and spread of drug resistance among bacteria, but when it comes to combat against drug or multidrug resistant bacteria, alternatives to traditional antibiotics are urgently needed. In this context, different strategies have been proposed as promising alternatives to antibiotics, including the use of cytokines and antimicrobial peptides (AMPs). Cytokines are small intercellular regulatory proteins that play a central role in initiating, maintaining, and regulating the innate immune response. Specifically, IFN-γ has a pivotal role in promoting protective immunity against infections. On the other hand, AMPs such as GWH1, are generally small cationic peptides with an amphipathic nature that have a broad-spectrum antibacterial, antifungal and antiviral activities, the ability to modulate the host immune response and a reduced possibility of inducing bacterial drug resistance. Despite their potential as anti-infective agents, cytokines and AMPs are subjected to several disadvantages that must be addressed prior to their possible biomedical application. In particular, low stability is a common drawback associated to these type of protein compounds. In this sense, different technologies and strategies have been applied in order to overpass this limitation and improve the efficiency of these drugs after administration. Most of these strategies consist on the vehicularization of these proteins or peptides into superior complexes, providing a protective environment and allowing the possibility of deliver them to the target site. The formation of these superior structures may be modulated by a direct rational design over the recombinant protein gene, such as que incorporation of certain peptides into the protein structure to generate building blocks for spontaneous self-assembling (soluble nanoparticles), or to obtain prone-to-aggregate proteins (inclusion bodies -IBs-). Although seemingly different, these small-scale complexes all originate from fundamental protein interactions and are driven by similar thermodynamic and kinetic factors finally leading to the formation of different proteins formats; superior structural arrangements that involve through directed or not directed interactions, the convergence of isolated protein forms. In this thesis, the nanoformulation of these molecules (cytokines and AMPs) into different protein formats including IBs and soluble self-assembling nanoparticles have been characterized and evaluated with the aim to develop therapeutic alternatives to antibiotics. In addition, some light has been shed on the forces that govern the aggregation process and how it can be modulated to promote the formation of IBs.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia

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24

Beaudoin, Maxime. "Caractérisation fonctionnelle et structurale d’une protéine alternative mitochondriale : AltMiD51". Mémoire, Université de Sherbrooke, 2017. http://hdl.handle.net/11143/10250.

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Contrairement à la vision classique des ARNms eucaryotes qui ne contiendrait qu’une seule séquence codante, de nombreuses évidences expérimentales montrent que ces ARNms contiennent plusieurs séquences codantes qui permettraient l’expression de plusieurs protéines différentes. Les ARNms sont donc multicodants et contiennent des cadres de lectures alternatifs (AltORFs pour alternative open reading frames). Ces ORFs alternatifs sont présents dans les régions non-traduites (UTRs) ou chevauchant le RefORF (cadre de lecture ouvert de référence) dans les cadres de lectures non-canoniques +2 et +3. Le protéome est donc plus complexe que ce que l’on pense. Toutefois, le rôle et la fonction de ces nouvelles protéines restent à être investigués. Au cours de mon projet de recherche à la maîtrise, j’ai commencé la caractérisation de la protéine alternative AltMiD51, codée dans le 5’UTR du gène bicistronique MIEF1/SMCR7L/MID51 et co-exprimée avec sa protéine de référence MiD51. Par des approches variées de biologie moléculaire, cellulaire et biochimique, j’ai d’abord démontré et confirmé la localisation cellulaire de la protéine AltMiD51 à la mitochondrie. Par la suite, j’ai pu démontrer que la présence d’AltMiD51 affecte significativement la morphologie mitochondriale en fragmentant celle-ci. De plus, j’ai pu davantage cibler la région qui contient l’information de sa localisation ainsi que son effet de fragmentation, soit seulement les 23 premiers acides aminés de sa séquence. J’ai également observé que cette région Nterminale (a.a.23) est encore plus efficace pour induire la fragmentation des mitochondries. J’ai pu démontrer que le motif protéique L-Y-R est essentiel pour l’activité de fragmentation. J’ai également validé l’interaction in vivo de AltMID51 avec sa protéine partenaire ACPM (Acyl carrier protein) dans des foci mitochondriaux. En conclusion, mes travaux à la maîtrise ont permis de mettre en évidence que la protéine AltMiD51 est un nouveau facteur impliqué dans la fission mitochondriale. Ces résultats ouvrent de nouvelles perspectives en ce qui concerne la caractérisation de nouvelles protéines alternatives et par leur contribution dans la biologie moléculaire de la cellule.
Abstract : Challenging the dogma that eukaryotic mRNAs contain a single coding sequence, an ever-growing number of studies highlight the possibility for these mRNAs to have several coding sequences, and thereby code for multiple proteins. Thus, eukaryotic mRNAs are multicoding and present alternative open reading frames (AltORFs). These alternative ORFs are present in the non-translated region (UTRs), and within or overlapping the RefORF (reference open reading frame) in non-canonical frames (+2 and +3). The proteome is indeed more complex than we initially thought. However, the role and biological function of these proteins remain to be elucidated. Over the course of my MPhil, I started characterizing the alternative protein AltMiD51, encoded in the 5’UTR of the bicistronic MIEF1/SMCR7L/MID51 gene, and coexpressed with its reference protein (MiD51). Using a wide range of molecular biology, cellular and biochemistry assays, I first demonstrated AltMiD mitochondrial localisation. I then proved AltMiD51 expression alters mitochondrial dynamics, enhancing a fragmented morphology. Moreover, I further characterized the sequence region responsible for AltMiD51 localisation and mitochondrial fragmentation, namely the first 23 amino acids. I also observed that this N-terminal region alone presents a stronger phenotype of mitochondrial fragmentation. In addition, I proved the L-Y-R domain is essential for AltMiD51 fragmentation activity, and I validated AltMiD51 in vivo interaction with ACPM (Acyl Carrier Mitochondrial Protein) within mitochondrial foci. Eventually, the work presented here highlighted AltMiD51 as a novel factor involved in mitochondrial fragmentation. These results shed light on new perspectives regarding the characterisation of alternative proteins and their contribution to the cellular metabolism.

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Silva, Thibério Carvalho da. "Hidrolisado proteico de resíduo de pescado na alimentação da tilápia do Nilo: digestibilidade e desempenho zootécnico". Universidade Estadual do Oeste do Paraná, 2014. http://tede.unioeste.br/handle/tede/3818.

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The aim of this study was to determine the apparent digestibility coefficient (ADC) of energy and nutrients from protein hydrolysate of fish waste (HFW) and the growth performance of larvae of Nile tilapia (Oreochromis niloticus) fed during HFW the sexual reversion. For the digestibility experiment , we used 120 juvenile Nile tilapia with an average weight of 100g, distributed in six tapered cylindrical tanks with a capacity of 90L, suitable for feces, where they remained for a period of seven days to adaptive diets and conditions experimental. The determination ADC and apparent digestible energy were made by indirect method, having been used 0.01% chromic oxide as an inert marker incorporated into the ration. For the performance experiment used 375 larvae were three days old (post-hatching), distributed in 25 aquariums 30L in completely randomized design with five treatments and five replications . Five diets were prepared based on vegetable ingredients, which were included 0, 2, 4, 6 and 8% HFW. The variables analyzed were: final weight, weight gain, specific growth rate, survival and flock uniformity. The digestibility values were: ADC of dry matter, 98.29%; ADC of crude protein, 99.28%; ADC gross energy, 99.13%, and apparent digestible energy of 6425.79 kcal.kg-1. The treatment effect (p<0.05) positively final weight and weight gain, with the best level of 4.75% and the specific growth rate, the best level recorded was 4.77%. We conclude that the HFW can be efficiently used for Nile tilapia. The growth performance was not affected by levels of incluisão HFW, however it is recommended to include 4.75% for this stage of the cultivation of tilapia .
O objetivo do trabalho foi determinar o coeficiente de digestibilidade aparente (CDA) da energia e nutrientes do hidrolisado proteico de resíduo de pescado (HPRP) e avaliar o desempenho zootécnico de larvas de tilápia do Nilo (Oreochromis niloticus) alimentadas com HPRP durante a fase de reversão sexual. Para o experimento de digestibilidade foram utilizados 120 juvenis de tilápia do Nilo com peso médio de 100g, distribuídos em seis tanques cônicos cilíndricos com capacidade de 90L, adequados para coleta de fezes, onde permaneceram por um período adaptativo de sete dias às dietas e condições experimentais. A determinação CDA e energia digestível aparente foram feitas por metodologia indireta, tendo sido utilizado 0,01% de óxido de crômio como marcador inerte incorporado à ração. Para o experimento de desempenho produtivo foram utilizadas 375 pós-larvas com três dias de idade (pós-eclosão), distribuídas em 25 aquários de 30L, em delineamento inteiramente casualisado, com cinco tratamentos e cinco repetições. Foram elaboradas cinco rações a base de ingredientes vegetais, as quais foram incluídas 0, 2, 4, 6 e 8% de HPRP. As variáveis analisadas foram: peso final, ganho de peso, taxa de crescimento específico, sobrevivência e uniformidade do lote. Os valores de digestibilidade encontrados foram: CDA da matéria seca, 98,29%; CDA da proteína bruta, 99,28%; CDA da energia bruta, 99,13%; e energia digestível aparente de 6425,79 kcal.kg-1. Os tratamentos influenciaram (p<0,05) positivamente o peso final e ganho em peso, sendo o melhor nível de inclusão de 4,75% e para a taxa de crescimento específico, o melhor nível indicado foi de 4,77%. Conclui-se que o HPRP pode ser eficientemente utilizado pela tilápia do Nilo. O desempenho zootécnico não foi prejudicado pelos níveis de incluisão do HPRP, indicando a inclusão de 4,75% em dietas para esta fase.

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Zhang, Meiling. "MOLECULAR DEFECTS OF MEF2 FAMILY PROTEINS AND NAC PROTEINS THAT BLOCK MYOGENESIS AND PROMOTE TUMORIGENESIS IN RHABDOMYOSARCOMA". OpenSIUC, 2015. https://opensiuc.lib.siu.edu/dissertations/1079.

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Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the molecular defects present in these cells that block myogenesis. We have found MEF2D is absent in RMS cell lines representing both major subtypes of RMS and primary cells derived from an embryonal RMS mice model. We have shown that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find MEF2D cannot bind to muscle specific gene promoters. Exogenous expression of MEF2D activates muscle specific luciferase constructs, upregulates p21 expression and increases muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility, anchorage independent growth in vitro, and tumor growth in vivo by xenograft assay. We also have found MEF2C is deregulated in rhabdomyosarcoma with the aberrant alternative splicing. We have shown that exon α in MEF2C is aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 being highly downregulated in RMS cells compared with normal myoblasts. We find that MEF2Cα1 is the ubiquitously expressed isoform which exhibits no myogenic activity and that MEF2Cα2, the muscle specific MEF2C isoform, is required for efficient differentiation. Compared with MEF2Cα2, MEF2Cα1 more strongly interacts with and recruits HDAC5 to myogenic gene promoters to repress muscle specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increases myogenic activity and promotes differentiation in RMS cells. We have also identified a serine protein kinase, SRPK3, which is downregulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage independent growth of RMS cells. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of the NAC complex in normal myogenesis and tumor progression in rhabdomyosarcoma cells. We found that the muscle specific subunit of the NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was induced in normal cells and downregulated in RMS cells, while BTF3, also known as NACβ, was induced in normal cells and severely downregulated in RMS cells. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated normal cells, and this association was dependent on the expression of BTF3. We further investigated the involvement of skNAC in RMS progression. We found that the muscle specific expressed methyltransferase Smyd1 was nuclear localized in RMS cells and its interaction partner skNAC was switched with corepressors (HDAC1 and TBX2). We also confirmed the expression of skNAC was regulated by the splicing factor kinase SRPK3 and overexpression of SPRK3 induced skNAC expression and muscle differentiation in RMS cells. We also confirmed the overexpression of BTF3 in patient RMS tumors and depletion of BTF3 induced apoptosis in RMS cells and decrease RMS cell survival. BTF3 depletion also sensitized TRAIL induced cell apoptosis in RMS cells. However, BTF3 played a different role in normal cells. Deletion of BTF3 in C2C12 cells does not induce cell apoptosis, which suggests BTF3 functions as an anti-apoptosis factor in RMS cells and could be used as a cancer specific therapeutic target in RMS cells.

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27

Chow, I.-Ting. "Functional study of ClpB95 and ClpB80, the alternative translation products of the E. coli clpB transcript /". Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9842.

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Ackelman, Jenny. "Alternative splicing and its regulation under normal and abnormal conditions". Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-56899.

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Cai, Yangjun. "Simple Alternative Patterning Techniques for Selective Protein Adsorption". University of Akron / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=akron1257386752.

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Decarli, Junior Antonio. "Hidrolisados proteicos na alimentação do jundiá (Rhamdia voulezi) em tanques-rede". Universidade Estadual do Oeste do Paraná, 2013. http://tede.unioeste.br:8080/tede/handle/tede/1532.

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Made available in DSpace on 2017-07-10T17:47:54Z (GMT). No. of bitstreams: 1 Junior_Antonio_Decarli.pdf: 458430 bytes, checksum: 7143459c6921785611288158636e97b3 (MD5) Previous issue date: 2013-04-12
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Current investigation evaluates the productive performance, carcass characteristics, chemical composition and biochemical parameters of catfish Rhamdia voulezi juveniles reared in cages and fed on diets with different meat hydrolysates. Three hundred and twenty juveniles, average initial weight 35.5 ± 0.3 g, distributed in sixteen 0.3 m3 hapas, were installed in four 4 m3 cages, in a randomized block design with four treatments and four replicates. Treatments included 60g pig liver hydrolysate/kg of diet, 40g tilapia carcass hydrolysate/kg of diet and 60g sardine hydrolysate/kg of diet. All treatments were isoproteic and isocaloric. Data were analyzed by ANOVA and then by Tukey´s test at 5% probability by SAEG statistical program. Results show that meat hydrolysates may be added to diets for the feeding of catfish
O objetivo do trabalho foi avaliar o desempenho zootécnico, as características da carcaça, a composição química e os parâmetros bioquímicos de juvenis de jundiá Rhamdia voulezi criados em tanques-rede e alimentados com diferentes hidrolisados cárneos incluídos na dieta. Para isso, foram distribuídos 320 juvenis com peso médio inicial de 35,5 ± 0,3 g em 16 hapas de 0,3 m3 de volume útil instalados em quatro tanques-rede de 4 m3, em um delineamento de blocos ao acaso com quatro tratamentos e quatro repetições. Os tratamentos constituíram-se da inclusão de 60 g de hidrolisado de fígado suíno/kg de ração, 40 g de hidrolisado de carcaça de tilápia/kg de ração e 60 g de hidrolisado de sardinha/kg de ração, sendo elas formuladas para serem isoproteicas e isoenergéticas. Os dados foram submetidos à Anova e posteriormente ao teste de Tukey ao nível de 5% de probabilidade utilizando o programa estatístico SAEG. Os hidrolisados cárneos podem ser adicionados em rações para a alimentação do jundiá

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31

Sheth, Mili. "Discovery and characterization of KNOX proteins lacking a homeodomain, produced by alternative splicing of KNAT1-like genes in gymnosperms and angiosperms". Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/31639.

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Kazak, Lawrence. "Mitochondrial targeting of nucleic acid transacting proteins via alternative translation initiation". Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607696.

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McQueen, Peter. "Alternative strategies for proteomic analysis and relative protein quantitation". Wiley-VCH, 2015. http://hdl.handle.net/1993/30850.

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The main approach to studying the proteome is a technique called data dependent acquisition (DDA). In DDA, peptides are analyzed by mass spectrometry to determine the protein composition of a biological isolate. However, DDA is limited in its ability to analyze the proteome, in that it only selects the most abundant ions for analysis, and different protein identifications can result even if the same sample is analyzed multiple times in succession. Data independent acquisition (DIA) is a newly developed method that should be able to solve these limitations and improve our ability to analyze the proteome. We used an implementation of DIA (SWATH) to perform relative protein quantitation in the model bacterial system, Clostridium stercorarium, using two different carbohydrate sources, and found that it was able to provide precise quantitation of proteins and was overall more consistent in its ability to identify components of the proteome than DDA. Relative quantitation of proteins is an important method that can determine which proteins are important to a biochemical process of interest. How we determine which proteins are differentially regulated between different conditions is an important question in proteomic analysis. We developed a new approach to analyzing differential protein expression using variation between biological replicates to determine which proteins are being differentially regulated between two conditions. This analysis showed that a large proportion of proteins identified by quantitative proteomic analysis can be differentially regulated and that these proteins are in fact related to biological processes. Analyzing changes in protein expression is a useful tool that can pinpoint many key processes in biological systems. However, these techniques fail to take into account that enzyme activity is regulated by other factors than controlling their level of expression. Activity based protein profiling (ABPP) is a method that can determine the activity state of an enzyme in whole cell proteomes. We found that enzyme activity can change in response to a number of different conditions and that these changes do not always correspond with compositional changes. Mass spectrometry techniques were also used to identify serine hydrolases and characterize their expression in this organism.
February 2016

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McDade, S. S. "Modulation of septin 9 protein levels by alternative splicing". Thesis, Queen's University Belfast, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411129.

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Stewart, Deborah. "P53 regulatory mechanisms by human papillomavirus (HPV) E6 and alternative splicing". Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85651.

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In normal cells, the p53 tumour suppressor induces cell cycle arrest or apoptosis in response to a variety of stresses, including DNA damage and ectopic oncogene expression. However, cellular pathways controlled by p53 are compromised in virtually all cancers. Defining the mechanisms regulating p53 activity in normal and tumour cells has therefore been a major priority in cell biology and cancer research.
In this study, we characterized two important regulatory mechanims of p53 activity: (i) Human papillomavirus (HPV) E6 interaction and (ii) alternative splicing. Recognized as the major etiological agents for cervical cancer, the oncogenic potential of HPVs correlates with their ability to target p53 for degradation. This study demonstrates that both p53 and HPV-18 E6 are exported from the nucleus when co-expressed, via a process that involves the C-terminal nuclear export signal (NES) of p53. However, neither nuclear export nor the p53 C-terminal NES is required for HPV-18 E6-mediated ubiquitination or degradation of p53.
This study also demonstrates that both low- and high-risk HPV E6 proteins are degraded by the ubiquitin-proteasome pathway, and thus provides an explanation for the low levels of E6 detected in cervical cancer cells.
Also reported in this study is a novel mechanism of p53 regulation arising through alternative splicing. This novel mRNA encodes a N-terminal deleted isoform of p53, termed p47. As demonstrated within, p47 does not supress cell viability but impairs both p53-mediated transcriptional activity and growth suppression. Interestingly, p47 increases both p53 monoubiquitination and nuclear export. We propose that p47 induces nuclear export of p53 by a mechanism involving monoubiquitination, as supported by recent findings from Li and colleagues (2003). The p47 protein also protects p53 from both Mdm2- and HPV-18 E6-mediated degradation. A number of cancers display abnormal localization of wildtype p53, and it will be important to examine the role of p47 in these tumours.
Taken together, the regulation of p53 activity by both HPV E6 and the alternative splice variant p47 involves alterations in p53 ubiquitination status, protein stability, and cell localization. Insight gained into these negative regulatory mechanisms may aid in the design of therapeutic strategies for reactivating wild-type p53 in HPV-associated and non-associated cancers.

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Lopez, Mejia Isabel Cristina. "Alternative splicing of LMNA gene : lessons from a new mouse model of Hutchinson-Gilfort progeria syndrome". Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20077.

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Le vieillissement est un processus complexe qui peut être influencé par des facteurs environnementaux et génétiques. Le syndrome progéroïde de Hutchinson-Gilford (HGPS ou progéria) fourni une preuve irréfutable de l'implication de l'épissage dans le processus de vieillissement. La progéria est une maladie due à une mutation hétérozygote silencieuse qui renforce l'utilisation d'un site 5' d'épissage interne dans l'exon 11 de l'ARN pré-messager LMNA, ce qui entraîne la production d'une protéine tronquée appelée «progérine». Le défaut d'épissage du gène LMNA a aussi lieu dans les cellules de personnes âgées, et la correction de ce défaut permet un sauvetage partiel des anomalies qu'il provoque. Ceci fait de l'ARN pré-messager LMNA une cible très attractive pour des thérapies ayant pour but de corriger l'épissage. Mes travaux de thèse ont montré que cette mutation silencieuse active un site d'épissage 5' dans l'exon 11 en changeant la structure de l'ARN. Ce changement de structure facilite l'interaction de la snRNP U1 avec le site d'épissage et permet ainsi sa modulation par les protéines SR SRSF1 et SRSF6. J'ai aussi participé à la caractérisation d'un nouveau modèle murin qui reproduit l'altération d'épissage des patients HGPS au niveau du gène Lmna souris. De façon surprenante, ce modèle récapitule tous les phénotypes du syndrome HGPS. Les souris homozygotes, dans lesquelles la plupart de la lamine A est convertie en progérine, ne vivent pas plus de 5 mois, alors que les souris hétérozygotes vivent autour d'un an et que les contrôles sauvages vivent deux ans. Étonnamment, des souris qui n'expriment ni la lamine A ni la progérine, mais uniquement de la lamine C, vivent plus longtemps que les souris contrôle, suggérant que la lamine A et la progérine, qui sont produites à partir du même transcrit, participent à la régulation de la durée de la vie. De plus, la caractérisation initiale des souris HGPS indique que l'expression de la progérine est délétère pour le tissu adipeux, établissant ainsi un lien inattendu entre l'épuisement du tissu adipeux et le vieillissement accéléré. Ce nouveau modèle murin est actuellement en train d'être utilisé pour des approches de modulation de l'épissage aberrant du gène LMNA avec des oligonucléotides antisense et des petites molécules chimiques
Aging is a complex cellular and organismal process that can be influenced by environmental as well as genetic factors. A striking proof-of-concept that splicing regulation plays an important role in the aging process is provided by Hutchinson-Gilford progeria syndrome (HGPS), a disease caused by a heterozygous silent mutation that enhances the use of an internal 5' splice site in exon 11 of LMNA pre-mRNA and leads to the production of a truncated protein called “progerin”. The LMNA splicing defect also occurs with increased frequency in cells from healthy aged individuals and correction of this defect leads to partial reversal of age-related dysfunction. This makes LMNA pre-mRNA an attractive target for splicing-correction therapies. During my PhD thesis I have characterized the splicing mechanism responsible for progerin production and demonstrated that this process is conserved from mouse to human. I have found that HGPS mutation changes the accessibility of the exon 11 internal 5' splice site, allowing its modulation by U1 snRNP and a subset of SR proteins, namely SRSF6 and SRSF1. I have also participated to the characterization of a new mouse model reproducing human HGPS splicing alteration in the mouse Lmna gene. Strikingly, this model recapitulates all phenotypic manifestations of HGPS. The homozygous mice, where most lamin A is converted to progerin, lived no longer than 5 months, whereas heterozygous mice lived in average one year and wild type littermates up to two years. Unexpectedly, mice expressing neither lamin A nor progerin, but only lamin C, lived longer than wild type littermates mice, suggesting that lamin A and progerin which are produced from the same transcript, control critical steps of lifespan. Furthermore, initial characterization of HGPS mouse model indicated that progerin expression is deleterious for adipose tissue, establishing an unexpected link between adipose tissue depletion and accelerated aging. The new mouse model is currently being used for pharmacological modulation of LMNA aberrant splicing by antisense oligonucleotides and small molecules

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Friesen, Erin. "Use of alternative feed ingredients and the effects on growth and flesh quality of Atlantic salmon (Salmo salar) and sablefish (Anoplopoma fimbria)". Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/2610.

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Aquaculture feeds, traditionally composed mainly of fishmeal and fish oil, currently represent the largest cost to fish farmers. With aquaculture growing at an average of 8.8% per year and limited supply of fishmeal and fish oil, suitable alternatives must be found. In addition to increasing sustainability and lowering production costs, the use of plant and/or animal ingredients has the potential to lower flesh levels of persistent organic pollutants (POPs) such as polychlorinated biphenyls. Fish oil and to a lesser extent fishmeal, are considered to be the largest source POPs in farmed fish. Using alternative feed ingredients however, can compromise fish growth and the flesh quality of the final product. Lipid sources including flaxseed oil, canola oil, poultry fat and the protein sources canola protein concentrate, soy protein concentrate and poultry by-product meal were examined as alternatives to fish oil and fishmeal in one on-farm field study and one laboratory feeding trial with Atlantic salmon (Salmon salar) and two laboratory feeding trials conducted on sablefish (Anoplopoma fimbria), a relatively new marine aquaculture species. The nutritive value of the alternative ingredients was assessed on the basis of fish growth performance, proximate composition, fatty acid composition and apparent digestibility coefficients. Sensory attributes were evaluated in the sablefish studies while flesh POP levels were determined in both species. The use of alternative dietary lipids showed no negative effects on fish performance. However replacement of fishmeal with plant proteins in some cases, negatively affected fish growth. Flesh levels of persistent organic pollutants were significantly decreased (p<0.05) with the use of alternative dietary lipids, and flesh levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also depressed. Activated carbon treated anchovy oil and finishing diets were examined in the Atlantic salmon laboratory feeding trial and were effective at lowering flesh POP levels while providing high levels of EPA and DHA. The use of alternative feed ingredients will soon be inevitable in aquaculture feeds. The current research shows alternative lipids and proteins can be incorporated successfully in sablefish and Atlantic salmon feeds with minimal effects on fish growth and quality.

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Rodríguez, Carbó Laia. "Characterization of alternative therapeutic sites on the androgen receptor and novel protein-protein associations". Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/386151.

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The androgen receptor (AR) is a ligand-activated transcription factor that plays a crucial role in the correct development, differentiation, and function of male reproductive organs. Alterations in the AR protein or in the AR signaling pathway result in pathologies such as prostate cancer (PCa), which is the fifth leading cause of cancer-related death in men in most western industrialized countries. AR represents the major clinical target in the treatment of PCa and, to date, all the FDA-approved drugs against the AR target the ligand-binding pocket of the receptor, competing with the natural hormones. Unfortunately, prolonged treatments with antiandrogens invariably fail, rendering them ineffective and resulting in the development of castration-resistant PCa. Thus, there is pressing need for more potent and selective novel AR antagonists capable of blocking the action of AR in tumors resistant to conventional antiandrogens. Structural and functional analysis highlighted the presence of another site on the ligand-binding domain (LBD) of AR named binding function-3 (BF-3). Importantly, the BF-3 pocket is a hot spot for mutations involved in PCa and androgen insensitivity syndromes, and some FDA-approved drugs have shown to bind at this site, evidencing its pharmacological potential. Our initial hypothesis was that BF-3 may be a protein-protein interaction site, which may play a physiological role in AR action. Therefore, the major goals of this PhD project have been to further determine the biological role of the newly described BF-3 regulatory surface and to identify and better characterize BF-3-interacting proteins. Firstly, we demonstrate that the newly identified BF-3 site in the human AR is highly conserved among the SR subclass, and that naturally occurring mutations associated with pathology and affecting the function of several NRs in vitro colocalize with their putative BF-3 sites. activation function-2 (AF-2) and BF-3 pockets were mutated in order to further elucidate the molecular mechanisms by which point mutations are linked to disease and determine the allosteric responses they induce in the receptor function. We demonstrate that mutations in the BF-3 pocket and residues lying between the AF-2 and BF-3 sites affect AR transactivation and coactivation by GRIP1, as well as AR N/C interaction and binding to NR corepressors NCoR and SMRT. Finally, as the shape and characteristics of the BF-3 pocket in the examined NRs suggest a possible role for protein-protein interactions, we performed several Y2H screens of both an adult human brain and prostate cDNA libraries to identify possible BF-3 novel interactors. In the presence of DHT, four new putative AR binders were identified: ARMC9, MAPK8IP1, Rab11FIP3, and Uba3. Moreover, Rab11FIP3 and Uba3 inhibited the AR LBD transactivation capacity, attenuated its coactivation by GRIP1 and disrupted the N/C interaction, suggesting that these proteins may be AR corepressors not previously described. Furthermore, we show that SRC3, ARMC9 and Uba3 are capable of binding to the AR LBD in the presence of antiandrogens. Our results suggest that targeting the BF-3 surface may open new promising alternatives to current therapeutics. Compounds that may affect allosterically NR function by binding to BF-3 open promising avenues to develop type-specific NR modulators.

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39

Miller, Becky M. "Functional analysis of Drosophila melanogaster muscle myosin heavy chain alternative domains /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2004. http://wwwlib.umi.com/cr/ucsd/fullcit?p3138951.

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Casanova, Pedro Trindade. "ALTERNATIVAS DE SUPLEMENTAÇÃO PARA RECRIA DE NOVILHAS DE CORTE EM PASTAGEM NATURAL VISANDO PESO PARA ACASALAMENTO". Universidade Federal de Santa Maria, 2016. http://repositorio.ufsm.br/handle/1/10904.

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Demand for beef is growing worldwide due to increased population and purchasing power, which leads to improvement in the diet of the population and consequently increased demand for meat products. In Rio Grande do Sul, livestock still has extensive production characteristics, being essential the increase of the production efficiency for better meet the growing demand for meat. Using forage management techniques coupled with the use of supplements, researchers seek alternatives to assist in the conservation and rational exploitation of pastures. The aim of this work was to reach 65% of adult weight (AW) at 22 months, with heifers of 18 months starting age and different initial weights (210 or 255 kg), reared in natural grasslands managed by rotational grazing method with two rest intervals (375 GD and 750 GD) which determined the treatments. The heifers received protein-energy supplementation and or protein-energy more corn. Herbage mass and canopy height were decreasing in the rest interval 375GD while in the 750 GD there was a maintenance of the mass and height r during most of the experimental period. The forage intake was similar between rest intervals, since the quality of the apparently consumed pasture had the lowest ADF content in the diet of heifers managed in the 750 GD which also dispended longer time in grazing activities, rumination and visited more stations per minute. The ultimate goal was achieved only in heifers managed in the rest interval 750 GD in order to conclude that to reach 65% of the PA, under the conditions of this experiment, the light heifers managed 375 GD rest interval must enter the second winter with at least 52% PA and heavy heifers 60% PA or extend supplementation for more than 21 days.
A demanda por carne bovina é crescente em nível mundial devido ao aumento da população e do poder aquisitivo, que leva a melhoria na dieta da população e consequentemente aumento na procura por produtos cárneos. No Rio Grande do Sul, a pecuária ainda tem características de produção extensiva, sendo indispensável o aumento na eficiência de produção para melhor atender à crescente demanda por carne. Utilizando técnicas de manejo forrageiro aliado ao uso de suplemento, pesquisadores buscam alternativas que auxiliem na exploração racional e conservacionista das pastagens. O objetivo com esse trabalho foi de atingir 65% do peso adulto (PA) aos 22 meses, em novilhas de corte com 18 meses de idade inicial e com diferentes pesos médios iniciais (210 e 255 kg), recriadas em pastagem natural manejada pelo método de pastoreio rotativo com dois intervalos de descanso (375 GD e 750 GD) que determinou os tratamentos. As novilhas receberam suplementação proteica-energética e ou proteica-energética mais milho. A massa de forragem foi decrescente no intervalo de descanso 375 GD enquanto que no de 750 GD houve uma manutenção da massa e a evolução da altura da forragem foi semelhante entre os intervalos de descanso. O consumo de forragem também foi semelhante entre os intervalos de descanso, já a qualidade do pasto aparentemente consumido apresentou menor teor de FDA na dieta das novilhas manejadas no 750 GD, que também dispenderam mais tempo nas atividades de pastejo, ruminação bem como visitaram mais estações por minuto. O objetivo final foi alcançado apenas nas novilhas manejadas no intervalo de descanso 750 GD de maneira a concluir que para chegar aos 65% do PA, nas condições desse experimento, as novilhas leves manejadas com 375 GD de descanso devem entrar no segundo inverno com pelo menos 52% do PA e as pesadas com 60% do PA ou estender a suplementação por mais pelo menos 21 dias.

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41

Voevodskaya, Nina. "Paramagnetic states of diiron carboxylate proteins". Doctoral thesis, Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-652.

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Melo, Thiago Anchieta de. "Efeito do extrato da alga marinha Ascophyllum nodosum e do fosfito de potássio na morfofisiologia do fungo Colletotrichum gloeosporioides, na indução de resistência em mangas \'Tommy Atkins\' contra a antracnose e em características físicas e químicas desses frutos". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/11/11135/tde-20032018-105839/.

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A mangicultura é uma das atividades mais importantes para a fruticultura brasileira. Dentre as variedades produzidas, o cultivar \'Tommy Atkins\', sem dúvida, é o mais expressivo. Após a colheita, a qualidade fisiológica das mangas, geralmente, é mantida pela integração de técnicas de controle físico e aplicação de moléculas com atividade biológica contra microrganismos, a exemplo dos fungicidas aplicados no controle do fungo Colletotrichum gloeosporioides, agente causal da antracnose, principal doença na fase de pós-colheita de mangas. Entretanto, atualmente há forte pressão da população para a utilização de moléculas que deixem nenhum ou o mínimo possível de resíduos em alimentos, especialmente os consumidos in natura. Vários produtos são vendidos no Brasil como biofertilizantes, mas, estes apresentam também, a capacidade de mitigar estresses bióticos e abióticos, inerentes da vida pós-colheita de frutas. Nesse contexto, pode-se citar o extrato da alga marinha Ascophyllum nodosum (Acadian®) e o fosfito de potássio (Phytogard®), utilizados em vários passos do processo de produção agrícola, mostrando respostas diversas sobre os vegetais tratados. Ambos os produtos apresentam baixa toxicidade ao homem e ao ambiente e não são fitotóxicos. Assim, com a ideia de gerar informação mais acertada acerca dos processos envolvidos a partir da utilização desses produtos, na cadeia produtiva da manga, este trabalho foi construído sobre três vertentes principais. A primeira parte, objetivou verificar o efeito in vitro do extrato da alga marinha A. nodosum e do fosfito de potássio sobre a morfofisiologia do fungo C. gloeosporioides isolado de mangas, variedade \'Tommy Atkins\'. Na segunda parte, o objetivo do trabalho foi avaliar o efeito do extrato da alga marinha A. nodosum e do fosfito de potássio, ambos aplicados em diferentes concentrações, sobre o parasitismo do fungo 1 em mangas \'Tommy Atkins\', na perspectiva da indução de resistência, na fase de pós-colheita desses frutos. Finalmente, na terceira parte do trabalho, objetivou-se verificar o efeito do extrato da alga marinha A. nodosum e do fosfito de potássio, ambos aplicados em diferentes concentrações, sobre características físicas e químicas de mangas \'Tommy Atkins\', na fase de pós-colheita. Como resultados da primeira parte do trabalho, observou-se que o extrato de algas induz o crescimento e a esporulação do fungo, inibindo, contudo, a germinação e a fixação de conídios produzidos pelo patógeno. O fosfito de potássio interfere no crescimento e esporulação do microrganismo e inibe a germinação e adesão de conídios produzidos por C. gloeosporioides. Os dois produtos alteram a permeabilidade seletiva da membrana plasmática da hifa e incrementam a atividade das enzimas β-1,3-glucanase e quitinase na estrutura. Entretanto, somente o extrato de algas interferiu no conteúdo total de proteínas da hifa, aumentando esse parâmetro. Os dois produtos diminuíram a atividade celulolítica de C. gloeosporioides. Na segunda parte, os resultados demonstraram que, tanto para o extrato de algas quanto para o fosfito de potássio, houve diminuição do tamanho da lesão, da velocidade de crescimento da lesão e da AACPD. Além disso, foram observados incrementos em todos os parâmetros bioquímicos analisados, o que indicou que os produtos têm efeito indutor de resistência em mangas. Finalmente, como resultados para a terceira parte do trabalho, foi evidenciado que tanto o extrato de algas quanto o sal de potássio, em todas as concentrações utilizadas, ajudaram na redução da perda de massa dos frutos, retardaram a diminuição do ângulo de cor da polpa (ângulo Hue) e a firmeza desta. Além disso, os produtos testados desaceleraram a perda de acidez da polpa e mantiveram elevados os valores de ácidos orgânicos, a exemplo do ácido cítrico; mantiveram abaixo do tratamento controle o conteúdo de sólidos solúveis (°Brix), mas não interferiram no total de carboidratos encontrados nas cascas dos frutos. Conclusivamente, o extrato de A. nodosum e o fosfito de potássio, retardam o amadurecimento e senescência de mangas na fase de pós-colheita, reduzem a severidade da antracnose nos frutos pela indução de resistência e ainda, apresentam efeitos diretos sobre o fungo C. gloeosporioides. Dessa maneira, os produtos podem ser utilizados como mantenedores da qualidade fisiológica de mangas \'Tommy Atkins\', pois minimizam os estresses de ordem biótica e abiótica relativos à vida pós-colheita dessas frutas.
Mango farming is one of the most important activities for Brazilian fruit growing. Among the varieties produced, the cultivar \'Tommy Atkins\' is undoubtedly the most expressive. After harvesting, the physiological quality of mangoes is generally maintained by the integration of physical control techniques and the application of molecules with biological activity against microorganisms, such as the fungicides applied in the control of the fungus Colletotrichum gloeosporioides, the causal agent of anthracnose, the main disease in the postharvest phase of mangoes. However, there is currently strong population pressure for the use of molecules that leave none or the least possible residues in food, especially those consumed in natura. Several products are sold in Brazil as biofertilizers, but also present the ability to mitigate biotic and abiotic stresses inherent of the postharvest fruit life. Ascophyllum nodosum seaweed extract (Acadian®) and potassium phosphite (Phytogard®), both used in several steps of the agricultural production process, can be mentioned in this context, showing different responses on treated plants. Both products have low toxicity to man and the environment and are not phytotoxic. Thus, in order to generate precise information about the processes involved in the use of these products, in the production chain of mango, this work was built on three main strands. The first part aimed to verify the in vitro effect of the A. nodosum seaweed extract and the potassium phosphite on the morphophysiology of the fungus C. gloeosporioides isolated from mangoes \'Tommy Atkins\'. In the second part, the objective of this work was to evaluate the effect of A. nodosum seaweed extract and the potassium phosphite, both applied in different concentrations, on the parasitism of the fungus C. gloeosporioides in mangoes \'Tommy Atkins\', from the perspective of induction of resistance in the postharvest phase of these fruits. Finally, in the third part of the work, the objective was to verify the effect of A. nodosum seaweed extract and of the potassium phosphite, both applied in different concentrations, on physical and chemical characteristics of \'Tommy Atkins\' mangoes in the postharvest stage. As results of the first part of this work, it was observed that the algae extract induces the growth and sporulation of the fungus; however, it inhibits the germination and adhesion of conidia produced by the pathogen. Potassium phosphite interferes with the growth and sporulation of the microorganism and inhibits the germination and adhesion of conidia produced by C. gloeosporioides. The two products alter the selective permeability of hypha plasma membrane and increase the activity of the enzymes β-1,3-glucanase and chitinase in the structure. However, only the algae extract interfered in the total protein content of the hypha, increasing this parameter. The two products decreased the cellulolytic activity of C. gloeosporioides. In the second part, the results demonstrated that, for both algae extract and potassium phosphite, there was a decrease in lesion diameter, lesion growth rate and AUDPC. In addition, increments were observed in all biochemical parameters analyzed, which indicated that the products have resistance-inducing effect on mangoes. Finally, as results for the third part of the work, it was evidenced that both the algae extract and the potassium salt, in all the concentrations used, helped to reduce the loss of mass of the fruits, delayed the decrease of pulp color angle (Hue angle) and the firmness of this. In addition, the products tested decelerated the loss of acidity of the pulp and maintained high values of organic acids, as citric acid; controlled soluble solids content in relation to the control (°Brix), but did not interfere in the total carbohydrate found in the fruit peels. Conclusively, the A. nodosum extract and potassium phosphite, delay the maturation and senescence of mangoes in the post-harvest phase, reduce the severity of the anthracnose in the fruits by the induction of resistance and also have direct effects on the fungus C. gloeosporioides. In this way, the products can be used to maintain the physiological quality of \'Tommy Atkins\' mangoes, since they minimize the biotic and abiotic stresses related to the postharvest life of these fruits.

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43

Hinman, Melissa N. "The Function and Regulation of Neurofibromatosis Type 1 Exon 23a Alternative Splicing". Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1390500738.

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Coleman, Jennifer Marie. "Assessing the Potential Use of Teff as an Alternative Grain Crop in Virginia". Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/77012.

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Teff (Eragrostis tef (Zucc.)) is an annual, warm-season cereal crop most notable for its gluten-free, nutrient-packed seed. Experiments were conducted in two regions of Virginia (Blacksburg and Steeles Tavern) in 2010 and 2011 to determine the grain production potential of two teff varieties (brown and white). Additionally, commercially purchased teff flour was evaluated for its suitability in producing a satisfactory baked product. Teff varieties were planted in early June and July at a seeding rate of 6 kg PLS ha??. Nitrogen fertilizer was applied at planting in the form of urea at a rate of 56 kg ha??. The experimental design was a randomized complete block with a two-way factorial treatment structure (variety and planting date) and four replications. Grain yield and nutritive value, straw yield and quality, and plant height were evaluated for each variety and planting date at Steeles Tavern in 2010. Due to failure in crop establishment and difficulties involved in threshing and processing the harvested crop, no data is available in 2010 or 2011 for Kentland or in 2011 for Steeles Tavern. In 2010 at Steeles Tavern, grain yield was significantly higher for the brown variety (367 kg ha??) compared to the white variety (97 kg ha??) for both planting dates. There was no significant difference in straw yield between varieties or planting dates with straw yield averaging 2645 and 2475 kg DM ha?? for brown and white varieties, respectively. Precipitation accumulation at Steeles Tavern was higher in 2010 (greater than 10 cm) during June and July compared to 2011 and the historic average. This may explain why the plots in 2010 were able to successfully establish and out compete weeds. In the lab, four types of baked products were tested to determine the suitability of teff for baked goods. Cakes, cookies, biscuits and bread were tested with varying treatments of teff: control (100% wheat flour) and 10, 20, 30, 40 and 100% teff flour. Each treatment was replicated three times for each product. Generally, bread and cake volumes decreased as the percent of teff increased. Teff flour was best suited for use in cookie and biscuit products compared to cakes and breads since cookies and biscuits require less leavening. Overall, both experiments (field and laboratory) demonstrated the potential of teff as an alternative grain crop in Virginia. However, additional research is needed to overcome problems associated with establishment, harvest, threshing and processing.
Master of Science

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45

Mason, Aaron Charles. "Functional analysis of an alternative Replication Protein A complex containing RPA4". Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/1020.

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Replication Protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism including, but not limited to, DNA replication, DNA repair and recombination. The `canonical' RPA is composed of three subunits (RPA1, RPA2, and RPA3). In addition to the three canonical subunits, there is a human homolog to the RPA2 subunit, termed RPA4, which can substitute for RPA2 in complex formation. The resulting RPA complex has been termed `alternative' RPA (aRPA). The normal function of aRPA is not known; however, previous studies have shown that it does not support S-phase progression in vivo. The goal of this thesis was to characterize the function of aRPA in DNA replication, DNA repair and recombination and profile its expression in human tissues. The studies presented in this thesis show that the aRPA complex has solution and DNA binding properties indistinguishable from the canonical RPA complex as determined by gel mobility shift assays. However, aRPA was unable to support DNA replication and inhibited canonical RPA function in a cell-free simian virus 40 system. aRPA inhibited both initiation and elongation of DNA synthesis in the SV40 system. Two regions of RPA4, the putative L34 loop and the C-terminal winged helix domain, were responsible for inhibiting SV40 DNA replication. The mechanism of SV40 DNA replication inhibition during initiation and elongation was characterized using assays for DNA polymerase α and DNA polymerase δ. aRPA was shown to have reduced interaction with DNA polymerase α and was not able to efficiently stimulate DNA synthesis by DNA polymerase α on aRPA coated single-stranded DNA. However, aRPA stimulated DNA synthesis by DNA polymerase δ in the presence of PCNA and RFC even though a reduced interaction was observed between aRPA and polymerase δ. The role of aRPA in DNA repair was also investigated. aRPA interacted with both Rad52 and Rad51 but had a reduced interaction with Rad51. However, aRPA was still able to stimulate Rad51-dependent strand exchange. aRPA also supported the dual incision/excision reaction of nucleotide excision repair. aRPA was less efficient in nucleotide excision repair than canonical RPA and this reduction was attributed to reduced interactions with the repair factor XPA. In contrast, aRPA exhibited higher affinity for damaged DNA than canonical RPA. The expression of RPA4 and RPA2 was determined by quantitative PCR in established cell lines, human normal tissues and human tumor tissue. RPA4 was shown to be expressed in all normal tissues examined but the level of expression was tissue specific. Additionally, RPA4 expression was decreased in all tumor tissues examined and was at the limit of detection in established cell lines. Taken together, the results presented in this thesis suggest that aRPA is a `non-proliferative' form of RPA that functions to maintain the genomic stability of non-dividing cells.

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46

Silva, Tarcila Souza de Castro. "Exigências em proteína e energia e avaliação de fontes proteicas alternativas na alimentação do cachara Pseudoplatystoma fasciatum". Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11139/tde-26042013-154047/.

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O cachara, Pseudoplatystoma fasciatum, é um siluriforme carnívoro da América do Sul. Apesar da importância da espécie para pesca e piscicultura, não há uma dieta específica e nem as exigências nutricionais determinadas. O objetivo deste projeto foi determinar a digestibilidade aparente da energia e proteína de diferentes alimentos de origem animal e vegetal e, com os valores da digestibilidade dos ingredientes, elaborar rações para determinar as exigências em proteína, energia e relação energia:proteína para o cachara. Para o ensaio de digestibilidade, 105 juvenis de cachara (82,35 ± 17,7 g; 23,04 ± 1,6 cm) foram distribuídos em 21 gaiolas cilíndricas plásticas de 80 L e alimentados até a saciedade aparente em duas refeições diárias (20h00min e 22h00min) com dietas teste obtidas a partir da adição de 0,1% de óxido crômico III e substituição de 30% de uma ração referência (RR; 46% proteína bruta; 4600 kcal energia bruta) pelos seguintes ingredientes: farinha de peixe, farinha de carne e ossos, farinha de vísceras, farinha de penas, farinha de sangue, farelo de soja, farelo de trigo, milho moído e glutenose de milho. Após a última refeição, os peixes eram transferidos para os aquários cônicos (200 L) acoplados a recipientes refrigerados para a coleta de fezes por sedimentação. Os melhores coeficientes de digestibilidade aparente da proteína (99,36%) e energia (86,25%) foram registrados para a farinha de vísceras de aves e farinha de carne e ossos, respectivamente, consideradas fontes alternativas adequadas para substituir com eficiência a farinha de peixe, ingrediente padrão para formulação de rações para o cachara. Em um segundo experimento foram determinados os melhores níveis de energia e proteína nas dietas para juvenis de cachara (53,6 ± 1,30 g e 20,1 ± 1,06 cm), distribuídos aleatoriamente em 75 gaiolas (210 L) alojadas em tanques de alvenaria (12 m3) com constante renovação de água e aeração e alimentados duas vezes ao dia (06h30m e 18h30m) por 60 dias com 25 dietas formuladas para conter cinco níveis de proteína digestível (32, 36, 40, 44 e 48%) e cinco níveis de energia digestível (3600, 3725, 3850, 3975 e 4100 kcal kg-1), em um delineamento inteiramente casualizado com um esquema fatorial 5 x 5 (n = 3). A energia e proteína dietética afetaram o ganho de peso, taxa de crescimento específico, consumo de ração, conversão alimentar, taxa de eficiência proteica, retenção de proteína, índice hepatossomático, índice lipossomático, índice viscerossomático, proteínas totais séricas e triglicerídeos no soro. A energia dietética afetou a retenção de energia pelo cachara, mas a retenção de fósforo e a composição do peixe inteiro não foram influenciadas pela dieta. Com os resultados é possível concluir que os níveis de 3600 kcal kg-1 de ED, 39% de PD e a relação ED:PD de 9,23 kcal g-1 garantem ótimo desempenho e retenção de nutrientes pelo cachara.
Striped surubim, Pseudoplatystoma fasciatum is a South American carnivore catfish of economic importance for fisheries and fish culture alike. However, in spite of its importance for the Brazilian aquaculture, there is no specific diet neither nutritional requirement determined for this specie. The aim of this study was thus determination of apparent digestibility coefficients of selected feedstuff and their use in diets for determination of protein, energy and energy:protein requirements of juvenile striped surubim. Juvenile striped surubim (82.35 ± 17.7 g and 23.04 ± 1.6 cm) were distributed in 21 cylindrical, plastic cages (80 L) and conditioned to a two daily meals (20h00m and 22h00m) feeding regimen on a practical, reference diet (RD) (460.0 g kg-1 crude protein (CP); 19.23 kJ g-1 gross energy (GE)). Test diets were obtained by adding 0.1% chromium III oxide and substituting 30% of one the following feedstuffs in RD: fish meal, meat and bone meal, poultry by-product meal, feather meal, blood meal, soybean meal, wheat bran, corn and corn gluten meal. After the last daily meal, fish were transferred to cylindrical-conical bottomed aquaria (200 L), coupled to refrigerated plastic bottles for feces collection by sedimentation. Best apparent digestibility coefficients of protein (99.36%) and energy (86.25%) were recorded for poultry by-product meal and meat and bone meal, respectively, so deemed ideal surrogate feedstuffs to fish meal, the standard protein source for the formulation and processing of diets for carnivore fish. For determination of best energy and protein level, juvenile striped surubim (53.6 ± 1.30 g and 20.1 ± 1.06 cm) were randomly distributed in 75 cages (210 L), housed in 12 m3 concrete tanks under constant water flow and aeration, and hand fed two daily meals (06h30m and 18h30m) for 60 days. Twenty-five diets were formulated to contain five levels of digestible protein (DP) (32, 36, 40, 44 and 48%) and five levels of digestible energy (DE) (3600, 3725, 3850, 3975 and 4100 kcal kg-1) in a randomized design, 5 x 5 factorial scheme (n = 3). The dietary energy and protein affected the weight gain, specific growth rate, feed intake, feed conversion rate, protein retention, hepatosomatic, liposomatic and viscerosomatic index, serum total protein and triglycerides, but energy retention was affected only by dietary energy. Phosphorus retention and whole body composition were not affected by diets. Estimated dietary requirement for the best performance and best nutrient retention of striped surubim were 3600 kcal kg-1 of DE, 39% of DP and a 9.23 kcal g-1 of DE:DP ratio.

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Vasconcelos, Luciana Guimarães. "Uso do concentrado proteico de soja para frangos de corte de 1 a 21 dias de idade". Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/3401.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Two experiments to evaluate the effect of the use of soy protein concentrate (SPC) in the diet of broilers from one to 21 days of age were performed. In the first experiment 600 broiler, males, of Cobb, from one to 40 days of age, were distributed in a completely randomized design (CRD) with four treatments, six replicates and 25 birds per experimental unit. Treatments consisted of four levels of inclusion of soy protein concentrate (0, 30, 60 and 90 g/kg) levels of pre-starter and starter diets. The parameters evaluated were: performance, enzyme production in the pancreas, gut integrity and immune parameters. In the second experiment, 144 broilers, males, of Cobb, from one to 21 days of age, distributed in CRD with four treatments, six replicates, and six birds per experimental unit. These birds were fed with experimental diets from 14 to 21 days of age and total excreta, to review the metabolization of rations, collected from 18 to 21 days old. The treatments were the same as in the first experiment. The results were submitted to ANOVA and Scott-Knott test; the polynomial regression; the nonparametric analysis (Kruskal-Wallis) and Bonferroni test; adopted was α = 0.05. The use of 30 and 90 g/kg SPC did not affect the weight gain, feed intake, feed conversion or the viability of the poultry. The use of 30 and 60 g/kg of SPC in the diet increased the activity of trypsin and the use of 90 g/kg increased amylase activity. The villus length and crypt depth in the small intestine increased with the use of 60 and 90 g/kg of SPC in the diet and the villous:crypt of the jejunum increased in the absence of SPC. The total leukocytes, eosinophils and lymphocytes and the dosage of immunoglobulin A (IgA) serum levels were not influenced by the SPC proposed in this study. The metabolization coefficient of dry matter (CMMS) increased linearly with the addition of SPC, although the metabolization coefficient of ash (CMCz) has reduced linearly.
Foram realizados dois experimentos com o objetivo de avaliar o efeito da utilização do concentrado proteico de soja (CPS) na dieta de frangos de corte de um a 21 dias de idade. No primeiro experimento 600 frangos de corte, machos, da linhagem Cobb, de um a 40 dias de idade, foram distribuídos em delineamento inteiramente casualizado (DIC), com quatro tratamentos, seis repetições e 25 aves por unidade experimental. Os tratamentos consistiram de quatro níveis de inclusão de concentrado proteico de soja (0, 30, 60 e 90 g/kg) na ração das dietas pré-inicial e inicial. Os parâmetros avaliados foram: o desempenho, a produção enzimática do pâncreas, a integridade intestinal e os parâmetros imunológicos. No segundo experimento foram utilizados 144 frangos de corte, machos, da linhagem Cobb, de um a 21 dias de idade, distribuídos em DIC, com quatro tratamentos, seis repetições e seis aves por unidade experimental. Estas aves foram arraçoadas com as dietas experimentais dos 14 aos 21 dias de idade e as excretas totais, para avaliação da metabolizabilidade das rações, coletadas do 18º ao 21º dia de idade. Os tratamentos estudados foram os mesmos do primeiro experimento. Os resultados foram submetidos à ANOVA e teste de Scott-Knott; à regressão polinomial; à análise não-paramétrica (Kruskal-Wallis) e teste de Bonferroni; adotou-se α = 0,05. A utilização de 30 a 90 g/kg de CPS não influenciou o ganho de peso, o consumo de ração, a conversão alimentar nem a viabilidade das aves. A utilização de 30 e 60 g/kg de CPS nas dietas aumentou a atividade de tripsina e a utilização de 90 g/kg aumentou a atividade de amilase. O comprimento de vilo e a profundidade de cripta do intestino delgado aumentaram com a utilização de 60 e 90 g/kg de CPS na ração e a relação vilo:cripta do jejuno aumentou na ausência de CPS. A contagem de leucócitos totais, eosinófilos e linfócitos e a dosagem de imunoglobulina A (IgA) séricas não foram influenciadas pelos níveis de CPS propostos neste estudo. O coeficiente de metabolizabilidade da matéria seca (CMMS) aumentou de forma linear com a inclusão do CPS, embora o coeficiente de metabolizabilidade das cinzas (CMCz) tenha reduzido linearmente.

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48

Tollervey, James Robert. "Understanding misregulation of alternative splicing in the human TDP-43 proteinopathies". Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609056.

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VIVARELLI, SILVIA. "New roles for RNA processing factors CFIm68 and SRPK2 highlight unexpected links in the control of mammalian gene expression". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2010. http://hdl.handle.net/10281/10747.

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The aim of the first work (presented in the Chapter 2) was to investigate the role performed by SRPK2 kinase in the regulation of alternative splicing in SH-SY5Y neuroblastoma cells after paraquat treatment (a complex I mitochondrial respiratory chain inhibitor). Alternative splicing is a versatile form of genetic control whereby a common precursor messenger RNA (pre-mRNA) is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. This process is particularly important in the nervous system and its essential nature is underscored by the finding that its misregulation is a common feature of human diseases, including neurodegenerative pathologies. Our approach to gain insight the regulation of neuron specific pre-mRNA splicing brought us to the characterization of SRPK2 kinase because this protein phosphorylates Serine/Arginine-rich domain (RS-domain)-containing proteins and it is expressed almost exclusively in the nervous system. In order to understand how SRPK2 intracellular localization and activity are regulated, in first instance we performed a mutational analysis. These mutants have been characterized by transient transfection in SH-SY5Y neuroblastoma cells. We analysed SRPK2 intracellular localization both under physiological condition and after generating stress through mitochondrial damage, since mitochondrial damage and oxidative stress are found in many neurodegenerative diseases. We also determined the effect of this stress treatment on SRPK2 phosphorylation and its nuclear translocation. We did this first by using the minigene E1A, an alternative splicing reporter system, and also by analysing both SR proteins intracellular localization and their phosphorylation status. This work showed that not only paraquat treatment increased the phosphorylated SRPK2 fraction, but also that a specific phosphorylation at its 581 residue could be connected with the nuclear translocation of SRPK2. Consequently, this nuclear translocation brought to a splicing change in the isoform ratio of the minigene reporter system. After the drug treatment we also observed a specific speckled enlarged pattern coupled with an increase in the phosphorylation level for the SR classical proteins, known targets of SR protein kinase 2. These findings supported a functional link between the nuclear translocation and the activity of this neuronal specific kinase. In the second line of our research (presented in the Chapter 3) we performed experiments assessing the function of the mammalian 3’ end processing factor CFIm68 in the mRNA export, thus confirming its action as an adaptor for TAP/NXF1 mRNA export receptor. In particular I helped to demonstrate that the tethering of CFIm68 promoted mRNA export by designing and performing an RNA FISH assay. I used a RNA-biotinylated probe that detected the intracellular localisation of an mRNA reporter construct co-transfected with the CFIm68 protein or control proteins. Therefore we observed an increase of the probe fluorescent cytoplasmic signal only in the presence of the overexpressed CFIm68 but not with other control proteins, observation confirmed by further Real Time PCR data. In the third line of our research (presented in the Chapter 4) we reported that CFIm68 was also involved in the 3’ end cleavage of mammalian histone transcripts (not polyadenylated) by interacting with the LSM11 U7 snRNP component both in vitro and in vivo thus increasing the efficiency of the 3’ end processing in vivo. In this context I performed the Bimolecular Fluorescence Complementation (BiFC) analysis. I co-transfected the CFIm68 and the LSM11 proteins (or its MPL loss-of-function mutant) fused respectively with the C-terminus and the N-terminus of a Venus-Yellow Fluorescent Protein. Thus I detected a nuclear fluorescent complementation in more than 90% of the cells (or not complementation with the MPL mutant counterpart). This data supported our whole characterized observation concerning the involvement of this mammalian cleavage factor in 3’ histone mRNAs processing.

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Taboski, Michael. "Human telomerase regulation by associated proteins and alternatively spliced mRNA variants". Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82434.

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Human telomerase is a ribonucleoprotein complex, minimally composed of a catalytic subunit, hTERT and an RNA component, hTR that contains the template used to synthesize telomere DNA. Telomerase is present in over 80% of all cancer cells. Therefore, an understanding of telomerase regulation is important for cancer research. This thesis work was focused upon the study of telomerase regulation by telomerase associated proteins and alternatively spliced hTERT mRNA variants. Tandem affinity purification (TAP) and immunopurifications with anti-hTERT antibody were used to visualize potential telomerase associated proteins. No specific proteins were identified in TAP-hTERT transfected cells. hTERT was purified from 293 telomerase positive cells, and proteins specifically associated with hTERT were visualized. Insertion 3 and 4 alternatively spliced hTERT mRNA variants were detected in telomerase positive cell lines by RT-PCR and Southern blot analysis. The gamma-deletion alternatively spliced hTERT mRNA variant was detected in Huh-7 liver cells. These results provide information and tools important for a greater understanding of telomerase.

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