Methods for comprehensive structural dynamic analysis generally employ input-output modal analysis with a mathematical model of structural vibration using excitation and response data. Recently operational modal analysis methods using only vibration response data have been developed. In this thesis, both input-output and operational modal analysis, in the presence of significant unmeasured excitations, is considered. This situation arises when a test structure cannot be effectively isolated from ambient excitations or where the operating environment imposes dynamically-important boundary conditions. The limitations of existing deterministic frequency-domain methods are assessed. A novel time-domain estimation algorithm, based on the estimation of a discrete-time autoregressive moving average with exogenous excitation (ARMAX) model, is proposed. It includes a stochastic component to explicitly account for unmeasured excitations and measurement noise. A criterion, based on the sign of modal damping, is incorporated to distinguish vibration modes from spurious modes due to unmeasured excitations and measurement noise, and to identify the most complete set of modal parameters from a group of estimated models. Numerical tests demonstrate that the proposed algorithm effectively identifies vibration modes even with significant unmeasured random and periodic excitations. Random noise is superimposed on response measurements in all tests. Simulated systems with low modal damping, closely spaced modes and high modal damping are considered independently. The accuracy of estimated modal parameters is good except for degreesof- freedom with a low response level but this could be overcome by appropriate placement of excitation and response measurement points. These observations are reflected in experimental tests that include unmeasured periodic excitations over 200% the level of measured excitations, unmeasured random excitations at 90% the level of measured excitations, and the superposition of periodic and random unmeasured excitations. Results indicate advantages of the proposed algorithm over a deterministic frequency domain algorithm. Piezoceramic plates are used for structural excitation in one experimental case and the limitations of distributed excitation for broadband analysis are observed and characterised in terms of actuator geometry and modal deformation. The ARMAX algorithm is extended for use with response measurements exclusively. Numerical and experimental tests demonstrate its performance using time series data and correlation functions calculated from response measurements.

No description available.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-02-27T16:48:15Z No. of bitstreams: 1 PGPV16MA210.pdf: 1614873 bytes, checksum: 236bab6cf01b676397429dd551089261 (MD5) / Made available in DSpace on 2018-02-27T16:48:15Z (GMT). No. of bitstreams: 1 PGPV16MA210.pdf: 1614873 bytes, checksum: 236bab6cf01b676397429dd551089261 (MD5) Previous issue date: 2016-07-26 / Capes / Cattleya coccinea and Coppensia ranifera are epiphytic orchids, which have ornamental potential. Understanding how is the behavior of those species on their performance environments, along with in vitro culture of research is fundamental to building solid plans for the use and conservation of natural resources. We evaluated the occurrence of C. coccinea in three installments in the conservation unit complex Serra da Farofa in Urupema, SC; and C. ranifera in a forest fragment of a particular property in Brunópolis, SC. the frequencies of orchids were analyzed in vertical strata, the points chains (north, south, east and west) and correlation with height and diameter. In micropropagation C. ranifera protocorms we were used germinated in vitro longitudinally sectioned (2 mm) in medium Knudson C stationary and gelled liquid, supplemented with thidiazuron (TDZ) (0, 1, 2, 4 and 8 uM) to induce protocorm-like structures (ESP). For regeneration of plantlets was used in medium lacking growth regulators culture. For C. coccinea were recorded in 1398 orchids distributed in 258 phorophytes. Drimys angustifolia corresponded to 65.1% of phorophytes and held 72.2% of orchids. The Middle Fuste of phorophytes concentrated 68.7%, and the North face took 40.0% of orchids. The positive correlation to the height of phorophyte (r = 0.93) and diameter classes (r = 0.95) depending on the frequency of orchids, where 7.6 m center class obtained 32% of orchids and phorophytes; and 13,4cm class center obtained 30% of orchids and phorophytes. C. To ranifera frequencies of orchids were analyzed in vertical layers, positions in the chains, and correlation with height and diameter. They sampled 473 individuals of C. ranifera distributed in 72 phorophytes. Lamanonia ternata recorded 34.7% of phorophytes and took 42.5% of orchids. The Middle Fuste of phorophytes concentrated 54.8% and 40.6% north face orchids. Positive correlation was found to the height of phorophyte (r = 0.25) and negative for diametric classes (r = -0.88) depending on the frequency of orchids, where 14.4m center class obtained 17% of orchids and of phorophytes; the 17.5cm class center obtained 20% of orchids and phorophytes. The ESP formation occurs by direct route, without callus. The use of the liquid culture medium is more efficient than the medium gelled with respect to the number of ESP formed per explant. In liquid culture medium the highest percentage of formation and more ESP per explant occurs with the addition of 4 uM TDZ. It was established an efficient method for in vitro propagation C. ranifera, using thin layer technique cell (TCL), the efficiency (conversion percentage) in plantlet regeneration gelled media is higher than in the liquid medium. The data indicate colonization capacity and the importance of conservation actions of orchid species / Cattleya coccinea e Coppensia ranifera são orquídeas epífitas, que possuem potencial ornamental. Entender como se dá o comportamento dessas espécies em ambientes de sua ocorrência, juntamente com pesquisa de cultivo in vitro é fundamental para construção de planos sólidos para o uso e conservação dos recursos naturais. Avaliou-se a ocorrência de C. coccinea em três parcelas na unidade de conservação Complexo Serra da Farofa, em Urupema, SC; e de C. ranifera em um fragmento de floresta de uma propriedade particular, em Brunópolis, SC. Foram analisadas as frequências de orquídeas em estratos verticais, aos pontos cadeias (norte, sul, leste e oeste) e correlação com a altura e diâmetro. Na micropropagação de C. ranifera foram utilizados protocormos germinados in vitro seccionados longitudinalmente (2 mm) em meio Knudson C líquido estacionário e gelificado, suplementado com thidiazuron (TDZ) (0, 1, 2, 4 e 8 M) para a indução de estruturas semelhantes a protocormos (ESP). Para a regeneração de plântulas foi utilizado meio de cultura isento de fitorreguladores. Para C. coccinea foram registradas 1398 orquídeas, distribuídas em 258 forófitos. Drimys angustifolia correspondeu a 65,1% dos forófitos e sustentou 72,2% das orquídeas. O Fuste Médio dos forófitos concentrou 68,7%, e a face Norte abrigou 40,0% das orquídeas. A correlação positiva para a altura do forófito (r=0,93) e classes Diamétricas (r=0,95) em função da frequência de orquídeas, onde centro de classe 7,6m obteve 32% das orquídeas e forófitos; e o centro de classe 13,4cm obteve 30% das orquídeas e forófitos. Para C. ranifera foram analisadas as frequências de orquídeas em estratos verticais, nas posições cadeias, e correlação com a altura e diâmetro. Foram amostrados 473 indivíduos de C. ranifera, distribuídas em 72 forófitos. Lamanonia ternata registrou 34,7% dos forófitos e abrigou 42,5% das orquídeas. O Fuste Médio dos forófitos concentraram 54,8% e a face Norte 40,6% orquídeas. Foi verificado a correlação positiva para a altura do forófito (r=0,25) e negativa para classes Diamétricas (r= - 0,88) em função da frequência de orquídeas, onde centro de classe 14,4m obteve 17% das orquídeas e dos forófitos; o centro de classe 17,5cm obteve 20% das orquídeas e forófitos. A formação de ESP ocorre por via direta, sem calo. A utilização de meio de cultura líquido é mais eficiente do que o meio gelificado com relação ao número de ESP formado por explante. Em meio líquido de cultura o maior percentual de formação e o maior número de ESP por explante ocorre com a adição de 4 M de TDZ. Estabeleceu-se um método eficiente de propagação in vitro para C. ranifera, utilizando a técnica de camada fina celular (TCL), a eficiência (percentual de conversão) na regeneração de plântula em meio gelificado é maior do que em meio líquido. Os dados indicam a capacidade de colonização e a importância de ações de conservação das espécies de orquídeas

No description available.

El virus de l'hepatitis C (VHC) provoca una hepatitis crònica que afecta a més de 170 milions de persones d'arreu del món. És un virus petit que es classifica dins de la família Flaviviridae i és un virus d'RNA de cadena positiva amb un genoma d'aproximadament 9.600 nucleòtids. A l'extrem 5' del genoma viral s'hi troba una regió no codificant (5'NCR) que comprèn els primers 341 nucleòtids i la seva funció està relaciona amb la traducció. Immediatament després hi ha una pauta de lectura oberta ORF que acaba en un únic codó d'aturada i codifica una poliproteïna de 3.010 aminoàcids. A continuació l'extrem 3' no codificant (3'NCR), que malgrat es desconeixen les seves funcions exactes, s'ha demostrat que és essencial per a la replicació vírica. La única poliproteïna generada és processada co- i postraduccionalment mitjançant proteases de l'hoste i víriques, donant lloc a les proteïnes estructurals (Core, E1 i E2-p7) i no estructurals (NS2-NS5B). Igual que la majoria de virus RNA, el VHC es caracteritza per tenir una taxa de mutació elevada. De fet, el genoma del virus no es pot definir com una única seqüència sinó per una població de variants molt relacionades entre sí. A aquesta manera d'organitzar la informació genètica se l'anomena quasiespècie viral i una de les seves implicacions principals és la facilitat amb què sorgeixen resistents al tractament. Els tractaments disponibles són llargs, cars, provoquen efectes secundaris considerables i només es resolen completament el 40% dels casos. Per aquesta raó es busquen altres solucions terapèutiques per combatre el virus entre les quals s'hi inclouen diferents estratègies. Una de les més innovadores i prometedores és la utilització de ribozims dirigits directament contra el genoma del virus. Aquest treball es centra en l'estudi de les noves estratègies terapèutiques basades en ribozims, concretament la ribonucleasa P. La ribonucleasa P és un ribozim que està present en tots els organismes ja que és l'enzim responsable de la maduració dels precursors d'RNA de transferència. El més interessant a nivell terapèutic és que s'ha demostrat que es pot dirigir la seva activitat cap a qualsevol RNA utilitzant una seqüència guia d'RNA que quan hibrida amb l'RNA diana, l'híbrid imita l'estructura secundària del substrat natural. En el cas del VHC, s'han estudiat ribozims dependents de seqüència (ribozims derivats d'RNAs satèl·lits i de viroides de plantes), sempre dirigits contra la regió més conservada del virus per evitar una disminució de l'eficiència del ribozim deguda a la variació de la diana. La ribonucleasa P és una endonucleasa d'activitat molt específica i es diferencia dels altres ribozims naturals en el sistema de reconeixement del substrat, reconeix elements estructurals i no de seqüència. L'objectiu final del treball és tallar in vitro l'RNA del VHC aprofitant la propietat que presenta aquest ribozim de reconèixer elements estructurals i no de seqüència ja que per a un mateix nombre de seqüències, el nombre d'estructures viables que pot adoptar l'RNA genòmic és molt més petit i per tant la variabilitat de la diana disminueix. S'han estudiat dos models d'RNasa P, la RNasa P humana guiada per seqüència guia externa (EGS) i l'RNA M1 de l'RNasa P d'E.coli unit a la seqüència guia per l'extrem 3' (ribozim M1GS). Abans però de dirigir el ribozim, s'han estudiat l'estructura i la variabilitat d'una regió del genoma del virus ja que s'ha descrit que són factors que poden limitar l'eficiència de qualsevol ribozim. Derivat d'aquests estudis s'aporten dades sobre accessibilitat i variabilitat d'una regió interna del genoma del virus de l'hepatitis C, la zona d'unió de la regió E2/NS2 (regió 2658-2869). L'estudi d'accessibilitat revela que la regió 2658-2869 del genoma del virus conté dominis oberts i tancats i que la transició entre uns i altres no és brusca si es compara amb altres regions d'estructura coneguda (regió 5' no codificant). Els resultats dels assajos in vitro amb els dos models de RNasa P mostren que s'ha aconseguit dirigir tant la ribonucleasa P humana com el ribozim M1GS cap a una zona, predeterminada segons l'estudi d'accessibilitat, com a poc estructurada i tallar l'RNA del virus. De l'anàlisi de mutacions, però, es dedueix que la regió estudiada és variable. Tot i dirigir el ribozim cap a la zona més accessible, la variació de la diana podria afectar la interacció amb la seqüència guia i per tant disminuir l'eficiència de tall. Si es proposés una estratègia terapèutica consistiria en un atac simultani de vàries dianes.D'altra banda i derivat d'un resultat inesperat on s'ha observat en els experiments control que l'extracte de RNasa P humana tallava l'RNA viral en absència de seqüències guia externes, s'ha caracteritzat una nova interacció entre l'RNA del VHC i la RNasa P humana. Per a la identificació de l'enzim responsable dels talls s'han aplicat diferents tècniques que es poden dividir en mètodes directes (RNA fingerprinting) i indirectes (immunoprecipitació i inhibicions competitives). Els resultats demostren que la ribonucleasa P humana, i no un altre enzim contaminant de l'extracte purificat, és la responsable dels dos talls específics observats i que es localitzen, un a l'entrada interna al ribosoma (IRES) i molt a prop del codó AUG d'inici de la traducció i l'altre entre la regió codificant estructural i no estructural. La ribonucleasa P és un dels enzims del metabolisme del tRNA que s'utilitza per identificar estructures similars al tRNA en substrats diferents del substrat natural. Així doncs, el fet que la ribonucleasa P reconegui i talli el genoma del VHC en dues posicions determinades suggereix que, a les zones de tall, el virus conté estructures semblants al substrat natural, és a dir estructures tipus tRNA. A més, tot i que el VHC és molt variable, els resultats indiquen que aquestes estructures poden ser importants per el virus, ja que es mantenen en totes les variants naturals analitzades. Creiem que la seva presència podria permetre al genoma interaccionar amb factors cel·lulars que intervenen en la biologia del tRNA,particularment en el cas de l'estructura tipus tRNA que es localitza a l'element IRES. Independentment però de la seva funció, es converteixen en unes noves dianes terapèutiques per a la RNasa P. S'ha de replantejar però l'estratègia inicial ja que la similitud amb el tRNA les fa susceptibles a l'atac de la ribonucleasa P, directament, en absència de seqüències guia externes. / Hepatitis C virus is a human pathogen causing chronic liver disease in 170 million people worldwide. The virus is classified within the family Flaviviridae. The RNA genome is single-stranded and functions as the sole mRNA species for translation. It comprises a 5'-untranslated region, which functions as an internal ribosome entry site, and a long open reading frame, which encodes a polyprotein precursor of about 3010 amino acids, that is cleaved into structural (core, envelope 1, envelope 2 and p7) and non-structural (NS-2, NS-3, NS-4 and NS-5) proteins; followed by a 3' non-coding region. Analyzing significant numbers of cDNA clones of hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. In the infected patient, a master (the most frequently represented sequence) and a spectrum of mutant sequences may be isolated at any given time during chronic infection. This manner of organizing genetic information, which characterizes most RNA viruses, is referred to as quasispecies. HCV resistance to treatment (either alone or in combination with ribavirin) is one of the most important clinical implications predicted by the quasispecies model suggesting the necessity to seek new therapies. HCV therapeutic strategies based on ribozyme cleavage are leading candidates. The ribozyme activity of Ribonuclease P (RNase P) is among proposed antiviral agents. RNase P is a ubiquitous cellular endonuclease and one of the most abundant and efficient enzymes in the cell. This enzyme is a ribonucleoprotein complex that catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA to generate the mature tRNA. Substrate recognition by the RNase P ribozyme does not rely on sequence requirements but on structural features of the RNA substrate. Custom-designed ribo-oligonucleotides, which hybridize with the target, called external guide sequences (EGSs), may provide the RNA structure which RNase P recognizes and cleaves in the hybridized complex. Recognition of structures instead of sequences may represent a great advantage in the fight against variable viruses because single or even double mutations in the target may be tolerated for RNase P recognition. One of the major aims of this work is to cleave HCV RNA using the RNase P ribozyme guided by EGS. To expand investigation of targeting in the HCV genome we assessed accessibility and low potential of variation of the target RNA since it is described that are crucial requirements for ribozyme therapy against viral infections. In the hepatitis C virus, the sequence of the 5' non coding region is conserved but the highly folded RNA structure severely limits the number of accessible sites. We have considered an internal genomic region whose sequence variation has been widely investigated. We have first mapped the accessibility of the genomic RNA to complementary DNAs within an internal genomic region. We performed a kinetic and thermodynamic study. Accordingly, we have designed and assayed four RNase P ribozymes targeted to the selected sites. Considerations on RNA structural accessibility and sequence variation indicate that several target sites should be defined for simultaneous attack. While performing targeting experiments on HCV RNA transcripts with RNase P we have found that, surprisingly, purified RNase P (peak activity) from HeLa cells cleaved HCV genomic RNA efficiently at two sites in the absence of EGSs. We report the techniques used to prove that the cleavage is specific to human RNase P (indirect methods: immunoprecipitation and competitive inhibition), and to show where cleavage occurs (direct method: RNA fingerprinting). We have confirmed that human RNase P is responsible for HCV RNA processing and that the two cleavages sites are in the IRES HCV domain, close to AUG initiator triplet, and in the E2/NS2 junction fragment (between structural and non structural coding region). To define cleavage by RNase P as a general property of HCV, viral sequences obtained from different patients were compared for RNase P cleavage accessibility. Cleavage was consistently observed in all sequences tested although with different efficiencies. Since RNase P recognizes and cleaves tRNA-like structures, we believe that such recognition by RNase P is an indication for the presence of two possible tRNA-like structures in the HCV genome. Comparison of such results at the two HCV RNase P cleavage sites should help us to understand in greater detail HCV substrate structure, tRNA mimicry, rules underlying recognition by human RNase P, and, in the particular case of the IRES motif, possible participation in translation. Whatever the role of such tRNA-like structures, such a strong tendency to maintain them might be important in the development of therapeutic strategies against the virus because they can represent highly susceptible targets for RNase P.

La synthèse d'une nouvelle famille d'oligomères de motifs contraints de dipeptides est décrite dans ce manuscrit. Les monomères utilisés sont des motifs 3(S)-amino-5-carbonylméthyl-2,3-dihydro-1,5-benzothiazépine-4(5H)-one (DBT), acide 2-aminométhyl-phényl-acétique (AMPA) et α-amino γ-lactames. La structure secondaire de ces édifices a été étudiée par spectroscopies RMN, IR, CD et RX. Nous avons montré tout d'abord que les oligomères de DBT sont capables d'adopter des structures stables et définies en ruban. En se basant sur ces structures, nous avons conçu de nouveaux systèmes beaucoup plus versatiles qui permettent de répartir diverses fonctions (basiques, acides, aromatiques) de part et d'autre de l'axe du ruban. Une stratégie de synthèse originale a été développée à cet effet. Elle consiste en la conversion directe de séquences peptidiques, incorporant des méthionines, en oligomères d'α-amino γ-lactames. Ils sont capables, au même titre que ceux de DBT, d'adopter des structures en ruban et de pénétrer dans les cellules. Enfin une étude in vivo chez la souris a montré le fort potentiel anti-tumoral d'un bioconjugué associant des oligomères d'AMPA à un inhibiteur de la Cathepsine D, enzyme lysosomale surexprimée et sécrétée par de nombreuses tumeurs solides. / The synthesis of a new type of constrained dipeptide motif oligomers is described. Monomers used are the (3S)-amino-5-(carboxylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (DBT), the 2-aminomethyl-phenyl-acetic acid (AMPA) and α-amino γ-lactams. The secondary structure of those architectures has been studied by NMR, IR, CD and X-ray spectroscopies. Firstly, we demonstrated that DBT oligomers are able to adopt stable and well defined ribbon like structures. Based on these structures, we designed new systems, far more versatile which are able to distribute various functions (basic, acidic, aromatic) on each side of the ribbon axis. An original strategy has been developed for this purpose. It consists in the direct conversion of peptidic sequences, incorporating methionine, in α-amino γ-lactams oligomers. They are able, as the DBT, to adopt ribbon like structures and to be internalized into cells. Finally, an in vivo study in mice showed the high anti-tumoral potency of a bioconjugate linking AMPA oligomers to an inhibitor of the cathepsin D, a lysosomal enzyme overexpressed and secreted by numerous solid tumors.