[ES] Las modificaciones químicas post-transcripcionales implican un nuevo nivel de modulación de la expresión génica. Al comienzo de esta Tesis, algunos componentes del complejo de metilación del nitrógeno en posición 6 de la adenosina (m6A) habían sido caracterizados en plantas. Sin embargo, a diferencia de mamíferos y levadura, ninguno de los 13 homólogos de AlkB (atALKBH1-10B) - potenciales desmetilasas (o erasers) - y las 13 proteínas de la familia YTH (ECT1- 11, AT4G11970 y CPSF30) - potenciales proteínas de reconocimiento de m6A (o readers) - identificadas en el genoma de Arabidopsis se habían caracterizado funcionalmente. Además, varios estudios describen la presencia de m6A en RNAs de virus de mamíferos y las diferentes funciones que desempeña esta modificación en la regulación de esas infecciones. No obstante, no se ha estudiado la posible implicación de este mecanismo molecular en las infecciones virales de plantas. El descubrimiento de la interacción entre la CP del virus del mosaico de la alfalfa (AMV) y una proteína de Arabidopsis (atALKBH9B) con homología a una eraser humana fue el punto de partida de esta Tesis. En este trabajo se confirma esta interacción, y se demuestra que atALKBH9B también puede reconocer los RNAs virales. Los resultados revelan que atALKBH9B tiene la capacidad de desmetilar m6A a partir de moléculas de RNA monocatenario in vitro. Esta proteína se acumula en gránulos citoplasmáticos que se colocalizan con siRNA bodies y se asocian a P-bodies, lo que sugiere que su actividad podría estar relacionada con el silenciamiento y/o degradación de mRNA. Por otro lado, ensayos preliminares muestran que los RNAs del AMV, el virus del mosaico del pepino (CMV), el virus de la arruga del nabo (TCV) y el virus del mosaico de la coliflor (CaMV) se metilan durante la infección en Arabidopsis. Además, para AMV y CMV, los resultados fueron corroborados por UPLC-PDA-Tof-MS y los sitios m6A a lo largo de los RNAs del AMV fueron identificados mediante MeRIP-seq. Los resultados presentados confirman que la relación m6A/A a lo largo de los RNAs virales aumenta en plantas atalkbh9b en comparación con las silvestres, mientras que la traducción y/o replicación se ven afectadas y el movimiento sistémico a los tallos florales está prácticamente bloqueado. A diferencia de la CP de AMV, la de CMV no interacciona con atALKBH9B por Y2H y, como ocurre con el resto de virus analizados (CMV, TCV y CaMV), su ciclo de infección no se ve afectado en plantas atalkbh9b. Además, la secuenciación de mRNA realizada en este trabajo revela que la infección por AMV induce algunos genes de Arabidopsis pertenecientes a la maquinaria m6A, MTA, MTB, VIR y ECT5. De acuerdo con el efecto antiviral dependiente de m6A para el AMV y teniendo en cuenta que ECT2, ECT3 y ECT4 fueron recientemente caracterizadas como readers citoplasmáticas, la supresión del módulo ECT2/ECT3/ECT5 aumenta significativamente los títulos sistémicos de AMV y CMV. El efecto antiviral de ECT2 sobre AMV parece estar modulado por su unión directa a los residuos de m6A presentes en los RNAs virales, ya que un mutante de ECT2 defectuoso en el reconocimiento de m6A pierde la actividad antiviral que sí presenta la proteína original y no es capaz de arrastrar RNAs virales in vivo. Por otro lado, acorde a la localización previamente descrita para ECT2 y ECT4 y la capacidad de ECT2 para experimentar una fase similar al gel in vitro, la expresión transitoria de ECT5 muestra un patrón citoplasmático con formación de agregados. Se propone que, como se ha descrito para las proteínas YTH de mamíferos, la interacción entre las ECTs y el RNA polimetilado (en este caso, RNA viral) promovería la formación de gránulos de estrés y, en consecuencia, reduciría las tasas de traducción y replicación viral. En resumen, en este trabajo se caracteriza la primera m6A eraser de plantas, atALKBH9B, y, por primera vez, se describe la influencia del mecanismo de metilación m6A en las infecciones virales de plantas. / [EN] Post-transcriptional chemical modifications entail a new level of gene expression modulation. At the beginning of this Thesis, some components of the N6-adenosine methylation (m6A) complex had been characterized in plants, whereas 13 homologs of AlkB (atALKBH1-10B) - putative demethylases (or erasers) - and 13 proteins of the YTH family (ECT1-11, AT4G11970 and CPSF30) had been identified in the Arabidopsis genome. However, unlike mammals and yeast, no functional roles had been described for any of these proteins. Besides, several reports have brought to light the presence of m6A residues in viral RNAs from mammalian viruses and the critical roles that this modification plays regulating viral infections. However, the potential relevance of this molecular mechanism on plant viral infections remained fully unexplored. The discovery of the interaction between the AMV CP and an Arabidopsis protein (atALKBH9B) with similarity to a human eraser was the starting point of this Thesis. Here, this interaction is confirmed and it is demonstrated that atALKBH9B can also recognize the viral RNAs. Furthermore, the obtained results prove that atALKBH9B has the capability of demethylating m6A from single-stranded RNA molecules in vitro. This protein was observed to accumulate in cytoplasmic granules that colocalize with siRNA-bodies and associate to P-bodies, suggesting that atALKBH9B activity could be related to mRNA silencing and/or decay processes. On the other hand, preliminary assays show that viral RNAs of AMV, Cucumber mosaic virus (CMV), Turnip crinkle virus (TCV) and Cauliflower mosaic virus (CaMV) become methylated during infection in Arabidopsis. Besides, for AMV and CMV, the results were corroborated by UPLC-PDA-Tof-MS and m6A sites along the RNAs of AMV were identified through MeRIP-seq approach. The results presented here confirm that m6A/A ratio along viral RNAs is increased in atalkbh9b plants compared to wild type, whereas translation and/or replication are impaired and systemic movement to the floral stems is practically blocked. In contrast to AMV, CMV CP does not interact with atALKBH9B by Y2H and, as it occurs with the rest of the assayed viruses (CMV, TCV and CaMV), its infection cycle is not affected in atalkbh9b plants. Furthermore, the mRNA-seq analysis performed in this Thesis reveals that some Arabidopsis factors belonging to the m6A machinery, MTA, MTB, VIR and ECT5 genes, are upregulated upon AMV infection. Consistent with the m6A-dependent antiviral effect for AMV and considering that ECT2, ECT3 and ECT4 were recently characterized as cytoplasmic m6A readers, mutations of ECT2/ECT3/ECT5 Arabidopsis module significantly increase AMV and CMV systemic titers. The antiviral effect of ECT2 on AMV seems to be modulated via its direct binding to the m6A residues presented in the viral RNAs, since an ECT2 mutant defective in m6A recognition loses wild type antiviral activity and is not able to pull down viral RNAs in vivo. On the other hand, according to the previous subcellular localization described for ECT2 and ECT4 and the ability of ECT2 to undergo gel-like phase in vitro, the transitory expression of ECT5 displays a cytoplasmic pattern with the formation of some aggregates. As found for mammal YTH proteins, the interaction between ECTs and poly-methylated RNA (in this case viral RNA) is proposed to promote the formation of stress granules and, consequently, reduce viral translation and replication rates. In summary, in this work, atALKBH9B is reported as the first m6A eraser identified in plants and, for the first time, it is described the influence of m6A methylation mechanism in plant viral infections. / [CA] Les modificacions químiques post-transcripcionals impliquen un nou nivell de modulació de l'expressió gènica. Al començament d'esta Tesi, s'havien caracteritzat alguns components del complex de metilació del nitrogen en posició 6 de la adenosina (m6A) en plantes. No obstant això, a diferència de mamífers i llevat, cap dels 13 homòlegs d'AlkB (atALKBH1-10B) - potencials desmetilases (o erasers) - i les 13 proteïnes de la família YTH (ECT1- 11, AT4G11970 i CPSF30) - potencials proteïnes de reconeixement de m6A (o readers) - identificades en el genoma d'Arabidopsis s'havien caracteritzat funcionalment. A més, diversos estudis han descrit la presència de residus m6A en RNAs de virus de mamífers i les diferents funcions que exercix esta modificació en la regulació de les infeccions virals. No obstant això, no s'ha estudiat la possible implicació d'este mecanisme molecular en les infeccions virals de plantes. El descobriment de la interacció entre la CP del virus del mosaic de l'alfals (AMV) i una proteïna d'Arabidopsis (atALKBH9B) amb homologia a una eraser humana va ser el punt de partida d'esta Tesi. En este treball es confirma esta interacció, i es demostra que atALKBH9B també pot reconéixer els RNAs virals. Els resultats revelen que atALKBH9B té la capacitat de desmetilar m6A a partir de molècules de RNA monocatenari in vitro. Esta proteïna s'acumula en grànuls citoplasmàtics que es colocalitzen amb siRNA bodies i s'associen a P-bodies, la qual cosa suggerix que l'activitat atALKBH9B podria estar relacionada amb els processos de silenciament i/o degradació de mRNA. D'altra banda, assajos preliminars mostren que els RNAs virals de l'AMV, el virus del mosaic del cogombre (CMV), el virus de l'arruga del nap (TCV) i el virus del mosaic de la coliflor (CaMV) es metilen durant la infecció en Arabidopsis. A més, per AMV i CMV els resultats van ser confirmats per UPLC-PDA-Tof-MS i els llocs m6A al llarg dels RNAs d'AMV s'identificaren mitjançant MeRIP-seq. Els resultats presentats confirmen que la relació m6A/A al llarg dels RNAs virals augmenta en les plantes atalkbh9b en comparació amb les silvestres, mentre que la traducció i/o replicació es veuen afectades i el moviment sistèmic a les tiges florals està pràcticament bloquejat. A diferència de la CP d'AMV, la de CMV no interacciona amb atALKBH9B per Y2H i, com ocorre amb la resta dels virus analitzats (CMV, TCV i CaMV), el seu cicle d'infecció no es veu afectat en plantes atalkbh9b. A més, la seqüenciació de mRNA realitzada en este treball revela que la infecció per AMV indueix alguns gens d'Arabidopsis pertanyents a la maquinària m6A, MTA, MTB, VIR i ECT5. D'acord amb l'efecte antiviral dependent de m6A per a l'AMV i tenint en compte que ECT2, ECT3 i ECT4 van ser recentment caracteritzades com readers citoplasmàtiques, la supressió del mòdul ECT2/ECT3/ECT5 augmenta significativament els títols sistèmics d'AMV i CMV. L'efecte antiviral d'ECT2 sobre AMV sembla estar modulat a través de la seua unió directa als nucleòtids m6A presents en els RNAs virals, ja que un mutant de la proteïna ECT2 defectuós en el reconeixement de m6A perd l'activitat antiviral que sí que presenta la proteïna original i no és capaç d'arrossegar RNAs virals in vivo. D'altra banda, d'acord amb la localització subcel·lular descrita prèviament per a ECT2 i ECT4 i la capacitat d'ECT2 per a experimentar una fase similar al gel in vitro, l'expressió transitòria d'ECT5 mostra un patró citoplasmàtic amb la formació d'agregats. Es proposa que, com s'ha descrit per a les proteïnes YTH de mamífers, la interacció entre les ECTs i el RNA polimetilat (en aquest cas, RNA viral) promouria la formació de grànuls d'estrès i, en conseqüència, reduiria les taxes de traducció i replicació viral. En resum, en este treball es caracteritza la primera m6A eraser de plantes, atALKBH9B, i, per primera vegada, es descriu la influència de l'mecanisme de metilació M6A en les infeccions virals de plantes. / Martínez Pérez, M. (2020). Involvement of the host RNA N6-adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155976

Natto and sprout soybeans are produced using small-seeded soybeans and their production is a high value alternative to grow grain soybeans for food in U.S. The development of soybean cultivars with improved natto and sprout quality is crucial for maintaining and increasing the soyfood market. However, there is insufficient information on sprout soybean characteristics. Therefore, the first objective of this study was to evaluate seed and sprout traits as potential selection criteria and study the storage effect on sprout quality. Seeds can be a vehicle for transmission of pathogens capable of causing human illness. That is why, the second objective was to identify seed-borne pathogens on a commercial soybean cultivar and to evaluate different seed decontamination treatments. Finally, seed coat deficiency is an undesirable trait for natto soybean seeds because it causes inferior appearance of the product. Thus, the third objective was to identify quantitative trait loci (QTL) underlying seed coat deficiency (SCD) and associated markers. Results showed that seed size, high-, average- and low-quality sprout percentage, hypocotyl thickness and length and sprout yield are the most important variables for breeding sprout cultivars; and one-year seed storage at room temperature reduced sprout quality. Fusarium, Alternaria and Diaphorte were the most frequent genera isolated from soybean seeds, and 2% calcium hypochlorite and 5% acetic acid were promising seed disinfection treatments. A stable QTL, qSCD20_1, was identified across two years explaining up to 25% of the variation of SCD; and eight molecular markers tightly linked and nearby qSCD20_1 were identified. Information presented will be helpful for sprout and natto soybean cultivar development. / Master of Science / Natto and sprout soybeans are produced using by small-seeded soybeans and their production is a high value alternative to grow grain soybeans for feed in U.S. The development of soybean cultivars with improved natto and sprout quality is crucial for maintaining and increasing the soyfood market. However, there is insufficient information on sprout soybean characteristics. Therefore, the first objective of this study was to evaluate important seed and sprout traits as potential selection criteria of soybean cultivars and study the storage effect on sprout quality. Seeds can be contaminated with fungi capable of causing human illness. So, the second objective was to identify fungus species associated with seeds of a sprout soybean cultivar and evaluate different seed disinfection treatments. Finally, seed coat deficiency is an undesirable trait for natto soybean seeds because it causes inferior appearance of the product. Thus, the third objective was to identify molecular markers associated with seed coat deficiency. Results showed that seed size, high-, average- and low-quality sprout percentage, hypocotyl thickness and length and sprout yield are the most important variables for breeding sprout cultivars; and one-year seed storage at room temperature reduced sprout quality. Fungal species that may cause human illness were isolated from sprout soybean seeds and calcium hypochlorite and acetic acid are promising seed disinfection treatment for reducing fungus incidence. Finally, eight molecular markers associated with seed coat deficiency were identified which may be potentially used in selecting natto soybean cultivars with low seed coat deficiency. Information presented will be helpful for sprout and natto soybean cultivar development.

Yes / Inter-arm differences (IAD) ≥10mmHg in systolic blood pressure (BP) are associated with greater incidence of cardiovascular disease. The effect of ethnicity and the white coat effect (WCE) on significant systolic inter-arm differences (ssIADs) are not well understood. Methods: Differences in BP by ethnicity for different methods of BP measurement were examined in 770 people (300 White British, 241 South Asian, 229 African-Caribbean). Repeated clinic measurements were obtained simultaneously in the right and left arm using two BP-Tru monitors and comparisons made between the first reading, mean of second and third and mean of second to sixth readings for patients with, and without known hypertension. All patients had ambulatory monitoring (ABPM). WCE was defined as systolic Clinic BP ≥10mmHg higher than daytime ABPM. Results: No significant differences were seen in the prevalence of ssIAD between ethnicities whichever combinations of BP measurement were used and regardless of hypertensive status. ssIADs fell between the 1st measurement (161, 22%), 2nd/3rd (113, 16%) and 2nd-6th (78, 11%) (1st vs 2nd/3rd and 2nd-6th, p<0.001). Hypertensives with a WCE were more likely to have ssIADs on 1st, (OR 1.73 (95% CI 1.04-2.86), 2nd/3rd, (OR 3.05 (1.68-5.53) and 2nd-6th measurements, (OR 2.58 (1.22-5.44). Non-hypertensive participants with a WCE were more likely to have a ssIAD on their first measurement (OR 3.82 (1.77 -8.25) only. Conclusion: ssIAD prevalence does not vary with ethnicity regardless of hypertensive status but is affected by the number of readings, suggesting the influence of WCE. Multiple readings should be used to confirm ssIADs. / This report presents independent research funded by the National Institute for Health Research (NIHR).

Doctor of Philosophy / Department of Civil Engineering / Mustaque Hossain / A Superpave asphalt mixture with 4.75-mm nominal maximum aggregate size (NMAS) is a promising, low-cost pavement preservation treatment for agencies such as the Kansas Department of Transportation (KDOT). The objective of this research study is to develop an optimized 4.75-mm NMAS Superpave mixture in Kansas. In addition, the study evaluated the residual tack coat application rate for the 4.75-mm NMAS mix overlay. Two, hot-in-place recycling (HIPR) projects in Kansas, on US-160 and K-25, were overlaid with a 15- to 19-mm thick layer of 4.75-mm NMAS Superpave mixture in 2007. The field tack coat application rate was measured during construction. Cores were collected from each test section for Hamburg wheel tracking device (HWTD) and laboratory bond tests performed after construction and after one year in service. Test results showed no significant effect of the tack coat application rate on the rutting performance of rehabilitated pavements. The number of wheel passes to rutting failure observed during the HWTD test was dependent on the aggregate source as well as on in-place density of the cores. Laboratory pull-off tests showed that most cores were fully bonded at the interface of the 4.75-mm NMAS overlay and the HIPR layer, regardless of the tack application rate. The failure mode during pull-off tests at the HMA interface was highly dependent on the aggregate source and mix design of the existing layer material. This study also confirmed that overlay construction with a high tack coat application rate may result in bond failure at the HMA interface. Twelve different 4.75-mm NMAS mix designs were developed using materials from the aforementioned but two binder grades and three different percentages of natural (river) sand. Laboratory performance tests were conducted to assess mixture performance. Results show that rutting and moisture damage potential in the laboratory depend on aggregate type irrespective of binder grade. Anti-stripping agent affects moisture sensitivity test results. Fatigue performance is significantly influenced by river sand content and binder grade. Finally, an optimized 4.75-mm NMAS mixture design was developed and verified based on statistical analysis of performance data.

Aim of this diploma thesis is both a comparison of three manuscripts which are copies of paintings of coats of arms of officials of the Land Court, which decorate spaces at Prague Castle, where Land Court met. Furthermore identification of the individual persons and coats of arms and their perception in the context of self-presentation of the nobility of Early Modern and finally prosopography of officials whose coats of arms are shown in the manuscripts.

As alterações vasculares são complicações clínicas secundárias a elevação da pressão arterial que podem comprometer a capacidade funcional e aumentar o risco de mortalidade. Um instrumento utilizado como marcador de doença arterial obstrutiva periférica que vem merecendo amplo interesse clínico e científico é o Índice Tornozelo-Braquial (ITB). Segundo diretrizes para a prática clínica, valores de ITB <=0,9 ou >=1,3 são considerados patológicos e associados a uma alta incidência de morbimortalidade cardiovascular. Este estudo descritivo e de corte transversal teve por objetivo identificar o risco cardiovascular em hipertensos do avental branco por meio da determinação do ITB com uso de esfigmomanômetros oscilométricos automáticos. Foi desenvolvido em um município localizado ao Nordeste do Estado de São Paulo, no período de agosto de 2010 a junho de 2011. Os participantes foram divididos em normotensos, hipertensos e hipertensos do avental branco, classificados de acordo com o diagnóstico médico e resultado da Monitorização Ambulatorial da Pressão Arterial (MAPA). As variáveis investigadas foram: idade, cor da pele, situação familiar conjugal, naturalidade, índice de escolaridade, profissão, peso, estatura, circunferência abdominal, pressão arterial em braços e tornozelos e ITB. O cálculo do ITB foi realizado pela relação da maior pressão arterial sistólica (PAS) da artéria tibial posterior com a maior pressão sistólica das artérias braquiais. As análises descritivas foram realizadas por meio do pacote estatístico StatisticalPackage for the Social Sciences - SPSS, versão 15.0. Utilizou-se análise de variância (ANOVA) para medidas repetidas e teste Tukey para comparações múltiplas das médias. O grau de relação linear nos escores de PAS e ITB foi verificado mediante a utilização do Coeficiente de Correlação de Pearson. Os resultados foram expressos como médias ± erros padrões das médias (EPM), e as diferenças consideradas estatisticamente significantes para p<0,05. Participaram do estudo 135 indivíduos, sendo 37% normotensos, 37% hipertensos e 26% hipertensos do avental branco. Em todos os grupos, a maioria dos participantes é do sexo feminino, de cor branca, vive com o cônjuge, é natural do estado de São Paulo, exerce atividades domésticas e tem ensino fundamental incompleto. Hipertensos do avental branco apresentam risco intermediário na análise de todas as variáveis clínicas estudadas. Apesar de não ter sido encontrada diferença significante na análise dos valores de ITB na comparação dos grupos, alterações compatíveis com doença arterial obstrutiva periférica e calcificação arterial foram observadas somente nos grupos hipertensão e hipertensão do avental branco. A análise do ITB de menor valor mostrou que 10% dos hipertensos e 5,7% dos hipertensos do avental branco apresentaram ITB<=0,9 e 6% dos hipertensos e 11,4% dos hipertensos do avental branco apresentaram ITB>1,3. Há correlação negativa entre os valores de PAS e ITB nos grupos hipertensão e hipertensão do avental branco. Estes achados remetem à premissa de que a hipertensão do avental branco não deve ser compreendida como uma condição benigna, sendo caracterizada por um quadro clínico que pode evoluir para hipertensão arterial estabelecida. A mensuração do ITB merece importância na abordagem clínica dos pacientes, devendo constituir um instrumento de avaliação do risco cardiovascular valorizado pelos profissionais na rotina dos serviços de saúde. / The vascular changes are secondary clinical complications of high blood pressure which can compromise the functional capacity and increase the risk of mortality. The Ankle-Brachial Index (ABI) is an instrument used as a marker of peripheral occlusive arterial disease which has attracted broad scientific and clinical interest. According to guidelines for clinical practice, ABI values <=0.9 or >=1.3 are considered pathological and associated with a high incidence of cardiovascular morbidity and mortality. This descriptive and cross-sectional study aimed at identifying cardiovascular risk in white coat hypertension by determining the ABI through the use of automatic oscillometric sphygmomanometers. The study was performed in a municipality located in the northeastern of the state of São Paulo, from August 2010 to June 2011. Participants were divided into normotensive, hypertensive, and white coat hypertensive subjects, classified according to the medical diagnosis and outcome of Ambulatory Blood Pressure Monitoring (ABPM). The variables investigated were: age, color of skin, marital family situation, nationality, level of education, occupation, weight, height, waist circumference, ankle-brachial blood pressure and ABI. The calculation of ABI was performed by the ratio between the higher systolic blood pressure (SBP) of the posterior tibial artery and the highest systolic brachial artery. Descriptive analyzes were performed using the Statistical Package for Social Sciences Statistical Package - SPSS, version 15.0. The analysis of variance (ANOVA) was used for repeated measures and Tukey test for multiple comparisons of means. The degree of linear relationship in the scores of SBP and ABI was verified by using the Pearson correlation coefficient. The results were expressed as means ± standard errors of the mean (SEM), and the differences were considered statistically significant at p<0.05. The study included 135 subjects, 37% normotensive, 37% hypertensive and 26% white coat hypertensive subjects. In all groups, most participants are female, white, live with the spouse, from the state of São Paulo, housewives and have incomplete elementary education. White coat hypertensive subjects have intermediate risk in the analysis of all clinical variables studied. Although no significant difference was found in the analysis of ABI values in the comparison of groups, changes consistent with peripheral occlusive arterial disease and arterial calcification was observed only in the groups with hypertensive and white coat hypertensive subjects. The analysis of the lower ABI value showed that 10% of hypertensive subjects and 5.7% of white- coat hypertensive subjects had ABI<=0.9 and 6% of hypertensive subjects and 11.4% of white-coat hypertensive subjects had ABI>1.3 . There is a negative correlation between SBP and ABI in the groups of hypertensive and white coat hypertensive subjects. These findings relate to the premise that white coat hypertension should not be understood as a benign condition, being characterized by a clinical condition that can lead to established hypertension. The measurement of ABI deserves importance in clinical management of patients and should be a tool for assessing cardiovascular risk valued by professionals in the routine of health services.

Seit einigen Jahren wird die Einkapselungstechnik für die Produktion künstlicher Samen oder zur Vorbereitung der Kryokonservierung genutzt. Das Ziel dieser Untersuchungen war es, Methoden zur Einkapselung von Dendranthema- und Rosa-Sprosssegmenten zu entwickeln und dadurch "künstliche Samen" herzustellen, die für die Konversion unter In-vitro- und Ex-vitro-Bedingungen geeignet sind. Es wurde mit den Dendranthema-Sorten `PS 27´, `Snowdon Weiß´, `Topfweiß´ und `Garden mum´ und mit der Rosensorte, Rosa hybrida `Kardinal´, gearbeitet. Von 3 bis 5 Wochen alten Sprosskulturen wurden 4 bis 5 mm lange Nodiensegmente entnommen und in der Gelmatrix eingekapselt. Sowohl das Alter der Sprosskulturen (Subkulturdauer) als auch die Position der Explantate am Spross hatten einen deutlichen Einfluss auf die Konversion. Die Konversion war bei den Sprossspitzen schneller und besser als bei den Nodiensegmenten. Bei Dendranthema konnte mit 5 Wochen alten Sprosskulturen gearbeitet werden. Bei den Rosen wurden bessere Ergebnisse mit Nodiensegmenten von 3 Wochen alten Sprosskulturen erzielt. Nach Optimierung der Gelmatrixkomponenten konnte für Dendranthema eine 100%ige Konversion unter sterilen Bedingungen erreicht werden, wenn der Gelmatrix (3% Na-Alginat) MS-Salze, Vitamine, 3% Saccharose und 1 mg/l IES zugesetzt wurden. Unter unsterilen Bedingungen erreichte die Sprossbildung 73%, wenn eine Zwei-Schicht-Kapsel mit einer nährstofffreien äußeren Schicht und Nährstoffen in der inneren Schicht verwendet wurde. Rosa hybrida `Kardinal´ hatte andere Ansprüche an die Nährstoffversorgung und die Zugabe von Wachstumsregulatoren als Dendranthema. Die Konversion der eingekapselten Nodiensegmente von Rosa hybrida `Kardinal´ erreichte auf einem Agar-Wasser-Medium fast 60%, wenn der Gelmatrix MS-Salze, Vitamine, 80 g/l bzw. 90 g/l Saccharose und 2 mg/l IBS zugesetzt wurden. Es wurden auch Nodiensegmente aus dem Gewächshaus direkt zur Einkapselung verwendet. Die Konversion dieser Nodiensegmente von Dendranthema `PS 27´ erreichte auf einem Agar-Wasser-Medium 75%, wenn der Gelmatrix MS + 4 mg/l IES zugesetzt wurden. Die eingekapselten Nodiensegmente von Dendranthema `PS 27´ konnten bei 4°C in einer MS + 1,0 mg/l IES-Lösung für 3 Monate gelagert werden. / Recently, the encapsulation technique is used for the production of artificial seeds or for cryopreservation. The aim of the present studies was to develop methods for encapsulation of Dendranthema- and Rosa-shoot segments to produce artificial seeds, that are suitable for the conversion under in vitro and ex vitro conditions. Dendranthema `PS 27´, `Snowdon Weiß´, `Topfweiß´ and `Garden mum´ and Rosa hybrida `Kardinal´ were used as plant material for the encapsulation. Nodal segments of about 4-5 mm length were taken as explants for the encapsulation. The conversion was influenced by the quality of the plant material used for encapsulation. The age of shoot cultures (subculture duration) and the position of explant on the shoot affected the conversion. The conversion of the shoot tips was faster and better than that of the nodal segments. With Dendranthema, 5 weeks old shoot cultures could be used, while in Rosa nodal segments of 3 weeks old cultures responded better. After optimization of the components of the gel matrix (3% Na-Alginat), 100% conversion could be recorded under sterile conditions for Dendranthema, when the gel matrix contained MS salts, vitamins, 3% sucrose and 1 mg/l IAA. The percentage of shoot formation reached 73% under non-sterile conditions, when a two-layer-capsule was used with a nutrient free cover layer and nutrients in the inner layer. Rosa hybrida `Kardinal´ had other nutritional and hormonal requirements compared with Dendranthema. The conversion frequency of the encapsulated shoot buds of Rosa hybrida `Kardinal´ reached almost 60% on agar-water-medium, when the gel matrix obtained MS salts, vitamins, 80 g/l or 90 g/l sucrose and 2 mg/l IBS. In this work, also shoot buds from the greenhouse were encapsulated directly. The conversion frequency of these encapsulated buds of Dendranthema `PS 27´ reached 75% on agar-water-medium, when the gel matrix contained MS + 4 mg/l IES. The encapsulated shoot buds of Dendranthema `PS 27´ could be stored for 3 months at 4°C in MS liquid medium.

Introdução: A hipertensão arterial apresenta controle insatisfatório em todo o mundo, fazendo-se necessária a busca de estratégias que incrementem seu controle. Neste contexto inserem-se a educação em saúde e a medida residencial da pressão arterial. Objetivos: Identificar o controle da hipertensão arterial em Unidades Básicas de Saúde antes e após implementação de programa educativo aos hipertensos; comparar o controle obtido pela medida casual com o controle pela medida residencial; e associar o controle com as demais variáveis do estudo. Casuística e Método: O estudo foi realizado com hipertensos de duas Unidades Básicas de Saúde da região oeste da cidade de São Paulo divididos em dois grupos, um que participou de programa educativo em hipertensão (grupo I) e outro que não participou (grupo II). A pressão arterial foi verificada antes e depois do programa educativo na unidade de saúde por enfermeiro e na residência pelo próprio paciente. Utilizou-se para medida da pressão arterial aparelho automático validado (OMROM-HEM 705 CP). O nível de significância adotado foi de 0,05. Resultados: Foram estudados 82 hipertensos com as seguintes características: sexo feminino (56,1%), cor branca (63,0%), com companheiro (68,0%), com ensino fundamental (70,7%), com renda familiar até 3 salários mínimos (56,1%) idade de 60,0±10,8 anos, índice de massa corporal (IMC) 29,4±5,4 Kg/m2 e circunferência da cintura 99,9±13,9 cm. Eram tabagistas 8,5% dos hipertensos, 24,4% consumiam bebida alcoólica, 40,7% foram considerados \"com presença\" de transtornos mentais comuns e 30,4% praticavam exercícios físicos. Quanto a atitudes frente ao tratamento 76,9 % relataram ter o hábito de medir a pressão, 74,4% afirmaram não interromper o tratamento e 75,6% referiram não faltar às consultas médicas. Foram classificados como não aderentes, pelo Teste de Morisky e Green 69,3% da amostra e 29,9% apresentaram moderada ou muita dificuldade de aderir ao tratamento. A MRPA dos pacientes que participaram do programa educativo (grupo I) apresentou significativo decréscimo ao término das intervenções (131,4±15,6 vs 127,3±17,4 para pressão sistólica; 79,2±12,2 vs 74,7±9,7 para pressão diastólica, p<0,05). O controle da pressão arterial aumentou nos dois grupos do estudo pela medida realizada na unidade de saúde (62,0% vs 71,0%, para grupo I e 40,0% vs 50,0% para grupo II). O controle pela medida da pressão na unidade de saúde foi similar ao controle residencial para hipertensos do grupo I (71,0% vs 68,3%) e menor do que o residencial para hipertensos do grupo II (50,0% vs 62,5%). O controle da pressão arterial avaliado na unidade de saúde se associou (p<0,05) com sexo feminino, renda salarial acima de três salários e prática de exercício físico. Valores mais elevadas da MRPA se associaram com menor renda salarial, presença de transtornos mentais comuns, não interrupção do tratamento, e moderada ou muita dificuldade de aderir ao tratamento. O efeito do avental branco foi mais expressivo nos pacientes que não participaram das intervenções educativas. Conclusão: O controle da pressão arterial aumentou após programa educativo para hipertensos e a MRPA pode ser usada para essa avaliação. A influência do observador foi mais expressiva em hipertensos do grupo II, em que não houve interação profissional-paciente / Introduction: The hypertension presents unsatisfactory control in the whole world, becoming necessary the search of strategies that develop its control. In this context it insert health education and home blood pressure measurement. Objectives: To identify the control of hypertension in Health Basic Units before and after implementation of educative program to hypertensive patients; to compare the control gotten for the casual measurement with the control for the home measurement; to associate the control with the variables studied. Material and Method: The study was carried out with patients of two Health Basic Units of the city of São Paulo divided in two groups, one that participated of educative program in hypertension (group I) and another who did not participate (group II). The blood pressure was measured before and after the educative program in the health unit for nurse and at home for the own patient. Validated automatic device was used for measuring blood pressure (OMROM-HEM 705 CP). The significant level adopted 0,05. Results: 82 patients with the following characteristics had been studied: feminine sex (56.1%), white (63.0%), married (68.0%), with primary school level (70.7%), with familiar income until 3 minimum wages (56.1%) age of 60,0±10,8 years, index body mass of 29,4±5,4 Kg/m2 and waist circumference of 99,9±13,9 cm. 8.5% were smokers, 24.4% alcohol consumers , 40.7% had been considered \"with presence\" of common mental disturbs and 30.4% practiced physical exercises. About the attitudes face to treatment 76.9% had told to have the habit to measure the pressure, 74.4% had affirmed not to interrupt the treatment and 75.6% had related not to absent to the medical visits. They had been classified as not adherent, by the Test of Morisky and Green, 69.3% of sample and 29.9% had presented moderate or much difficulty to adhere to the treatment. The HBPM of the patients who had participated of the educative program (group I) presented significant decrease to the ending of the interventions (131,4±15,6 versus 127,3±17,4 for systolic pressure; 79,2±12,2 vs 74,7±9,7 for diastolic pressure, p< 0,05). The control of blood pressure increased in the two groups of the study by measurement carried through in the health unit (62.0% vs 71.0%, for group I and 40.0% vs 50.0% for group II). The control by pressure measurement in the health unit was similar to the home control for patients of group I (71.0% versus 68.3%) and minor of home one for patients of group II (50.0% versus 62.5%). The control of the blood pressure evaluated in the health unit was associated (p< 0,05) with feminine sex, wage income above of three salaries and pratice of physical exercise. Higher values of HBPM was associated with minor wage income, presence of common mental disturbs, interruption of the treatment, and moderate or much difficulty to adhering to the treatment. The effect of the white coat was bigger in the patients who had not participated of the educative interventions. Conclusion: Hypertensive control increased after educative program for hypertensive patients and HBPM can be used for this evaluation. The influence of the observer was more intense for patients of group II, which did not have interaction professional-patient

TCV (Turnip crinkle virus) coat protein is required for the resistance response in Arabidopsis thaliana Di-17 plants. An aspartate to asparagine mutation at amino acid four of the coat protein is sufficient to result in resistance-breaking. To determine the essential chemical properties responsible for the induction of resistance, a series of site-directed mutants were produced. Serine as well as asparagine at amino acid four induces systemic disease on both Di-3 and Di-17 plants; however, replacement of aspartate with glutamate retains the ability to induce the HR (hypersensitive response) and resist TCV infection with rapid and strong induction of PR-1 gene. These data suggest that the negative charge at the fourth amino acid of the coat protein is critical for the induction of resistance. Taken together with other mutagenesis research, the N-terminus of the coat protein appears to be the sole viral recognition element. The A. thaliana TIP protein is suggested to be involved in resistance, mainly through its C-terminus. Interestingly, one of the resistance-breaking mutants (D4N) produces a HR on Di-3 plants that are normally susceptible. The Di-3 TIP protein has several differences from the Di-17 TIP. To detect whether the delayed HR is related to interaction between Di-3 TIP and D4N mutation, a yeast two-hybrid assay was attempted. Interactions have not yet been detected. There are a number of possible explanations.

X-ray crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules at atomic resolution. Crystallography can reliably provide the answer to many structure related questions, from global folds to atomic details of bonding. Crystallographic techniques find wide applications in understanding macromolecular assembly, enzyme mechanism, mode of activation of enzymes, substrate-specificity, ligand-binding properties, domain movement etc. The knowledge of accurate molecular structures is also a prerequisite for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents. The current thesis can be broadly divided into two major parts. The first four chapters deal with assembly studies that have been carried out on Sesbania mosaic virus and the next two chapters describe the structure and function of a stationary phase survival protein, SurE from Salmonella typhimurium. In both studies X-ray crystallographic techniques have been used extensively for the structural studies. Viruses are obligate parasites with a proteinaceous capsid enclosing the genetic material. For genetic economy, several copies of capsid proteins self assemble to form complex virus capsids. Due to their intricate symmetric structures, viruses are considered as minute marvels of molecular architecture and study of virus structures serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes at near-atomic resolution, and is thus an extraordinarily powerful tool for understanding the function of these biological systems. Protein-protein interactions, protein-nucleic acid interactions, metal-ion mediated interactions, interactions between capsid proteins and auxillary or scaffolding proteins and particle maturation or post processing of capsid protein subunits are various elements that play a role in capsid assembly. Many structural and sequential motifs have been proposed as important conformational switches of capsid assembly. A functional analysis of these motifs by way of mutations in the capsid protein and structural studies of these mutants can provide further insight into capsid assembly pathways. Interaction between capsid protein subunits can determine the size and robustness of the capsid. Analysis of protein-protein interactions can help in understanding the principles of self-assembly. Arresting capsid assembly by disrupting intersubunit interactions and trapping the assembly intermediates will be helpful to delineate the changes that happen in capsid protein during the course of assembly and understand assembly pathways. Sesbania mosaic virus (SeMV) is a plant virus with a positive sense single-stranded RNA genome and belongs to the Sobemovirus genus. The protein and nucleic acids of SeMV can be separated and reassembled in vitro. Also, expression of the coat protein (CP) gene of SeMV in E. coli leads to the formation of virus like particles (VLPs). Therefore, SeMV is an excellent model system to study the assembly pathways that lead to the formation of complex virus shells. Earlier structural and functional studies on the native virus and the recombinant capsid protein and its various mutants have revealed the following: SeMV is a T=3 virus with chemically identical A-, B- and C-subunits occupying quasi equivalent positions in the icosahedral asymmetric unit of the virus particle. The A-type subunits form pentamers at the five-fold, and the B- and C- type subunits form hexamers at the icosahedral three-fold axes. The amino terminus of the polypeptide is ordered from residue 72 in the A- and B- subunits whereas it is ordered from residue 44 in the C-subunit. The disordered segment in all the subunits has an arginine rich motif (N-ARM). The segment ordered only in C-subunits has a -annulus structure that promotes intersubunit interactions at the quasi six-fold and a -segment (A). The virus is stabilized by protein-protein, protein–RNA and Ca2+ mediated protein-protein interactions. Virus like particles (VLPs) formed by the expression of full length CP encapsidate 23 S E. coli rRNA and CP mRNA. Expression of a deletion mutant lacking the N-terminal 65 residues (rCP∆N65) which results in the removal of the N-ARM, the -annulus and the A leads to the formation of stable T=1 particles. The -annulus, which was earlier believed to be an important molecular switch controlling the assembly of T=3 VLPs was found to be dispensable. The N-ARM, though important for RNA encapsidation, was not essential for capsid assembly . Depletion of Ca2+ ions led to slight swelling of virus particles and significantly reduced stability. Extensive studies on the VLPs suggested that the assembly is most likely initiated by the dimers of the capsid protein. Following a brief account of the historical highlights in the field of structural virology, a review of current literature on the available crystal structures of viruses and various assembly studies on viruses that have been carried out with emphasis on role of nucleic acid mediated interactions, protein-protein interactions and role of specific residues and ion-mediated interactions in assembly are presented in Chapter I of the thesis. A separate section in this chapter deals with the disassembly experiments that have led to the formation of smaller oligomers of spherical viruses. This chapter also gives an account of the earlier work that has been carried out on SeMV, which is the model system of study for the present thesis. Chapter II describes in detail the structural studies on the β-annulus deletion mutant of SeMV. A unique feature of several T = 3 icosahedral viruses is the presence of a structure called the β-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T = 3 capsid assembly. In order to examine the importance of the β-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48–59 involved in the formation of the β-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Δ48–59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of size close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6Å resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the β-annulus. Therefore, the β-annulus is not essential for T = 3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the β-annulus could be closely similar. Chapter III begins with a detailed description of the interfacial residue mutations that have been carried out in SeMV with the aim of disrupting assembly and trapping an assembly intermediate. These mutations were performed in rCP as well as rCP∆N65 gene. Among these, a single point mutation of a Trp 170 to a charged residue (either Glu or Lys) arrested virus assembly and resulted in stable dimers of the capsid protein. The chapter also gives an account of the biophysical characterization of these mutants. rCP∆N65 dimer mutants showed a characteristic 230 nm peak in CD spectral studies which may be due to the interactions of a stretch of aromatic residues in the capsid protein. The isolated dimers were more susceptible to trypsin cleavage compared to the assembled capsids due to the exposed basic amino terminus. Thermal melting studies showed that the isolated dimer mutants were much less stable when compared to the assembled capsids, probably due to the loss of intersubunit interactions and Ca2+ mediated interactions. The structure of one of the isolated dimer mutant- rCP∆N65W170K was solved to a resolution of 2.65Å. Chapter IV describes the crystal structure analysis of the rCP∆N65W170K mutant dimer and compares its structure with the dimers of native virus, T=3 and T=1 VLPs. A number of structural changes occur especially in the loop and interfacial regions during the course of assembly. The dimer in solution was “more relaxed” than the dimer that initiates assembly. Ca2+ ion is not bound and consequently the C-terminal residues are disordered. The FG loop, which interacts with RNA, was found to be flexible and adopts a different conformation in the unassembled dimer. The present thesis also deals with the structural and functional studies of a phosphatase, SurE, the stationary phase survival protein from Salmonella typhimurium. Chapter V provides a general introduction on Salmonella, which is a mesophilic food borne pathogen, its general features, classification and stress responses. This chapter also gives an account of stationary phase in bacteria and stress responses. A brief description about phosphatases and their classification is also presented in this chapter. Following this, a review of the current literature on the structural, biochemical and functional role of stress related proteins and phylogenetic and enzymatic studies of various homologues of SurE are described in detail. Chapter VI deals with the detailed crystal structure analysis of SurE, the first stationary phase survival protein from a mesophilic organism. SurE, of Salmonella typhimurium forms part of a stress survival operon regulated by the stationary phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. Here we report the X-ray crystal structure of SurE from Salmonella typhimurium (St SurE). The protein crystallized in two forms- orthorhombic F222 and monoclinic C2. The two structures were determined to resolutions of 1.7Å and 2.7Å, respectively. The protein exists as a domain swapped dimer. The residue Asp 230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal-ion dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg2+ present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate- para- Nitro Phenyl Phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg2+. Thermal denaturation studies showed that St SurE is much less stable compared to its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free energy. The thesis concludes with a brief summary of the entire work that have been presented and future prospects. The various crystallographic, biochemical and biophysical techniques employed in the investigations are described under the section experimental techniques in Appendix I and the NCS matrices used in the structure solution of the β-annulus deletion mutant are listed in Appendix II.