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.

Covering 30% of the land surface and storing 45% of terrestrial carbon, forest ecosystems play a major role in global biogeochemical cycles and climate. Despite the importance of forests, responses and feedbacks of forests to global change agents remain among the least understood processes in predicting future global change scenarios. We use the temperate forests biome of the eastern US as a case study to ask several general questions about tree physiology and ecology to inform key knowledge gaps relevant to predicting how forests will respond to future global change. Trees invest significant amounts of carbon into support tissues, defense, and storage. To begin, we examine the process of tree carbon storage, as measured by nonstructural carbohydrates (NSCs) by evaluating the extent that NSCs vary as a function of tree life history strategy, physical traits, and phylogeny. We find that NSCs vary largely at broad taxonomic scales, and across study sites. This suggests that a broad-based approach to studying NSCs is needed if they are to effectively inform ecosystem models. Next, we use annual tree ring increments to determine the spatial scales controlling variation in tree growth. We find that individual variability is the largest control on growth, explaining 27% of variability – and primarily explained by tree size, canopy position, and species. Regional-scale variability is the next most dominant, explaining 13% of variability – half of which is explained by changes in species composition across the region. Growth and mortality are important demographic processes responsible for large, and potentially rapid, changes to the terrestrial carbon cycle. In the last chapter, we explore the extent that NSCs explain growth and mortality. We find that stressed trees have significantly lower NSC concentrations than living trees and dead trees have the lowest concentrations. We also find that the strength and direction of the NSC – growth relationship varies greatly by species This dissertation contributes to our understanding of the processes driving tree growth and NSC storage dynamics, as well as the extent to which NSCs drive tree demographic processes across eastern US forests.

Submitted by MARCELLO BONTEMPI PIZZI null (marcello.pizzi@gmail.com) on 2016-09-30T19:50:12Z No. of bitstreams: 1 Marcello_Bontempi_Pizzi_Dissertacao_UNESP_Desfolhas_Sucessivas.pdf: 7157176 bytes, checksum: 6cac889ca8ee38013ebd76f802772f05 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-10-04T17:29:25Z (GMT) No. of bitstreams: 1 pizzi_mb_me_bot.pdf: 7157176 bytes, checksum: 6cac889ca8ee38013ebd76f802772f05 (MD5) / Made available in DSpace on 2016-10-04T17:29:25Z (GMT). No. of bitstreams: 1 pizzi_mb_me_bot.pdf: 7157176 bytes, checksum: 6cac889ca8ee38013ebd76f802772f05 (MD5) Previous issue date: 2016-08-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A produtividade de plantios de eucalipto no Brasil foi triplicada nas últimas quatro décadas graças, principalmente, à pesquisa intensiva, investimentos em tecnologia silvicultural, clonagem e melhoramento genético. Atualmente, existem diversas pesquisas relacionadas ao gênero Eucalyptus, mas são poucas as que abordam o tema estresse biótico por ser uma avaliação complexa e de difícil planejamento e execução. Em Piracicaba, em um sítio experimental com alta sanidade e sobrevivência, foram induzidas uma, duas e três desfolhas sucessivas, retirando-se manualmente todas as folhas da copa das árvores de 12 clones de eucalipto de extrema relevância à silvicultura brasileira. As desfolhas ocorreram aproximadamente aos 15, 19 e 23 meses de idade das árvores, e a avaliação final de perda de produtividade ocorreu aos 27 meses. O crescimento das árvores foi monitorado semanalmente, sendo possível ver o efeito das desfolhas ao longo do período. Os clones de eucalipto escolhidos para este estudo, apesar de pertencerem ao mesmo gênero, mostraram resultados bastante diferentes. O impacto das desfolhas no crescimento das árvores foi imediato e prolongado, com o crescimento próximo à zero por 50 a 120 dias após a desfolha. As desfolhas causaram redução de crescimento em DAP (Diâmetro à Altura do Peito), altura total, volume e biomassa de madeira. As árvores não desfolhadas acabaram dominando as desfolhadas. Isso causou o estiolamento das árvores desfolhadas e a redução da homogeneidade das parcelas. Aos 27 meses, as árvores que sofreram uma desfolha apresentaram biomassa de madeira 48% inferior ao das árvores que não sofreram desfolhas (variando entre 24 e 57% para clones). Duas desfolhas sucessivas causaram redução média de 59% de biomassa (variando entre 49 e 72%) e três desfolhas sucessivas causaram perdas médias de 68% (variando entre 57 e 80%). / The Brazilian Eucalyptus productivity has tripled in the last four decades, mainly because of intensive research, development of forestry and genetics. Nowadays, there are several studies related to the Eucalyptus genus, but only a few address biotic stress, as the subject is of complex evaluation, difficult planning and execution. In Piracicaba, in an experimental site with high health and survival rate, one, two and three successive defoliations were induced, manually removing all the canopy leaves of 12 extremely relevant clones to Brazilian forestry. The defoliations occurred in approximately 15, 19 and 23 months after planting and the final evaluation of productivity occurred at 27 months. Tree growth was weekly monitored, and the effect of defoliation over time was visible. The eucalypts clones chosen for this study, despite belonging to the same genus, showed quite different results. The impact of canopy defoliation on tree growth was immediate and prolonged, with growth close to zero for 50 to 120 days following defoliation. Defoliation caused growth reduction in DBH (Diameter at Breast Height), total height, volume and wood biomass. Defoliated trees were etiolated as the non-defoliated were the dominant trees in the plot. Defoliation reduced plot homogeneity. One year after the first defoliation, the 27-month-old trees that have undergone one defoliation showed 48% lower wood biomass, in comparison with non-defoliated trees (ranging from 24 to 57% by clones). Two successive defoliations caused an average reduction of 59% of biomass (ranging from 49 to 72%) and three successive defoliations caused 68% of losses (ranging from 57 to 80%).

1 Viruses are submicroscopic obligate parasites that depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. In the recent times, many new RNA viruses have evolved that possess the potential threat to plants and animals. One among them is Tospovirus (Family Bunyaviridae) which has severely affected the agricultural productivity in India. One of the Tospoviruses GBNV is a major challenge of crop production in south India. Tospoviruses shares several features such as morphology, genome structure and organization with members of other genera in the family Bunyaviridae. Virus particles are 80–120 nm in diameter. The genome includes three RNAs referred to as large (L), medium (M) and small (S). The L RNA is in negative-sense while the M and S RNAs are ambisense. The L RNA codes for the RNA-dependent RNA polymerase (RdRp), and the M RNA for the precursor of two glycoproteins (GN and GC) and a non-structural protein (NSm). The S RNA codes for the N protein and another non-structural protein (NSs). Tospovirus infection is an emerging threat for agricultural productivity in India. Therefore, biochemical and molecular characterization of these viruses is essential for developing various strategies for control of these diseases. 2 Present thesis deals with biochemical characterization of nonstructural protein, NSs of GBNV. 3 A review of literature on Tospovirus genome organization, replication, transcription, translation and assembly is presented in Chapter I. This chapter also includes the recent work on all the proteins encoded by the tospoviruses. 4 The objectives of the present study are as follows; a. Cloning, expression, purification and biophysical characterizations of rNSs. b. Analysis of its NTPase/dATPase activity c. Demonstration of nucleic acid 5’ phosphatase activity d. Characterization of nucleic acid unwinding activity of rNSs 5 The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in bacteria, protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology are described in chapter II 6 NSs of TSWV was shown to be suppressor of gene silencing (PTGS) in 2002. Since then there has been no further work on this protein. Till date neither in vitro nor in vivo study of NSs of any tospovirus has been carried out in detail. To gain insight into the biochemical function of rNSs, the NSS gene was cloned, overexpressed in E.coli and purified. The NSS gene, was cloned into pRSET-C vector. 7. Chapter 3 deals with cloning, overexpression, purification and biophysical characterization of GBNV NSs in terms of secondary structure analysis as well as its interaction with siRNA and ssRNA. The results provide the evidence that rNSs was successfully expressed in E.coli and purified (Fig. 3.1). Molecular mass of purified rNSs was confirmed by MALDI TOF, which gave the molecular mass of expected size 51.5 kDa (Fig. 3.2) Circular dichroism study revealed that rNSs has negative ellipticity peak at 215 and 223 nm typical of a globular protein. The protein had an emission maximum at 340 nm (Fig 3.3 B) when exited at 280 nm, which reflects that rNSs is well folded. Thermal melting study (Fig 3.3 C) showed rNSs had a reasonably high Tm (65°C). So overall, spectral study suggested that purified rNSs was soluble, well folded and thermally stable and could be used for further biochemical assay. The oligomeric status of the protein was determined by size exclusion chromatography to be trimeric (156 kDa, Fig 3.5). Purified rNSs was used to raise the polyclonal antibodies in rabbit. The antiserum could detect rNSs specific band only in IPTG induced sample not in uninduced sample (Fig 3.6). 50% binding was observed at 100 ng/ml of antigen showing that these antibodies were of high affinity (Fig 3.7 B). Further, the 50% binding was observed at 1:34000 dilution of the antiserum, which suggests that high titer antibodies against rNSs were obtained (Fig 3.7 A). 8 Further, the RNA binding property of rNSs was examined. Synthetic 21 bp siRNA and in vitro transcribed 100 nt ssRNA was used to analyze the RNA binding property of rNSs. Indeed rNSs was able to bind with 100 nt ssRNA (Fig 3.8 A) or 21 nt siRNA in a protein concentration dependent manner (Fig 3.8 B). The binding however did not require presence of divalent cation such as Mg 2+ (Fig 3.8 C). In order to understand the biological function of rNSs, its interaction with the structural protein, NP by ELISA was investigated. rNSs could interact with the NP protein (Fig 3.9) . Further 15 amino deletions from C terminus of NP did not affect its interaction with rNSs protein (Fig 3.9), which suggest that the C terminal 15 amino acid residues of NP are not essential for interaction with rNSs in vitro. 9. Sequence analysis of GBNV NSs revealed the presence of Walker motifs A (GxxxxGKT) and B (DExx) in its primary structure (Fig 4.2). The proteins that possess the Walker motifs A and B exhibit ATPase activity. Therefore, the purified rNSs was tested for its ability to hydrolyze ATP in the absence and presence of poly(A) (chapter IV). rNSs could hydrolyze [γ-32P] ATP in a concentration-dependent manner (Fig. 4.3 A). Further, ATPase activity was stimulated in presence of poly(A) (Fig. 4.3 B). Quantitative analysis of reaction product suggested that the reaction was linear in the presence of poly(A) upto 1.6 µg of rNSs (Fig. 4.3 C). 10. The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase, a positive control used in the assay. In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, purified in same way as rNSs, did not show the release of phosphate, suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate could be seen when immunodepleted rNSs was used suggesting that the activity was inherent to the protein and was not due to bacterial contamination (Fig 4.3 lane 7). Time course analysis of ATPase activity revealed that the reaction is linear up to 25 mins (Fig 4.4). Further, pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig 4.5 A) and the temperature optimum was at 25 °C(Fig 4.5 B). Most of the known viral ATPases require the divalent cation for their activity. The rNSs exhibited the optimum ATPase activity between 2-2.5 mM of MgCl2. The reaction was inhibited by increasing concentration of EDTA demonstrating the requirement of Mg2+ for ATP hydrolysis (Fig. 4.7). Further, the ATPase activity of rNSs was inhibited by increasing concentrations of non-hydrolyzable analog of ATP (Fig. 4.8) and was not inhibited by AMP (Fig 4.9) suggesting that rNSs is not a nucleotidyl phosphatase and is a true ATPase. Limited proteolysis of rNSs suggested that core domain was 23 kDa in size and could catalyze ATP hydrolysis (Fig. 21 and 4.22). 11. Interestingly rNSs not only cleaved ATP rather it could hydrolyze all rNTPs as well as dATP (Fig 4.10). Kinetic parameters were determined for its enzymatic activity. Comparison of the kinetic constants of rNSs NTPase activity revealed little variation, suggesting that the rNSs has a broad substrate specificity (Fig 4.10- 4.15 and table 4.1). 12. To assess the role of amino acids in Walker motif A and B (Fig. 4.16) site specific mutants K189A and D159A were generated ( Fig 4.17) confirmed by sequencing, overexpressed in E.coli and purified (Fig. 4.18). Point mutation in Walker motif B (D159A) reduced the ATPase activity (Fig 4.19) where as point mutation in Walker motif A (K189A abolishes the activity (Fig 4.19). 13. Chapter V deals with the nucleic acid 5’ phosphatase activity of rNSs. Experimental evidence presented in this chapter clearly shows that rNSs can cleave the single phosphate from the ssDNA, ssRNA, dsRNA and dsDNA. Nucleic acid 5’ phosphatase activity of rNSs was inhibited by AMP and ATP (Fig 5.2 and Fig 5.3). Interestingly the K189A mutant rNSs was as active as wild type rNSs where as D159A mutant showed slightly reduced activity (Fig 5.7 C). 14. As mentioned earlier, rNSs was shown to possesses the RNA stimulated NTPase/dATPase activity, a hallmark of all known helicases. Therefore, its nucleic acid unwinding activity was examined using dsDNA and dsRNA as a substrate. rNSs was able to unwind the dsDNA as well as dsRNA in a ATP dependent manner (chapter VI, Fig. 6.1 and 6.5 respectively). ATP and Mg2+ are essential cofactors for the unwinding activity (Fig. 6.1). While the unwinding activity could be observed with ATP and to some extent with dATP, all other NTPs and dNTPs failed to support the helicase function of rNSs (Fig 6.2) Further experimental evidence suggested that rNSs is a bidirectional helicase (Fig. 6.3). D159A mutation in Walker motif B resulted in reduced helicase activity where as K189A mutation in walker Motif A completely abolished the DNA as well as RNA helicase activity of rNSs (Fig. 6.6 and Fig 6.7 respectively). Therefore, mutational analysis clearly suggests that helicase activity is an intrinsic property of rNSs. 15. In conclusion rNSs of GBNV is multifunctional enzyme. This is the first report on the demonstration that rNSs is an non canonical ATP dependent helicase in the Bunyaviridae family. In addition to being a suppressor of PTGS, NSs may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. This new research finding on NSs might pave way for further studies on its role in viral replication and transcription.

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Diploma thesis deals with design of a flood protection a controlling structures with respect to problém of inner waters in the area of interest. The river reaches assessed are located mostly in the urban area of the city Letovice, more precisely from the junction of the Svitava and Křetínka river. The reaches are at kilometer 59,608 – 61,520 of the Svitava and kilometer 0-1,752 of the Křetínka river including the race between them. Program MIKE 11 (developed by Danish hydraulic institut) was used to calculate the water levels as nonuniform flow.

Cranberry (Vaccinium macrocarpon Ait.) is a woody perennial crop that can remain productive for decades. Competition for resources between cranberries and weeds can depress cranberry farm yields, resulting in large annual crop losses. Renewed interest in reducing chemical inputs into cranberry systems has provided the motivation to evaluate methods, such as flame cultivation (FC), as potential nonchemical options for weed control. Also known as thermal weeding, FC exposes plants to brief periods of high temperature that causes the water in the plant tissue to expand rapidly, rupturing plant cells and leading to necrosis. Various FC methods have been used successfully in annual crops as both a preemergence and postemergence weed control, but few scientific reports have been published on the use of FC on perennial weeds in a woody perennial crop system. Dewberry (Rubus spp.), sawbrier (Smilax glauca), and common rush (Juncus effusus) are cranberry weeds that are difficult to control, spread quickly and can cause significant crop loss. Flame cultivation may be an effective non-chemical means for controlling these weeds in cranberry systems. FC would ideally be used as a spot treatment for weeds growing in the cranberry canopy, as well as on larger non-production areas where cranberry vines are not as abundant, such as bog edges, ditches, and dikes. Using FC to treat weeds within the cranberry canopy will likely cause localized damage to cranberry plants immediately surrounding the weeds, thus cranberry response to FC is also of interest. The following experiments were designed to examine the response of weeds and cranberry plants to FC. Perennial plants rely on reserves of nonstructural carbohydrates (NSC) for growth and survival, thus the efficacy of FC treatments to weeds will likely be impacted by the timing and frequency of treatments as they relate to the specific carbohydrate cycles of targeted weeds, such as dewberry. An additional experiment studied the seasonal fluctuations of NSC in dewberry roots. Cranberry growers were also surveyed on their past experiences with FC, as well as their willingness to adopt FC if proven an effective method for controlling weeds.

Controlled Rocking Steel Braced Frames (CRSBFs) have been developed as a high-performance structural solution to resist seismic forces, due to their ability to minimize structural damage and self-centre the structure back to its original position after an earthquake. A CRSBF is intentionally allowed to uplift and rock on its foundation, which acts as the nonlinear mechanism for the system rather than member yielding and buckling. While the CRSBF is in the rocking phase, the response of the system is controlled by prestressing which anchors the frame to the foundation and energy dissipation devices which are engaged by uplift. Although CRSBFs have shown promising structural performance, an assessment of the overall effectiveness of this system must also consider the performance of nonstructural components which have a significant impact on the safety and economic performance of the system. The purpose of this thesis is to compare the performance of nonstructural components in buildings with CRSBFs to their performance in a conventional codified system such as a buckling restrained braced frame (BRBF), while also investigating which design parameters influence nonstructural component demands in CRSBFs. The responses of various types of nonstructural components, including anchored components, stocky unanchored components that slide, and slender unanchored components that rock, are determined using a cascading analysis approach where absolute floor accelerations generated from nonlinear time-history analyses of each structural system are used as input for computing the responses of nonstructural components. The results show that the trade-off of maintaining elastic behaviour of the CRSBF members is, in general, larger demands on nonstructural components compared to the BRBF system. The results also show that the stiffness of the frame and vibration of the frame in its elastic higher modes are the main influencers for nonstructural component demands in buildings with CRSBFs, while energy dissipation has a minimal impact. / Thesis / Master of Applied Science (MASc) / Controlled Rocking Steel Braced Frames (CRSBFs) have been proposed as a high-performance structural system that resists earthquake forces on buildings. This system has the ability to minimize damage to structural members and self-centre the building back to its original position after an earthquake, two characteristics that are typically not achieved by current conventional systems. However, an assessment of the CRSBF’s overall effectiveness cannot be limited to the consideration of only the structural skeleton, as the performance of nonstructural components (e.g. architectural elements, mechanical and electrical equipment, furnishings, and building contents) that are not part of the structural skeleton can have a significant impact on the safety and economic performance of earthquake resisting systems. This thesis compares the demands on nonstructural components in buildings with CRSBFs to their demands in a more conventional system during earthquake motions. The results show that the trade-off for avoiding damage to structural members in the CRSBFs is often higher demands on the nonstructural components.

Structuraj investigations on the Ddi1 (DNA-damage inducible protein 1) of Leishmania major and on the rotavirus nonstructural protein NSP4 were carried out. Ddi1 belongs to the ubiquitin receptor family of proteins. One of its domains is similar to the retroviral aspartic proteinases. It has been shown that this domain is the target of HIV-protease inhibitors that were being used in the treatment of AIDS and it was observed that these drugs effectively controlled opportunistic diseases caused by many parasitic protozoa such as Leishmania and Plasmodium species. The retroviral protease-like domains present in Ddi1 proteins of these organisms were identified as the targets of these drugs. Structural studies on Ddi1 from L. major have been carried out, in an attempt to provide a platform for the design of anti-protozoal compounds. Rotavirus NSP4, the first viral enterotoxin to be identified, is a multifunctional glycoprotein that plays critical roles in viral pathogenesis and morphogenesis. As part of an ongoing project on the structural characterization of NSP4, we determined the structure of the diarrhea-inducing region of this protein from the rotavirus strain MF66. Chapter 1 presents an overview of Ddi1 and NSP4 of the rotavirus with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Structural studies on the retroviral protease-like domain of Ddi1 (Ddi1-RVP) of L. major is presented in Chapter 3. Apart from this domain, Ddi1 of L. major also has a ubiquitin-associated and ubiquitin-like domains whereas P. falciparum has only the ubiquitin-associated domain. Activity of the full length Ddi1 of L. major and the retroviral protease domain of P. falciparum using an HIV protease substrate was shown to be inhibited by an HIV protease inhibitor, saquinavir. Binding of saquinavir to the proteins was also confirmed by Biolayer Interferometry studies. The crystal structure of the retroviral protease domain of L. major Ddi1 has been determined. It forms a homodimeric structure similar to that of HIV protease and the reported structure of the same domain from Saccharomyces cerevisiae. The loops in Ddi1-RVP are similar to the 'flap' regions of the HIV protease which close-in upon substrate/inhibitor binding; they are visible in the electron density maps, unlike the case of the S. cerevisiae protein. Though the native form of the domain shows an open dimeric structure, normal mode analysis reveals that it can take up a closed conformation resulting from relative movements of the subunits. The present structure of Ddi1-RVP of L. major with the defined 'flap'-like loops will be helpful in the design of effective drugs against protozoal diseases, starting with HIV protease inhibitors as the lead compounds. Chapter 4 describes the structural investigations carried out on the diarrhea-inducing region of the nonstructural protein NSP4 of the rotavirus strain MF66 which forms an α-helical coiled-coil structure. Crystal structures of a synthetic peptide and of two recombinant proteins spanning this region showed parallel tetrameric organization of this domain with a bound Ca2+ ion at the core. Subsequently, we determined the structure of NSP4 from a different strain as a pentamer without the bound Ca2+ ion. This new structure provides more insights into understanding some of the functions of NSP4 such as the release of ions into the cytoplasm and binding to the double-layered particle (DLP). We also established conditions responsible for these structural transitions. The crystal structure of the coiled-coil domain of NSP4 presented in this chapter shows an entirely different structure which is an antiparallel tetramer. This explains our failure to determine the structure by the molecular replacement method using known oligomers. The structure was solved by the Sulphur-SAD method using diffraction data collected with Cr Ka radiation. The study reveals that the structural diversity of NSP4 is not limited. We could relate sequence variations and pH conditions to the differences in oligomeric assemblies. Surface properties of the domain suggest that the new form is likely to interact with different sets of proteins compared to those that interact with the parallel tetramers or pentamers. Further investigations are needed to establish this property.