Deoxyribonucleic acid and ribonucleic acid under physiological condition are polyanions composed of heterocyclic bases linked through sugar phosphate backbone. Due to Watson-Crick base pairing, DNA exists in double-helical form between two antiparallel strands of nucleic acid. Different conformations of DNA is possible among which the B-DNA form is considered to be the most common, and it is a right-handed double-helix with base pairs stacked at the center. There are two well-defined grooves termed as major and minor grooves, each has characteristic width and depth. Most of the DNA binding proteins generally approach DNA through the major groove, while small molecules such as drugs, antitumor antibiotics,1 their synthetic analogue,2 carcinogens,3 and the transition metal complexes4 interact with DNA through minor groove. The nucleic acids function in the storage and transfer of genetic information. The function of cell expressions of proteins, synthesis of all bio-materials are directly or indirectly governed by the nucleic acid present in the body. Not only that, the origin of many diseases lie behind the structural modification or alterations in nucleic acids occur beyond our control.5 There are different drugs both natural and synthetic which are important in antibiotic chemotherapy, act against these diseases by interacting with DNA. Now to understand the actual mechanism of many diseases, how drugs interact with DNA and its specificity, binding sites of DNA, we need to develop molecules that modify or interact with biological molecules and such molecules can probe various structural aspects and type of interaction of macromolecular association complexes. One of such probe is the DNA cleaving agent. The potential scope of the utility of these compound is enormous and ranges from the creation of synthetic restriction enzymes for use by molecular biologists to the development of chemotherapeutic agents (Fe(BLM), calicheamicin) that may be effective against a variety of neoplastic diseases. They can also act as a structural probe (e.g. Fe(EDTA)2), drug / protein-DNA footprinting agent and affinity cleaving agent.

HMG-CoA reductase (HMGR) catalyzes the rate controlling step in cholesterol production. This enzyme is highly expressed in the liver where it is subject to extensive hormonal and dietary regulation. This study was undertaken to examine the occupancy and function of the hepatic HMGR promoter in regards to insulin and sterol regulation. HMGR protein and mRNA are substantially decreased in diabetic animals and rapidly restored by administration of insulin. Nuclear run-on assays revealed that HMGR transcription was substantially reduced in the diabetic rats, and fully restored within two hours after insulin treatment. In vivo footprinting revealed several areas of protein binding as shown by dimethyl sulfate protection or enhancement. The CRE was heavily protected in all conditions - including diabetes, cholesterol feeding, or statin treatment. Striking enhancements in footprints from diabetic animals were observed at -142 and at -161 (in the SRE). Protections at a newly ident ified NF-Y site at -70/-71 were seen in normal animals, and not in diabetics. This proximal NF-Y site was found to be required for efficient HMGR transcription. CREB-1 was able to bind the HMGR CRE in vitro, and to the promoter in vivo. The data supports an essential role for CREB in transcription of hepatic HMGR, and identifies at least two sites where in vivo occupancy is regulated by insulin. The technique of in vivo electroporation was utilized to perform the first functional analysis of the HMGR promoter in live animals. Analysis of a series of deletion constructs showed that deletion of the region containing the cyclic AMP response element (CRE) at -104 to -96 and the newly identified NF-Y site at -70 resulted in marked reduction of promoter activity. Possible sterol regulation of the promoter was investigated by raising tissue cholesterol levels by feeding cholesterol, or by inhibiting cholesterol synthesis with a statin (lovastatin). It was found that HMGR promoter constructs r esponded to lovastatin, in agreement with previous findings in cultured cells. This work sheds light on the regulation of the HMGR promoter in the liver, whose expression is a key determinant of serum cholesterol levels- a major risk factor for cardiovascular disease.

The purpose of this thesis is to assess and compare the energy consumption and carbon footprints of two, online and offline, major movie rental services in Canada. The thesis is divided into two parts that represent two papers that are being published. The comprehensive literature survey provides the state-of-the-art in E-Commerce carbon footprinting with a new categorization framework. The model development and application compares the energy consumption and carbon footprints of the two business models via a systems approach and the Economic Input Output Life Cycle Assessment (EIO-LCA) model and evaluates environmental performances. The portions of the logistics chains that were different in the two business models were analyzed and processes that were common were excluded. Regarding findings, the analyses conclude that the online movie rental service has lower carbon footprint than the offline one. We suggest practical implications for policy makers, government, businesses, and customers in movie rental industry. / This thesis contains two papers, recently presented at international conferences and accepted for publication in recognized journals.

Nickel is an essential transition metal for the virulence and survival of Helicobacter pylori in the acidic human stomach. The nickel– and proton– dependent transcriptional regulator HpNikR is important for maintaining nickel homeostasis inside the cytosol by regulating multiple H. pylori genes. A previous ChIP-sequencing experiment with H. pylori G27 and HpNikR identified two novel genes currently annotated as putative iron-transporters, HpG27_866 and HpG27_1499. In vitro DNA-binding assays with the promoter sequences of the two genes revealed nickel-dependent HpNikR binding with an affinity of ~10-7 M. The recognition site of HpNikR was identified on HpG27_1499 by footprinting assays, which loosely correlates with the HpNikR pseudo-consensus sequence. Furthermore, HpG27_1499 transcription showed nickel-dependent repression in WT H. pylori, and no changes in an isogenic ΔnikR strain. These data suggest that HpG27_1499 could be a nickel importer that is regulated by HpNikR in a nickel-responsive manner.

Nickel is an essential transition metal for the virulence and survival of Helicobacter pylori in the acidic human stomach. The nickel– and proton– dependent transcriptional regulator HpNikR is important for maintaining nickel homeostasis inside the cytosol by regulating multiple H. pylori genes. A previous ChIP-sequencing experiment with H. pylori G27 and HpNikR identified two novel genes currently annotated as putative iron-transporters, HpG27_866 and HpG27_1499. In vitro DNA-binding assays with the promoter sequences of the two genes revealed nickel-dependent HpNikR binding with an affinity of ~10-7 M. The recognition site of HpNikR was identified on HpG27_1499 by footprinting assays, which loosely correlates with the HpNikR pseudo-consensus sequence. Furthermore, HpG27_1499 transcription showed nickel-dependent repression in WT H. pylori, and no changes in an isogenic ΔnikR strain. These data suggest that HpG27_1499 could be a nickel importer that is regulated by HpNikR in a nickel-responsive manner.

This thesis reports on the research undertaken to reduce the water usage within manufacturing through modelling and improving the water efficiencies at both process and production system levels. The primary objectives of this research are: to develop a framework which classifies the various water usages within a manufacturing facility, to define a number of efficiency ratios to highlight the water inefficient activities, and to develop a decision support tool to aid with the selection of the most effective solutions for reduction of water usage within manufacturing applications. The research undertaken in the past three years is divided into four main parts. The first part reviews the relevant literature on water availability and distribution, the role of water in manufacturing, and relevant legislations and policies governing the water usage in manufacturing industries. The review also includes assessment of current water flow modelling and wastewater management tools and technologies. The second part introduces a Manufacturing Water Usage framework which classifies the water usage within a manufacturing facility as production-related and non-production-related water. The Production Water, which is the main focus of this research, is further classified as Process and System Water required to produce a product. Process Water is defined as the water used directly by the production processes, whereas System Water is defined as the water used to support the operation of a process and/or to maintain the production equipment. The framework also incorporates the definition of a number of Water Efficiency Ratios to determine the water critical processes. The third part of the thesis describes the implementation of this framework within a water simulation model as the main engine for a water reduction decision support tool. The final part of the thesis demonstrates the utilisation of this tool to support the decisions aimed at reducing water within a real food production line. In summary, the research has concluded that the consideration of water reduction scenarios within the manufacturing industry requires a detailed understanding of where and how water is used at production process level, and utilisation of this knowledge to develop a series of proactive approaches based on product/process redesign and radical operational planning improvements.

Orientador: Fabio Cesar Gozzo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-13T13:49:01Z (GMT). No. of bitstreams: 1 Iglesias_AmadeuHoshi_D.pdf: 8088319 bytes, checksum: 8f6da7c324d46a34a7ce5354e6670443 (MD5) Previous issue date: 2009 / Resumo: A Espectrometria de Massas (MS) é hoje a principal técnica de caracterização de estrutura primária de proteínas devido às vantagens intrínsecas da técnica. As técnicas de cross-linking e footprinting visam desfrutar de tais vantagens para obtenção de informações estruturais de complexos protéicos. Nesse projeto foram realizados estudos de fragmentação de peptídeos contendo como cross-linker o DSS, reagente mais utilizado para esse propósito. Os mecanismos de fragmentação foram propostos baseados na dissociação de peptídeos modelos sintetizados para gerar espécies que mimetizassem experimentos com proteínas. Esses estudos permitiram a identificação de íons marcadores, que foram posteriormente utilizados em experimentos de Varredura de Íons Precursores para auxiliar na detecção de peptídeos modificados. Para realização dos experimentos de footprinting, foi desenvolvida uma linha de luz no LNLS. Posteriormente, foi proposto um novo método de quantificação de cinética de oxidação baseado nos dados de LC-MS, levando em consideração todos os produtos de oxidação formados. Esses métodos foram utilizados no estudo de interação das proteínas Tif34 e Tif35 do fator de iniciação de tradução de Saccharomyces cerevisiae. Os resultados obtidos indicam que Tif34 apresenta dois possíveis sítios de interação para a proteína Tif35 / Abstract: Mass Spectrometry (MS) is the most important tool for analyses related to protein primary structure. Cross-Linking and footprinting aim to bring those advantages to gain insights in the spatial structure of protein complexes. In this work the fragmentation of peptides containing DSS, the most used cross-linker, was studied. Fragmentation mechanisms were proposed based on the dissociation of model peptides which were synthesized in order to generate species that would resemble species found in experiments with proteins. Diagnostic ions were identified which allowed the use of Precursor Ion Scan to detect those species. In order to perform footprinting experiments, a new beamline was developed at the LNLS. A new method for quantitation of oxidation kinetics was developed based on LC-MS analysis, which considers every single oxidation product that can be generated. Those methods were thereafter used in the study of the interaction between Tif34 and Tif35, proteins which compose a translation initiation factor of Saccharomyces cerevisiae. The results indicate that Tif34 presents two different sites for the interaction with Tif35 / Doutorado / Quimica Organica / Doutor em Ciências

Achieving a reduction in greenhouse gas emissions has become a key challenge facing global society and its economies. Despite this, tourism policy and strategic planning rarely acknowledge carbon mitigation as a strategic objective and tourism as a sector is rarely recognised in low-carbon plans. This situation represents a substantial challenge, as tourism and travel have a high-carbon impact and carbon mitigation is hindered by lack of carbon data, and a continued drive for economic growth. The purpose of this thesis was to investigate the effectiveness of carbon footprinting and scenario modelling to help examine the opportunities and challenges for implementing low-carbon tourism pathways in destinations, and to consider how the opportunities could be enabled. The 'REAP Tourism' footprint tool was used to investigate the carbon impact of visitors to destinations across South West England. The purpose was to estimate emissions, suggest a baseline footprint and offer alternative growth and mitigation scenarios of how tourism could more effectively reduce emissions. Through participatory workshops, evaluation questionnaires and semi-structured interviews, stakeholders identified the limitations and benefits of carbon modelling and the challenges and opportunities for a transition towards low-carbon tourism in destinations. The results demonstrated that the carbon footprint was a useful and informative indicator. The baseline data and scenarios provided a basis for constructive low-carbon dialogue with tourism stakeholders, which helped to challenge current thinking and facilitate the co-creation of ideas and potential interventions. A range of low-carbon opportunities and challenges were identified relating to the cultural, political and structural components of tourism governance. A conceptual low-carbon transition framework is proposed, to illustrate the opportunities. Stakeholder dialogue and debate, informed by quantitative and qualitative data, is central to the framework. Cultural, political and structural opportunities for change are also identified. Further investigation is needed to test the framework and examine the levels of influence and capabilities of different types of tourism stakeholders. The use of integrated environmental-economic indicators to inform national and local tourism policy and strategy, also require application. This thesis contributes to an emerging body of knowledge on the governance of low-carbon destinations, from a practical, methodological and conceptual basis.

Responsible water management in an era of globalised supply chains needs to consider both local and regional water balances and international trade. In this paper, we assess the water footprints of total final demand in the EU-27 at a very detailed product level and spatial scale - an important step towards informed water policy. We apply the multi-regional input-output (MRIO) model EXIOBASE, including water data, to track the distribution of water use along product supply chains within and across countries. This enables the first spatially-explicit MRIO analysis of water embodied in Europe's external trade for almost 11,000 watersheds world-wide, tracing indirect ("virtual") water consumption in one country back to those watersheds where the water was actually extracted. We show that the EU-27 indirectly imports large quantities of blue and green water via international trade of products, most notably processed crop products, and these imports far exceed the water used from domestic sources. The Indus, Danube and Mississippi watersheds are the largest individual contributors to the EU-27's final water consumption, which causes large environmental impacts due to water scarcity in both the Indus and Mississippi watersheds. We conclude by sketching out policy options to ensure that sustainable water management within and outside European borders is not compromised by European consumption.

Metabolomics approaches have recently been used to understand the complex molecular interactions of biological systems. One popular area in which these methods are being developed is to understand the biochemical changes during abiotic and biotic stresses; for example, how a cell may respond to a drug. Since metabolites are the end products of gene expression, these can be used to indicate the result of the activities and interaction of the cell or organism with its environment. The investigation of the level and compositional changes of metabolites against metabolic stresses such as chemotherapeutic treatment (drug exposure) are required to understand more fully abiotic perturbation to biological systems. The aim of this project was to understand the metabolic effect that the anti-viral drugs indinavir and lopinavir (currently used by HIV patients) have on HPV-related cervical cancer cell lines by measuring changes in metabolism using a wide range of analytical techniques; including Fourier transform infrared (FT-IR) and Raman spectroscopies, and gas and liquid chromatography-mass spectrometry (GC and LC-MS). The analyses and interpretation of the large volumes of complex multidimensional data generated by metabolomics approaches were performed with a combination of multivariate data analysis techniques such as principal components analysis (PCA) and canonical variates analysis (CVA), as well as univariate approaches such as N-Way analysis of variance (ANOVA). By combining biochemical imaging, metabolite fingerprinting and footprinting, and metabolite profiling, with multi- and uni-variate analyses, the actions and effects of the anti-viral drugs were investigated. FT-IR spectroscopy was initially used to generate global biochemical finger- and foot-prints, and Raman spectroscopy was employed to investigate intracellular distribution of metabolites, and other cellular species, as well as the localisation of drug molecules within cells. FT-IR spectroscopy ascertained that the intra- and extra-cellular metabolomes were being directly influenced in a fashion that correlated with increasing anti-viral dosing; these effects were phenotypic rather than measurements of the drug level. Raman imaging spectroscopy indicated that the indinavir but not lopinavir was being compartmentalised within the cell nucleus, but only in HPV early protein 6 (E6) expressing cells. This observation was further confirmed by fractionation of cell samples into nuclear and cytoplasmic fractions and assessing the indinavir concentrations via LC-MS. Finally, LC-MS and GC-MS metabolite profiling were employed to investigate changes in the intracellular metabolome in response to the anti-viral compounds across a range of physiologically relevant concentrations and in the presence and absence of the E6 oncoprotein. General effects of both anti-viral compounds included the regulation of metabolites such as glutathione, octenedionoic and octadecenoic acids, which may be involved in stress related responses, reduced levels of sugars and sugar-phosphates indicating a potential arrest of glycolysis, and reduced levels of malic acid indicating potential decreased flux into the TCA cycle; all indicating that central metabolism was being reduced. Finally, LC-MS based quantification indicated that in the presence of E6, lopinavir was actively removed from the cell, whereas the indinavir intracellular concentration increased concomitantly with the level of dosing. These investigations have revealed that metabolomics approaches are an apt tool for the study of anti-viral effects within cell cultures, but improvements need to be made with respect to the major limitation of metabolite identification.