Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-11Bitstream added on 2014-06-13T19:42:38Z : No. of bitstreams: 1 takeda_aas_dr_botib_parcial.pdf: 198000 bytes, checksum: dde840aa4960be4dc4599e18c0694b14 (MD5) Bitstreams deleted on 2014-12-19T18:32:42Z: takeda_aas_dr_botib_parcial.pdf,Bitstream added on 2014-12-19T18:33:33Z : No. of bitstreams: 1 000636567.pdf: 1314615 bytes, checksum: 216d1e404d6340f6787609cd55ed585c (MD5) / A Importina-α (ImpA) participa da via clássica de importação nuclear reconhecendo seqüências de localização nuclear (NLS) presentes em proteínas que apresentam atividades no núcleo. Almejando mais informações a respeito do mecanismo de reconhecimento para importação nuclear, nesse trabalho foram efetuados experimentos de expressão, purificação e cristalografia de raios-X da ImpA de Mus musculus com o peptídeo NLS da proteína de reparo de DNA Ku70 e peptídeos TMNLS, T52NLS, frutos de bibliotecas de peptídeos NLS específicos para isoformas da ImpA de Mus musculus e Homo sapiens, respectivamente. O peptídeo TMNLS, conforme esperado, ligou-se à ImpA de maneira similar ao NLS do antígeno T da SV40. Já o peptídeo não clássico T52NLS, obtido de uma biblioteca de peptídeos NLS para a isoforma 5 de Homo sapiens, acomodou-se ao sítio principal da ImpA de maneira satisfatória, indicando que essa seqüência também apresenta afinidade à isoforma de Mus musculus. Complementar à esse resultado, foi constatada a ligação do T52NLS ao sítio secundário da ImpA e, pela primeira vez, foi observada a ligação de um peptídeo monopartido de maneira alternativa, paralela à ImpA, ao contrário da posição anti-paralela, convencional. Surpreendentemente, resultados do complexo ImpA-NLS de Ku70 indicaramno como monopartido, e ligando-se a ImpA de maneira similar à versão fosforilada do peptídeo NLS do antígeno T da SV40. Posições específicas nos sítios de ligação foram confirmadas como essenciais, bem como resíduos da ImpA conservados nessas regiões, indicando a importância das interações intermoleculares nesses sítios. Adicionalmente foram obtidas informações relevantes, como a importância de resíduos de prolina nos NLSs e a não obrigatoriedade de regiões altamente básicas para o reconhecimento de um NLS pela ImpA / The Importin-α (ImpA) plays a role in the classic nuclear import pathway, recognizing proteins that contain nuclear localization sequences (NLS), which have activities in the nucleus. Aiming additional information about the mechanism of nuclear import recognition, in this work, the expression, purification and X-ray crystallography experiments were performed with ImpA from Mus musculus and NLS peptides from the DNA repair protein Ku70 and the peptides TMNLS and T52NLS obtained from NLS peptide libraries specific for ImpA isoforms of Mus musculus e Homo sapiens, respectively. The peptide TMNLS, as expected, bound to the ImpA in a similar way to SV40 antigen T NLS. However, the non classical peptide T52NLS, obtained from a NLS peptide library for isoform 5 of Homo sapiens, which accomodation into the major site of ImpA was acceptable, showed that this sequence has affinity to the Mus musculus isoform. There was also the binding of T52NLS in the minor site of ImpA and for the first time the binding of a monopartite peptide in an alternative way was observed. It was parallel to the ImpA in spite of the conventional nonparallel position. Surprisingly the results of the ImpA- Ku70NLS complex indicated the peptide as monopartite and bounded to the ImpA similarly to the phosphorilated version of the SV40 antigen T NLS. Specific positions in the binding sites were confirmed as essentials, and conserved residues of ImpA in these regions were highlighted, indicating the importance of intermolecular interactions in these sites. Additional information was obtained like the importance of proline residues in NLS sequences and the non-mandatory highly basic regions for a NLS recognizing by ImpA. Keywords: Importin-!, nuclear import, NLS, X-ray crystallography, Ku70, TMNLS, T52NLS

Master of Agribusiness / Department of Agricultural Economics / Tian Xia / Between the years 2008 and 2017, pasta imports in Kenya grew at a rate of 16.12%. This is from data obtained from Kenya National Bureau of Statistics. Growth is projected to continue and can be attributed to factors such as population growth which grew from 38 million to 50 million for that period of time. This population is projected to keep growing and double by the year 2050. GDP per capita grew 58% from US$ 916 to US$ 1,455 between the years 2008 to 2016. This has resulted to growth of middle-class families and increased their purchasing power. Changing lifestyles has led to increased number of dual-income families where both husband and wife work, creating food habits that call for fast and convenient foods. Without domestic production, Kenya imports all its pasta. With the increased demand, it might be the right time for import substitution. This research seeks to find out if it is economically feasible to commercially produce and market pasta in Kenya. The research begins by simulating investment in a small scale pasta production plant in Kenya, followed assessing costs and revenues expected in the seven-year life of the investment. It involves starting up the plant, producing, marketing, and distributing the resulting pasta goods. It is funded through a Kenyan Shilling (Ksh.) 12 million loan from a local bank, payable in seven years at a 14% interest rate. From costs and revenues projections, cash flows are determined. The project’s feasibility is determined by evaluating the cash flows using both Net Present Value (NPV) and Internal Rate of Return (IRR) methods. Using the NPV method, the project has a positive NPV of Ksh.1,286,282.00. When using the IRR method it has an IRR of 45% which is above the cost of capital. It is concluded that the project is feasible. It is also noted that both semolina cost (main pasta production raw), and pasta sales price are important feasibility drivers. NPV’s sensitivity to both is therefore determined. From the analysis, the project remains feasible at all proposed pasta sales prices, as long as semolina cost does not exceed Ksh.56/Kilogram.

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Call number: LD2668 .T4 1950 L65 / Master of Science

M.Com. (Economics) / Please refer to full text to view abstract

Abstract The resilience of trade balances of the major industrialised economies such as the US and Japan to changes in their exchange rates following the switch from fixed to floating exchange rate regimes, triggered interest in the exchange rate pass-through relationship. Because of the importance of the pass-through issue particularly in economic policy formulation, a sizeable literature has developed over recent years. Comprehensive surveys of this literature include Menon (1995), Goldberg and Knetter (1997) and McCarthy (2002). However, not much attention has been paid to the comparison of the methodologies for estimating exchange rate pass-through. This research report aims to address this imbalance by comparing some of the exchange rate pass-through estimation methodologies via a Monte Carlo simulation study, based on the South African data set. The econometric results reported in this research report suggest that the Johansen type VECMs are superior to polynomial distributed lag models, exchange rate pass-through to South Africa’s import prices is incomplete (around 78%) and that the speed of adjustment to long-run equilibrium is low, about 7 per cent of disequilibrium in the previous month is corrected in the current month. We conclude that if we are not sure about the unit root properties of the data (as is normally the case), then the ARDL precedure is the appropriate model for empirical work.

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. High risk HPV types, including HPV 16, can cause cervical carcinomas upon infecting squamous basal epithelial cells. The HPV E2 protein is a multifunctional protein that regulates viral DNA replication and expression of a large number of cellular and viral genes, including the E6 and E7 viral oncogenes. Previous research in the Moroianu lab has identified a novel alpha-helical nuclear localization signal (NLS) in the C-terminal domain of HPV 16 E2 protein (75). Here, we focused on continuing the dissection of the HPV 16 E2 NLS and on identification of the nuclear import mechanism used by this protein. We identified several residues in the C-terminal domain of HPV 16 E2 (327KHK329) and within the NLS (K299, C300) that enhance the function of the NLS. Additionally, we determined that dimerization of the C-terminal domain plays an important role in the nuclear import of HPV 16 E2 as a mutation that disrupted it led to a significant decrease in the nuclear localization of the protein. We discovered that importin 11 karyopherin is a nuclear import receptor for HPV 16 E2. Our data suggest a nuclear import mechanism for HPV 16 E2 whereby UbcM2/UBE2E3 E2-type ubiquitin-conjugating enzyme acts as an adapter to bind HPV 16 E2 to importin 11 karyopherin for its nuclear import. This is a previously undescribed nuclear import mechanism which may have implications for the control of HPV 16 E2 functions. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Orientador: Marcos Roberto de Mattos Fontes / Banca: Mario Sergio Palma / Banca: Maria Celia Bertolini / Banca: Ivan De Godoy Maia / Banca: Ney Lemke / Resumo: A Importina-α (ImpA) participa da via clássica de importação nuclear reconhecendo seqüências de localização nuclear (NLS) presentes em proteínas que apresentam atividades no núcleo. Almejando mais informações a respeito do mecanismo de reconhecimento para importação nuclear, nesse trabalho foram efetuados experimentos de expressão, purificação e cristalografia de raios-X da ImpA de Mus musculus com o peptídeo NLS da proteína de reparo de DNA Ku70 e peptídeos TMNLS, T52NLS, frutos de bibliotecas de peptídeos NLS específicos para isoformas da ImpA de Mus musculus e Homo sapiens, respectivamente. O peptídeo TMNLS, conforme esperado, ligou-se à ImpA de maneira similar ao NLS do antígeno T da SV40. Já o peptídeo não clássico T52NLS, obtido de uma biblioteca de peptídeos NLS para a isoforma 5 de Homo sapiens, acomodou-se ao sítio principal da ImpA de maneira satisfatória, indicando que essa seqüência também apresenta afinidade à isoforma de Mus musculus. Complementar à esse resultado, foi constatada a ligação do T52NLS ao sítio secundário da ImpA e, pela primeira vez, foi observada a ligação de um peptídeo monopartido de maneira alternativa, paralela à ImpA, ao contrário da posição anti-paralela, convencional. Surpreendentemente, resultados do complexo ImpA-NLS de Ku70 indicaramno como monopartido, e ligando-se a ImpA de maneira similar à versão fosforilada do peptídeo NLS do antígeno T da SV40. Posições específicas nos sítios de ligação foram confirmadas como essenciais, bem como resíduos da ImpA conservados nessas regiões, indicando a importância das interações intermoleculares nesses sítios. Adicionalmente foram obtidas informações relevantes, como a importância de resíduos de prolina nos NLSs e a não obrigatoriedade de regiões altamente básicas para o reconhecimento de um NLS pela ImpA / Abstract: The Importin-α (ImpA) plays a role in the classic nuclear import pathway, recognizing proteins that contain nuclear localization sequences (NLS), which have activities in the nucleus. Aiming additional information about the mechanism of nuclear import recognition, in this work, the expression, purification and X-ray crystallography experiments were performed with ImpA from Mus musculus and NLS peptides from the DNA repair protein Ku70 and the peptides TMNLS and T52NLS obtained from NLS peptide libraries specific for ImpA isoforms of Mus musculus e Homo sapiens, respectively. The peptide TMNLS, as expected, bound to the ImpA in a similar way to SV40 antigen T NLS. However, the non classical peptide T52NLS, obtained from a NLS peptide library for isoform 5 of Homo sapiens, which accomodation into the major site of ImpA was acceptable, showed that this sequence has affinity to the Mus musculus isoform. There was also the binding of T52NLS in the minor site of ImpA and for the first time the binding of a monopartite peptide in an alternative way was observed. It was parallel to the ImpA in spite of the conventional nonparallel position. Surprisingly the results of the ImpA- Ku70NLS complex indicated the peptide as monopartite and bounded to the ImpA similarly to the phosphorilated version of the SV40 antigen T NLS. Specific positions in the binding sites were confirmed as essentials, and conserved residues of ImpA in these regions were highlighted, indicating the importance of intermolecular interactions in these sites. Additional information was obtained like the importance of proline residues in NLS sequences and the non-mandatory highly basic regions for a NLS recognizing by ImpA. Keywords: Importin-!, nuclear import, NLS, X-ray crystallography, Ku70, TMNLS, T52NLS / Doutor

<p>As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase.</p><p>In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system.</p><p>We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.</p>

As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase. In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system. We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.