京都大学 / 新制・課程博士 / 博士(医学) / 甲第24784号 / 医博第4976号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 髙折 晃史, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-26T11:35:14Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CasasNobresBelem.pdf: 12136323 bytes, checksum: f9867efb91d90c01b1e96c9da1458bd6 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-06-02T16:42:41Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CasasNobresBelem.pdf: 12136323 bytes, checksum: f9867efb91d90c01b1e96c9da1458bd6 (MD5) / Made available in DSpace on 2017-06-02T16:42:41Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_CasasNobresBelem.pdf: 12136323 bytes, checksum: f9867efb91d90c01b1e96c9da1458bd6 (MD5) Previous issue date: 2013-08-30 / Este trabalho investiga as Casas Nobres construídas em Belém na segunda metade do século XVIII e início do século XIX, identificadas, selecionadas e analisadas com base em pesquisas bibliográficas, documentais e iconográficas. Utiliza-se como recorte geográfico o centro histórico da cidade citada onde estão inseridos os bairros da Cidade Velha e da Campina, correspondentes aos primitivos núcleos que conformavam o espaço urbano de Belém, antiga sede da Capitania e do Estado do Grão-Pará, durante o modelo administrativo de Colônia na América portuguesa. O conhecimento sobre a tipologia de edifício residencial Casa Nobre, e posterior identificação de exemplares no centro histórico de Belém, visa revelar a sua ocorrência em solos amazônicos num contexto social, econômico e político específico do Antigo Regime português. Três edificações foram selecionadas como estudo de caso: 1) o Solar do Barão de Guajará; 2) a Casa Rosada; 3) e a Casa das Onze Janelas. Buscou-se, em princípio, a genealogia na Casa Nobre portuguesa com a compreensão dos valores simbólicos e as variações semânticas ligadas à sua morfologia, assim como o conhecimento da sua arquitetura. Posteriormente, foi investigada a arquitetura residencial da Nobreza no cenário colonial brasileiro, sistematizando as principais características a partir de princípios encontrados na sua gênese portuguesa. Por fim, realizaram-se as análises das três Casas Nobres selecionadas, pautadas no conhecimento legado pelos estudos inicias das suas congêneres portuguesas e luso-brasileiras, estabelecendo entre elas, analogias e reconhecendo as especificidades locais. O resultado da pesquisa fornece uma base de conhecimento de um gênero específico de construção residencial, a Casa Nobre, o que viabiliza a identificação de outros exemplares em Belém, ou em outras localidades no Estado do Pará, em contextos urbanos ou rurais, tornando possíveis, também, intervenções mais embasadas, quando estas são necessárias para a sua preservação, pois, é preciso conhecer o que se restaura. / This paper investigates the Noble Houses built in Belém in the second half of the eighteenth and early nineteenth century, identified, selected and analyzed based on literature, documentary and iconographic research. The historic city center of the city mentioned is used as a spatial area where the neighborhoods of Cidade Velha and Campina are inserted. They correspond to the primitive nuclei which shaped the urban space of Belém, former headquarters of the Province and State of Grão-Pará during the Administrative Model Colony in Portuguese America. Knowledge about the type of residential building Noble Houses and subsequent identification of specimens in the center of Belém is to reveal their occurrence in the Amazonian soil in a social, economic and political context specific to the Portuguese Ancient Regime. Three buildings were selected as case study: 1) Solar do Barão de Guajará, 2) the Casa Rosada, 3) and the House of Eleven Windows. At first, the genealogy in the Noble Portuguese House was searched, with an understanding of the symbolic values and semantic variations related to their morphology, as well as the knowledge of its architecture. Subsequently, we investigated the residential architecture of the Aristocracy in the Brazilian colonial settings, systematizing the main features from principles found in Portuguese genesis. Finally, the three selected Noble Houses were analyzed, guided by the knowledge of initial studies of its Portuguese and Luso-Brazilian congeners, establishing analogies among them and recognizing local specificities. The result of this research provides a knowledge basis for a specific genre of residential construction, the Noble House, which enables the identification of other specimens in Belém, and in other locations in the state of Pará, in urban or rural areas, making it also possible more informed interventions when these are necessary for its preservation, for we must know what we restore.

Objectives The study aims to describe the socio-demographic characteristics, clinical outcomes of the patients in the Gauteng public sector roll-out programme and establish the association between these. There are contradictory results from international studies on these associations, in the absence of SA results. Methods This is a retrospective cohort, exploratory, secondary data, record review study and a comparison between two sites. Routinely collected socio-demographic data and clinical data were used to establish the impact of socio-demographic factors on response to HAART. This was collected for patients who enrolled from April 2004 to August 2004. Chris Hani Baragwanaath (CHB) had 494 records, Helen Joseph (HJ) had159 records collected. Exposure variables (age, sex, marital status, education level, residential area, employment, baseline viral load and baseline cd4 count). Outcome variables were (CD4 and Viral load at 3 months, 6 months and 12 months). Data Analysis T tests were used for comparing means; logistic regression was used to find the effect of ordered exposure variables and binary outcome. Chi square and fishers exact were used to find frequencies and association between the categorical variables. Regression was used to find the association between the continuous exposure variables and the continuous outcome variables. In a multivariate model, to assess the effect of the exposure variables to the outcome variables Multivariate regression was used. Statistical significance was assessed at the 5% significance level, giving 95% confidence interval. Results The majority of the patients (653) were female, African, unemployed and were literate. At CHB, at the end of the first year, three quarters were still on treatment however; just under a fifth (19%) had died. The majority responded well to treatment and had a mean baseline CD4 count of 58.9cells/mm3 (CHB) and 78.4cells/mm3 (HJ) and mean CD4 count of 245 (CHB) and 268 (HJ) after 12 months. increasing age, and being widowed, lowers the immunological response. Employment, education, sex and had no impact on response. Conclusion • There is positive virological and immunological response to HAART in Gauteng ARV roll-out programme despite the low socio economic status of the majority of the patients. • Provision of free antiretroviral drugs and access to the disability grant has assisted in mitigating the effects of HIV/ AIDS on the socio-economically disadvantaged. • The elderly and the widowed might need close monitoring as their response appears to be lower than the others. • The group with no schooling is not well represented in this sample; the question is whether the HIV/AIDS prevention messages and treatment is accessible for this group. This needs further research.

The genome of Escherichia coli harbors several catabolic operons involved in the utilization of a wide variety of natural compounds as carbon sources. The chitobiose (chu) operons of E.coli Is involved in the utilization of chitobiose(disaccharide of N-acety1-D-glucosamine) and cellbiose (disaccharide of glucose) derived from the two most abundant naturally occurring carbon sources on earth, chitin and cellulose respectively. The operon consists of the chbBCARFG genes coding for transport, regulation and hydrolysis functions required to utilize these compounds; the chuyBCA genes code for a multi-subuni PTS transporter ; the chuR codes for a dual function repressor/activator of the operon; the chbF codes for a phospho-glucosidase and the chbG codes for a protein of unknown function. The chu operon Is regulated by three transcription factors; NagC, a key regulator of the nag genes involved in amino sugar metabolism; ChbR, a dual function operon-specific regulator; and CRP_cAMP. The operon is repressed by NagC and ChbR in the absence of catabolic substrate. In the presence of chitobiose, expression is induced by the abrogation of NagC-mediated repression by GlcNAc-6-P generated by the hydrolysis of chitobiose-6-P and subsequent activation of transcription by ChbR and CPR-cAMP. Wild type E.coli connot utilize cellbiose due to the inability of cellbiose to induce expression from the operon. The simultaneous presence of a loss of function mutation in nagC and a gain –of-function mutation in chbR is necessary and sufficient to allow cellbiose to induce expression and confer on E.coli the ability to utilize cellbiose. The activation step by ChbR and CPR-cAMP requires an inducer that is recognized by ChbR. The chemical identity of the inducer and the mechanism of transcriptional activation by ChbR and CPR-cAMP are not understood. The studies described in the chapter 2 shows that chbG is essential for the utilization of the acetylated sugars chitobiose and chitotriose while it is dispensable for the sugars lacking the acety1group such as cellobiose and chitosan dimer, a disaccharide of N-glucosamine. ChbG is produced as a cytosolic protein and removes one acety1 group from chitobiose and chitotriose thus shows a mono-decetylase activity. Taken together, the observing suggest that ChbG deacetylates chitobiose-6-P and chitotriose-6-P producing the mono-decetylated from of the sugars. The deacetylateion is necessary for their recognition both as inducers by ChbR to activate transcription along with CRP-cAMP and as substractes by phosop-glucosidase ChbF. Cellobiose positive(Cel+) mutants carrying nagC delection and different gain-of-function mutations in chbR are independent of chbG for induction by chitobiose suggesting that the mutations in ChbR can allow it to recognize the acetylated form of chitobiose-6-P. Despite normal induction, the mutants to grow on chitobiose without chbG are consistant with the requirement of deacetylation for hydrolysis by ChbF. The prediction active site of chbG was validated by demonstrating the loss of chbG function upon alanine substitution of the putative metal binding residues. Vibro cholerace ChbG can complement the function of E.coli ChbG indicating that ChbG is conserved in both the organisms. The studies presented in chapter 3 address the mechanism of transcriptional activation of the chb operon by ChbR and CPR-cAMP. ChbR and CPR-cAMP function in a synergistic manner in response to the induction signal. The synergy is not because of their cooperative binding to the DNA. The role of CRP as a class I activator via the known mechanism involving interaction between the Activation region1 (AR1) and the C-terminal domain of the alpha subunit of RNA polymerase (CTD) was not crucial for the chb operon. A direct interaction between the two activators in virto was observed. Based on these results and the close spacing of the synergy is due to interaction between the two regulators bound to DNA that is enhanced in the presence of the inducer, binding about an optimal confirmation in ChbR required to interact with RNA polymerase. ChbR contacts different residues in the subunit in response to cellbiose and chitobiose; whereas it utilizes the known residues in the presence cellbiose, it appears to require different and unknown residues for induction in the presence of chitobiose. In conclusion, the studies reported in chapter 2 and 3 provide an understanding of the regulation of the chitin oligosaccharides utilization in E.coli at different levels. The broad implications of these studies and possible future directions are discussed in chapter 4. ChbG is an evolutionary conserved protein found in both prokaryotes and enkayotes including humans. ChbG homologs have been implicated in inflammatory bowel disorders in humans and development in metazoans. Therefore, the studies on chbG described in this thesis have been broader significance.

Escherichia coli has the ability to respond to stress such as starvation in a very efficient manner. Under conditions of starvation wherein a novel substrate is provided as a sole nutritional source, Spontaneous mutants arise in a population of E.Coli that are able to utilize this novel carbon Many generic systems, upon mutational activation, have been shown to allow E.coli to Grow on novel substrates. . Wildtype E.coli is not able to utilize cellobiose, a disaccharide of glucose, as a carbon source. However after prolonged incubation with cellobiose as a sole carbon source, spontaneous Cel+ mutants can be isolated. The Cel+ derivatives have mutations in the chb operon involved in the utilization of N-N-diacetylchitobiose, a disaccharide of N-acetyl glucosamine. The chb operon of E.coli is comprised of six ORFs (chbBCARFG) with a ~200bp regulatory region (chbOP); chbBCA encode the IIB, IIC and IIA domains of the PTS-dependent permease respectively, chbR encodes for a dual function activator/repressor, chbF encodes the phopho-chitobiase and chbG codes for a protein of unknown function. It has been shown that the three proteins ChbR, CAP and NagC regulate the expression of the chb operon. ChbR along with CAP activates the chb operon in the presence of chitobiose. In the absence of the inducer, ChbR, along with NagC, represses the chb operon. Activation of the chb operon allowing utilization of cellobiose was earlier shown to occur either via insertion of IS1, IS2 or IS5 into the regulatory region (chbOP) upstream of the transcription start site or by base substitutions in chbR. Comparison of the chb operon sequence obtained from various Cel+ mutants with E.coli K12 genome sequence showed many differences. These differences were clustered in both the permease (chbBCA) as well as the enzyme (chbF) of the chb operon, suggesting that mutations are needed in all the ORFs of this operon in order to alter the specificity of E.coli towards utilization of cellobiose. The main objective of this thesis is to elucidate the mechanism of mutational activation of the chb operon of E.coli to allow utilization of cellobiose. These studies have shown that two classes of mutations, those that abrogate repression by NagC and those that alter the regulation by ChbR, together are necessary and sufficient to confer a Cel+ phenotype to E.coli. These studies also show that the wildtype permease and phospho-â -glucosidase are able to recognize and cleave cellobiose. Initial experiments were designed to study the role of independent mutational events of either insertion within the regulatory region or loss-of-function of chbR in conferring E.coli a Cel+ phenotype. The single mutational event of either the insertion within the regulatory region chbOP that disrupts the strong NagC binding site (mimicking an IS element) or knockout of chbR did not confer on E.c oli a Cel+ phenotype. However the presence of the artificial insertion within chbOP accelerated the process of obtaining Cel+ mutants suggesting a positive role for insertion elements. The apparent inability of the chbR knockout strain to mutate to Cel+ suggested that chbR is essential for acquisition of a Cel+ phenotype. Reporter gene assays showed that the presence of an insertion within chbOP enhances the promoter activity marginally. The role of chbR as a repressor was further ascertained by increased promoter activity seen from wildtype chbOP-lacZ fusion in a chbR knockout strain. A marginal enhancement in promoter activity in the presence of cellobiose in a strain carrying a wildtype chbR as compared to chbR knockout strain suggested an additional positive role of chbR. The inability of cellobiose to produce an inducing signal necessary for activation by wildtype ChbR protein suggested that gain-of-function mutations within chbR locus might play a crucial role in acquisition of cellobiose utilization phenotype by E.coli. The chbR clones obtained from various Cel+ mutants could activate transcription from the chb promoter at a higher level in the presence of cellobiose. However this activation was seen only in a strain carrying disruptions of the chromosomal nagC and chbR loci. These transformants also showed a Cel+ phenotype on the MacConkey cellobiose medium suggesting that the wildtype permease and enzyme upon induction could recognise, transport and cleave cellobiose, respectively. This was confirmed by cloning the wildtype genes encoding the permease and phospho-â -glucosidase under a heterologous promoter (Plac). The wildtype E.coli strain transformed with a plasmid carrying the genes could utilize cellobiose efficiently. Large scale isolation of Cel+ mutants was undertaken. Variation in the ability of cellobiose utilization was observed among the different mutants. Several Cel+ mutants retained the ability to utilize chitobiose. Cel+ mutants lacking insertions within chbOP contained a loss-of-function mutation at the nagC locus. The sequencing of the chbR locus from Cel+ mutant strains showed a single basepair change at the DNA level translating into a single amino acid change when compared to the Cel- counterpart. Nucleotide sequence of chbR obtained from two Cel+ natural isolates of E.coli also showed a single base mutation. The chbR clones from the two mutants, when transformed into a strain carrying disruptions at the chromosomal nagC and chbR loci, conferred it a Cel+ phenotype. Initial characterization of one of the mutant ChbR (N238S) was carried out. Reporter assays in a strain containing a wildtype copy of chbR at the genomic locus and a disruption of nagC showed that the wildtype ChbR is dominant over the mutant ChbR (N238S). The biochemical investigations of the wildtype and mutant ChbR (N238S) were undertaken. Wildtype ChbR showed non-specific binding to chbOP that could not be competed out by excess cold DNA. DNaseI protection assays confirmed that wildtype ChbR formed a relatively nonspecific complex with chbOP as compared to mutant ChbR (N238S). Finally DNaseI footprinting experiments showed that mutant ChbR (N238S) binds the specific direct repeat within chbOP better than the wildtype protein. These results indicated that mutant ChbR (N238S) has lost its ability to repress transcription by its inability to bind chbOP non-specifically. In addition, the mutant ChbR (N238S) has acquired the ability to activate transcription in the presence of cellobiose. This could be partly mediated via enhanced binding of the mutant ChbR (N238S) to the specific DNA binding site within chbOP in contrast to its wildtype counterpart. To conclude, this work has shown that acquisitive evolution of E.coli towards utilization of cellobiose in laboratory conditions alters the regulation of the chb operon and allows it to acquire new metabolic capability for utilizing cellobiose under selective pressure.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-26T11:24:23Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_RuaDrAssis.pdf: 101446120 bytes, checksum: cb2b8efc58b1acd94a79760e23048f7c (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-06-02T12:54:57Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_RuaDrAssis.pdf: 101446120 bytes, checksum: cb2b8efc58b1acd94a79760e23048f7c (MD5) / Made available in DSpace on 2017-06-02T12:54:57Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_RuaDrAssis.pdf: 101446120 bytes, checksum: cb2b8efc58b1acd94a79760e23048f7c (MD5) Previous issue date: 2013-03-19 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este trabalho investiga a transformação da paisagem da Rua Dr. Assis, no Bairro da Cidade Velha, na cidade de Belém, a partir da releitura das edificações classificadas como “renovação” pela Lei 7.709 de 1994, responsável pela preservação e proteção do Patrimônio Histórico, Artístico, Ambiental e Cultural. A categoria “renovação” enquadra os imóveis sem interesse à preservação, onde em seu lugar pode ser construída uma nova edificação. Com isso, esses lotes presentes no conjunto histórico “abrem a guarda” para a dinâmica de transformação intrínseca à cidade. A paisagem transfigurada, que escapa à “estética patrimonial” passa a consolidar uma nova “Cidade Velha”- espaço gênese de Belém, que juntamente com o bairro da Campina formam o Centro Histórico. Utilizando como recorte a Rua Dr. Assis - logradouro inserido no bairro da Cidade Velha – e com base no método da etnografia de rua, é feita uma incursão nesta paisagem que guarda na materialidade e na memória de moradores e comerciantes, o processo de transformação do tecido urbano tombado. Assim, o (re)conhecimento deste espaço, visa trazer à tona a relação material-imaterial eclipsada pela categorização da legislação de 1994. / This research investigate Dr. Assis street landscape transformation – located in Cidade Velha neighborhood of Belém – based on rereading of buildings its classified as "renewal" by the Law 7.709 of 1994. This law is responsible for Belém’s protection and preservation Historic, Artistic, Environmental and Cultural Heritage. The “renewal” category fits the properties with no interest of preservation, which can be replaced by new ones. Thus these lots in the historic center, “opens custody” to the dynamics of transformation, a city's intrinsic characteristic. The transfigured landscape which escapes from the “cultural heritage aesthetic” consolidates a new “Cidade Velha” – Belém’s genesis district – which along with Campina are the city’s Historic Center. Based on street ethnography method, an incursion is made into this landscape, which keeps in its, the building’s materiality and in the memories of the local residents and traders, the transformer process of the protected urban fabric. Thus, the space (re) cognition, aims to bring out the material-immaterial relation, eclipsed by the legislation category of 1994.

The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-02-20T15:55:04Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AlemFormasFuncoes.pdf: 22805037 bytes, checksum: 8e46d17180893a9dcd41929302ef86ac (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-03-02T16:08:34Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AlemFormasFuncoes.pdf: 22805037 bytes, checksum: 8e46d17180893a9dcd41929302ef86ac (MD5) / Made available in DSpace on 2017-03-02T16:08:34Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_AlemFormasFuncoes.pdf: 22805037 bytes, checksum: 8e46d17180893a9dcd41929302ef86ac (MD5) Previous issue date: 2014-10-31 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Atualmente, muitas cidades brasileiras sofrem com uma crescente perda da capacidade de manter vivo e atrativo seus centros históricos, em meio às transformações de uso do solo provenientes da dinâmica da cidade contemporânea. Apreender o centro histórico de uma cidade, identificando valores e/ou significados da configuração espacial e de seus elementos da paisagem em meio ao movimento da sociedade, é uma primeira etapa de análise, além de servir como base para planos e projetos urbanísticos e sociais que visem não só a conservá-lo, mas a integrá-lo à vida contemporânea. O presente trabalho considera o Centro Histórico de Belém (CHB), na Amazônia brasileira, como um espaço social dotado de forma e conteúdo. Os processos de intervenção urbana neste centro histórico têm corroborado com diversas práticas espaciais que o modificam. Esses processos de intervenção, na maioria das vezes, estão mais voltados para uma gestão da forma e da função do que para uma gestão social dos centros históricos. Ao não considerar esse espaço social autoexplicativo, esta pesquisa utiliza as categorias socioespaciais (estrutura, processo, função e forma), que explicam a produção social do espaço, para apreender as relações dialéticas entre forma física e ações ao longo da história. O objetivo geral da pesquisa foi analisar a configuração espacial do CHB a partir da gestão preservacionista de seus elementos da paisagem, buscando apreender os significados das formas criadas e as alterações de suas funções no decorrer do tempo, a partir da compreensão tanto de suas estruturas (de onde elas surgiram, o contexto histórico) quanto dos processos que foram responsáveis pelo surgimento das mesmas. Constatou-se, a partir das intervenções no CHB, que a forma espacial e sua função são os elementos que mais são considerados como base para planos e projetos urbanísticos para essa área, secundarizando outros elementos formadores desse espaço, tais como as relações sociais. Nesse sentido, o argumento central sustentado no decorrer da pesquisa foi que as categorias socioespaciais (estrutura, processo, forma e função), que ajudam a explicar a origem da paisagem urbana atual da cidade de Belém, na maioria das vezes, não são utilizadas como base em leis, planos e projetos urbanísticos para o CHB. Com isso, a gestão do tipo preservacionista não dá conta da história da totalidade espacial deste centro histórico, de maneira que a apropriação dos valores e/ou significados está materializada por uma gestão da forma e da função, secundarizando as estruturas e os processos que deram origem a essas mesmas formas e funções do CHB. / Nowadays, many Brazilian cities suffer from a growing disability of keeping alive and attractive its historic areas caused by land use transformations deriving from the dynamism of contemporary times. Learning a city's historic area, identifying its values and/or spacial cofiguration meanings and its landscape components in the middle of a society movement, is the first step of analysis, in addition to it serving as a base for social and urban plans and projects that look not only to preserve it but to integrate it to a contemporary life style. This present work takes into consideration the “Historic Center of Belem” (HCB), in the Brazilian Amazon, as a social space containning shape and content. The intervention process on this historic center has been changing it through many spacial actions. Those intervention processes, most of the time, are more focused on the management of shape and function than the social management of its historic centers. By not taking in consideration this auto explaining social space, this research utilizes social and spacial categories (structure, process, function and shape) which explain the space social production, in order to learn dialectical relations between physical shape and actions during history. The general objective of this research was to analize the HCB spacial configuration from the preservationist management of its landscape elements, trying to learn the meaning of created shapes and the changes of its uses along the time, from the understanding of both its structures (where they were coming from, the historic context) and the processes that were responsible for their origins. We realized from the interventions on the HCB, that the spacial shape and its use are the most considered elements of bases for this area’s plans and urban projects, leaving behind other components and elements of this space, such as social relations. In this case, the central argument sustained during this research was that the social space categories (structure, process, shape and use) that helped explain the current origin of Belem’s urban landscape, which most of the time, is not taken into consideration as base for new laws, plans and urban projects on the HCB. With that in mind, the preservationist kind of management cannot handle the total spacial history of this historic center, in a way that the value appropriation and/or meaning is materialized by a shape and use management, leaving behind the structures and processes that gave birth to these same shape and functions of the HCB.

Conventional 2-level inverters have been quite popular in industry for drives applications. It used pulse width modulation techniques to generate a voltage waveform with high quality. For achieving this, it had to switch at high frequencies and also the switching is between 0 and Vdc. Also additional LC filters are required before feeding to a motor. 3-phase IM is the work horse of the industry. Several speed control techniques have been established namely the V/f control technique and for high performance, vector control is adopted. An electric drive system comprises of a rectifier, inverter, a motor and a load. each module is a topic by itself. This thesis work discusses the novel inverter topologies to overcome the demerits of a conventional 2-level inverter or even the basic multilevel topologies, for an electric drive. The word ‘multilevel’ itself signifies that inverter can generate more than two levels. The idea was first originated by Nabae, Takahashi and Akagi to bring an additional voltage level so that the waveform becomes a quasi square wave. This additional voltage level brought additional benefits in terms of reduced dv/dt and requirement of low switching frequency. But this was not without any cost. The inverter structure is slightly more complicated than a 2-level and also required more devices. But the advantage it gave was superior enough to such an extent that the above topology (popularly known as NPC) has become quite popular in industry. This topology was later modified to equalize the semiconductor losses among switches by replacing the clamping diodes with controllable switches and such topologies are popularly known as Active NPCs (ANPCs) because of the replacement of diodes with active switches. 3-level flying capacitors were then introduced where the additional voltage level is provided using charged capacitors. But this capacitor voltage has to be maintained at its nominal value during the inverter operation. An additional floating capacitor, which is an electrolytic capacitor is needed for this. Increasing the number of electrolytic capacitors reduces the reliability of the inverter drive since they are the weakest link in any inverters and its count has to be kept to the minimum. By using a H-bridge cell in each of the three phases, three voltage levels can be easily obtained.This is commonly known as Cascaded H-bridge (CHB) multilevel inverter. The above three topologies have been discussed with respect to generation of three pole voltage levels and these topologies are quite suited also. A higher number of voltage levels will reduce the switching frequency even lesser and also the dv/dt. On increasing the number of levels further and further, finally the inverter need not do any PWM switching and just generating the levels is sufficient enough for a good quality waveform and also low dv/dt. But when the above topologies are scaled for more than three voltage levels, all of them suffer serious drawbacks which is briefly discussed below. The diode clamped inverter (known as NPC if it is 3-level), when extended to more than three levels suffers from the neutral point balancing issue and also the count of clamping diodes increase drastically. FC inverters, when extended beyond 3-level, the number of electrolytic capacitors increases and also balancing of these capacitors to their nominal voltages becomes complicated. In the case of multilevel CHB, when extended beyond 3-level, the requirement of isolated DC sources also increases. To generate isolated supplies, phase shifting transformer and 8, 12 or 24 pulse diode rectifier is needed which increases the weight , size and cost of the drive. Therefore its application is limited. In this thesis, the aim is to develop a novel method to develop a multilevel inverter without the drawbacks faced by the basic multilevel topologies when scaled for higher number of voltage levels. This is done through stacking the basic or hybrid combination of these basic multilevel topologies through selector switches. This method is experimentally verified by stacking two 5-level inverters through a 2-level selector switch (whose switching losses can be minimized through soft cycle commutation). This will generate nine levels.Generating 9-levels through scaling the basic topologies is disadvantageous, the comparison table is provided in the thesis. This is true for any higher voltage level generation. Each of the above 5-level inverter is developed through cascading an FC with a capacitor fed H-bridge. The device count can be reduced by making the FC-CHB module common to the selector switches by shifting the selector switches between the DC link and the common FC-CHB module. Doing so, reduces the modular feature of the drive but the device count can be reduced. The FFT plot at different frequencies of operation and the switching losses of the different modules-FC, CHB and the selector switches are also plotted for different frequencies of operation. The next step is to check whether this method can be extended to any number of stackings for generation of more voltage levels. For this, a 49-level inverter is developed in laboratory by stacking three 17-level inverters. Each of the 17-level inverter is developed by cascading an FC with three CHBs. When there are 49 levels in the pole voltage waveform, there is no need to do any regular PWM since the output waveform will be very close to a sine wave even without any PWM switching. The technique used is commonly known in literature as Nearest Level Control (NLC). This method of stacking and cascading has the advantage that the FC and the CHB modules now are of very low voltages and the switching losses can be reduced. The switching losses of the different modules are calculated and plotted for different operating frequencies in the thesis. To reduce the voltages of the modules further, a 6-phase machine has been reconfigured as a 3-phase machine, the advantage being that now the DC link voltage requirement is half of that needed earlier for the same power. This further reduces voltages of the modules by half and this allows the switches to be replaced with MOSFETs, improving the efficiency of the drive. This topology is also experimentally verified for both steady state and transient conditions. So far the research focussed on a 3-phase IM fed through a stacked MLI. It can be observed that a stacked MLI needs as many DC sources as the number of stackings. A 6-phase machine apart from reduced DC link voltage requirement, has other advantages of better fault tolerant capability and better space harmonics. They are serious contenders for applications like ship propulsion, locomotive traction, electric vehicles, more electric aircraft and other high power industrial applications. Using the unique property of a 6-phase machine that its opposite windings always draw equal and opposite current, the neutral point (NP) (formed as a result of stacking two MLIs) voltage can be balanced. It was observed that the net mid point current drawn from the mid point can be made zero in a switching interval. It was later observed that with minimal changes, the mid point current drawn from the NP can be made instantaneously zero and the NP voltage deviation is completely arrested and the topology needs only very low capacity series connected capacitors energized from a single DC link. This topology is also experimentally verified using the stacked 9-level inverter topology discussed above but now for 6-phase application and experimental results are provided in the thesis. Single DC link enables direct back to back conversion and power can be fed back to the mains at any desired power factor. All the experimental verification is done on a DSP (TMS320F28335) and FPGA (Spartan 3 XCS3200) platform. An IM is run using V/f control scheme and the above inverter topologies are used to drive the motor. The IGBTs used are SKM75GB123D for the stacked 9-level inverter in the 3-phase and 6-phase experiments. For the 49-level inverter experiment, MOSFETs-IRF260N were used. Both steady state and transient results ensure that the proposed inverter topologies are suitable for high power applications.

The genetic systems involved in the utilisation of aromatic β-glucosides in E. coli consist of the bgl, asc, and chb operons and the locus bglA encoding phospho-β-glucosidase A. The bgl and asc operons are known as cryptic or silent systems since their expression is not sufficient for utilisation of these sugars in wild type strains of E. coli. Their transcriptional activation by different classes of mutations confers a Bgl+ phenotype to the mutant. The maintenance of cryptic genes without accumulating deleterious mutation in spite of being silent is an evolutionary puzzle. Several observations have suggested the possibility that these genes may be expressed under specific physiological conditions conferring a fitness advantage to the organism. The main aim of this study was to investigate the possible role of aromatic β-glucoside catabolic systems of E. coli in combating nutrient stress and microaerobic growth conditions. The results presented in Chapter 2 address the evolution of aromatic β-glucoside catabolic systems when exposed to a novel β-glucoside as the sole substrate. The results indicate that the bgl opeon, the primary system involved in the utilisation of the aromatic β-glucosides arbutin and salicin, is also involved in esculin utilisation. In the absence of bglB encoding the enzyme phospho-β-glucosidase B, activation of the silent asc operon enables esculin utilisation. The bglA gene encoding phospho-β-glucosidase A specific for arbutin, can undergo successive mutations to evolve the ability to hydrolyse esculin and salicin sequentially when bglB and ascB are absent. The Esc+ and Sal+ mutants retain their arbutin+ phenotype, indicating that the mutations enhance the promiscuity of the enzyme. Sequencing data indicate that the first step Esc+ mutant carries a four base insertion within the promoter of the bglA gene that results in enhanced transcription of bglA. RT-PCR studies confirm that both the steady-state levels as well as the half-life of the bglA mRNA are enhanced in the mutant. This is further corroborated by the observation that overexpression of wild type bglA in the parent strain using a multicopy plasmid confers an Esc+ phenotype. The second step Sal+ mutant carries a point mutation within bglA ORF, a thymine to guanine transversion at position 583 (T583G) of the bglA gene, resulting in an amino acid change from cysteine to glycine at position 195 (C195G) of the BglA ORF close to the active site. Presence of a plasmid carrying the T583G mutation, introduced by site-directed mutagenesis, results in a Sal+ phenotype, confirming the role of the transversion in conferring the Sal+ phenotype. Based on docking studies, the positioning of salicin into the substrate binding site of the mutant BglA enzyme is different compared to wild type BglA due to the loss of stearic hindrance for the binding of salicin when C195 is replaced by the smaller amino acid glycine in the mutant protein. These observations indicate that under conditions of nutrient deprivation, exposure to novel substrates can result in the evolution of new metabolic capabilities by the sequential modification of a pre-existing genetic system. In the case of one novel substrate, the mutation results in the overexpression of the hydrolytic enzyme, while in the case of the second substrate, a mutation close to its active site increases its substrate specificity. Results presented in Chapter 3 specifically deal with the involvement of the bgl operon under low levels of oxygen. Earlier observations have shown that there is a 22 fold enhancement in the expression of the bgl operon under anaerobic condition. The present results provide evidence that bgl expression has a physiological role under low levels of oxygen and in addition suggest a possible mechanism for the overexpression of the bgl operon that involves the ArcAB two component system known to mediate regulation under microaerobic and static conditions. Transcription studies using a lacZ reporter fused to the wild type bgl promoter show that there is enhanced transcription from the bgl promoter under microaerobic and static conditions in the presence of arcA encoding the response regulator compared to that in its absence. The positive effect of arcA on the expression of the bgl operon is dispensable in the absence of H-NS since presence or absence of arcA does not change the expression of the bgl operon in an hns-null background, implying that the involvement of ArcA is via antagonizing H-NS. Competition experiments indicate that there is growth advantage associated with the activated allele of the bgl operon under low levels of oxygen since Bgl+ strains carrying the activated allele of the bgl operon as well as strains expressing BglG constitutively can out-compete wild-type strains. Presence of the wild type arcA allele results in a strong growth advantage compared to its absence under static conditions but not aerobic condition. The bgl operon seems to be one of the possible downstream targets of ArcA under static condition since absence of the bgl operon results in a modest reduction of the growth advantage (GASP) phenotype conferred by arcA. The up-regulation of the bgl operon is likely to enable the cells to scavenge available nutrients from their niche more efficiently. These experiments also show that the GASP phenotype associated with BglG constitutive strains under static conditions involves downstream genes that are different from oppA known to be one of the downstream targets during aerobic growth. It is possible that under low level of oxygen, the bgl operon is regulating a different set of downstream genes involving a different mechanism. In summary, the results of this investigation show that the aromatic β-glucoside catabolic systems in E. coli play a role in the generation of new metabolic capabilities via mutations in pre-existing genetic systems as well as through changes in gene expression patterns. The mechanisms outlined in this study are likely to be of broader significance applicable to microbial evolution under stress in general.