O imprinting genômico é um processo regulado epigeneticamente que faz com que os alelos sejam expressos de acordo com a sua origem parental. No cromossomo 11 (11p15.5), existem duas regiões controladoras de imprinting (ICR1 e ICR2), que controlam a expressão de genes marcados (imprinted). Os padrões de metilação dessas regiões podem ser alterados pela dissomia uniparental (DUP), que ocorre quando parte de ou um cromossomo inteiro do mesmo par de homólogos é herdado de somente um genitor. Erros mitóticos podem gerar mosaicismo com uma linhagem de células com DUP e a outra biparental. As síndromes de Silver-Russell (SSR) e Beckwith-Wiedemann (SBW) são doenças de alterações do imprinting genômico, envolvendo os cromossomos 7 (SSR) e 11 (SSR e SBW). A Hemihiperplasia Isolada (HHI) parece corresponder a uma forma mais leve da SBW.. No presente trabalho, foi realizada uma varredura in silico para busca de novos microssatélites nos cromossomos 7 e 11, e selecionados seis do tipo tetra ou pentanucleotídeos, no cromossomo 7, e 12, no cromossomo 11. O perfil de metilação nas ICRs foi verificado por três técnicas distintas: MS-MLPA, DESM-RT e por uma nova estratégia desenvolvida neste trabalho denominada DESM-QFPCR. Foram avaliados 32 pacientes com SBW, 16 HHI, 20 com SSR e seus pais, quando disponíveis, além de um paciente com fenótipo aparentemente normal com cariótipo 46,XX/46,XY e cuja placenta apresentou displasia mesenquimal placentária (DMP) a qual está associada à SBW. Os novos marcadores apresentaram alta taxa de heterozigose (média de 70%), e ausência das características indesejáveis dos dinucleotídeos predominantemente utilizados para detecção de DUP. Seis marcadores estão entre genes controlados pelas ICRs 1 e 2. A DUP paterna do cromossomo 11 (DUPpat Cr11), sempre restrita a 11p15.5, foi responsável por 13% dos casos de HHI e 19% dos de SBW. As alterações estruturais foram confirmadas por minissequenciamento quantitativo de SNPs e por MS-MLPA. Um paciente apresentou duplicação paterna abrangendo ambas as ICRs. Uma deleção não descrita anteriormente no gene CDKN1C foi observada em uma paciente e sua mãe. Para os pacientes com DUPpat Cr11, foram investigados microssatélites em 13 autossomos e nos cromossomos sexuais para detecção de mosaicismo global. Apenas o paciente com DMP apresentou mosaicismo [células androgenéticas (25-30%) e biparentais], sugerindo evento de dupla fertilização. Nos pacientes com SSR, foi observada hipometilação na ICR1 em 25% dos casos. Para a SBW, foi observada hipermetilação na ICR1 e hipometilação na ICR2 em 6% e 42% dos casos, respectivamente. Os casos com DUPpat Cr11 apresentaram alteração de metilação em ambas as ICRs. As frequências de alterações (epi) genéticas encontradas foram semelhantes às previamente descritas na literatura para as SBW, SSR e HHI. Neste trabalho, foi desenvolvida uma nova técnica para estudo de metilação do DNA de ICRs e testados marcadores microssatélites inéditos na região 11p15, que quando comparados com metodologias mais tradicionais de avaliação, como DESM-RT e MS-MLPA, mostraram elevada correlação dos resultados. Os achados mostram a complexidade da etiologia das doenças estudadas no presente trabalho e os dados moleculares serão imprescindíveis para o aconselhamento genético adequado para cada caso em particular e suas famílias. / Genomic imprinting is a epigenetically regulated process where the alleles are expressed in terms of their parental origin. On chromosome 11 (11p15.5) there are two regions controlling imprinting (ICR1 and ICR2), which control imprinted gene expression. The methylation patterns in these regions may be altered by uniparental disomy (UPD), which occurs when part or whole chromose is inherited from only one parent. Mitotic errors can lead to mosaicism with a cell line with DUP and other, biparental. The Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) are diseases of abnormal genomic imprinting, involving chromosomes 7 (SSR) and 11 (SRS and BWS). The Isolated Hemihiperplasia (IHH) seems to correspond to a milder form of the SBW. In the present study, we performed an in silico scan to search for new microsatellites on chromosomes 7 and 11, and selected six tetra- and/or pentanucleotides on chromosome 7, and 12 on chromosome 11. The pattern of methylation in ICRs was verified by three different techniques: MS-MLPA, DESM-RT and a new strategy developed in this work called DESM-QFPCR. We evaluated 32 patients with BWS, HHI 16, with 20 SSR and their parents, when available, and one patient with apparently normal phenotype with karyotype 46, XX/46, XY and whose placenta showed placental mesenchymal dysplasia (PMD) which is associated with SBW. The new markers showed a high heterozygosity rate (average 70%), and absence of undesirable characteristics of dinucleotides, predominantly used for detection of DUP. Six markers spans genes controlled by the ICRs 1 and 2. The paternal UPD for chromosome 11 (UPDpat Cr11), all restricted to 11p15.5, was responsible for 13% of cases of HHI and 19% of the SBW. Structural changes were confirmed by quantitative SNaPshot sequencing of SNPs and MS-MLPA. One patient had paternal duplication encompassing both ICRs. A not previously described deletion in the gene CDKN1C was observed in one patient and her mother. For patients with DUPpat Cr11, microsatellites were investigated in 13 autosomes and sex chromosomes to detect wide mosaicism. Only patients with DMP showed mosaicism [androgenetic cells (25-30%) and biparental], suggesting double fertilization. In patients with SRS, ICR1 hypomethylation was observed in 25% of cases. For BWS, ICR1 hypermethylation and in ICR2 hypomethylation were observed 6% and 42% of cases, respectively. All cases with UPDpat Cr11 presented abnormal methylation in both ICRs. The (epi) genetic change frequencies were similar to those previously described in the literature for BWS, SRR andIHH. In the present work, we developed a new technique to study DNA methylation of ICRs and tested novel microsatellite markers in the 11p15 region, which showed high correlation of results, when compared with more traditional methods such as RT-DESM and MS-MLPA. The results show the complex etiology of these diseases and the molecular data are essential for appropriate patient and families genetic counseling.

O Hipogonadismo hipogonadotrófico isolado (HHI) congênito é uma síndrome clínica rara causada por defeito na produção ou secreção do hormônio liberador de gonadotrofinas (GnRH) pelo hipotálamo ou por resistência hipofisária à ação do GnRH. O HHI é mais prevalente em homens e cerca de 50% a 60% dos indivíduos afetados apresentam anosmia ou hiposmia associada, caracterizando a síndrome de Kallmann. Diversos genes já foram associados à patogênese do HHI congênito, porém, a maioria dos casos ainda permanece sem diagnóstico molecular definido. Até recentemente, a identificação das causas genéticas dos pacientes com HHI era realizada por sequenciamento de genes candidatos, empregando a técnica de Sanger. No entanto, com o número crescente de genes descritos nos últimos anos, esse processo vem se tornando impraticável. Novas metodologias de sequenciamento (sequenciamento paralelo em larga escala) foram desenvolvidas permitindo a genotipagem simultânea de diversas regiões, com maior velocidade e menor custo relativo. O atual projeto foi desenvolvido com o objetivo de rastrear genes candidatos em pacientes portadores de HHI congênito utilizando-se o sequenciamento paralelo em larga escala, visando ampliar o conhecimento genético do HHI. Realizamos o sequenciamento paralelo em larga escala (SPLE) de 130 pacientes com HHI congênito utilizando um painel contendo 36 genes relacionados ao HHI. Inicialmente, identificamos 104 variantes, potencialmente patogênicas em 77 pacientes (59,2%). Após a filtragem inicial, foi realizada uma análise individualizada de cada variante e com isso foram mantidos 41 (31,5%) pacientes com variantes classificadas como patogênicas ou provavelmente patogênicas. Os genes KAL1, FGFR1, CHD7 e GNRHR foram os mais frequentemente afetados. Esses resultados confirmam a importância dos genes classicamente associados ao HHI congênito. Destaca-se a alta prevalência de variantes no CHD7 (10,8%), gene bastante extenso, levando à dificuldade técnica de sequenciá-lo pelos métodos tradicionais, até então sem estudos nessa coorte. O CHD7 é um gene originalmente associado à complexa síndrome de CHARGE, porém, nos últimos anos vem sendo cada vez mais associados ao HHI congênito. Dentre os resultados, ressaltamos a identificação de uma mutação inédita no gene GNRH1, causa rara de HHI, e a identificação de variantes deletérias no gene IGSF10, recentemente descrito associado ao atraso puberal, mas sem papel claro no fenótipo de HHI, em dois pacientes que tiveram reversibilidade do hipogonadismo. Variantes provavelmente patogênicas em genes com poucas descrições ou até mesmo sem relatos de associação ao fenótipo de HHI (SPRY4, FLRT3, IGSF1, NSMF, SOX10 e OTX2) também foram identificadas nessa coorte, ampliando nosso conhecimento genético do HHI. A oligogenicidade, previamente descrita com prevalência de 2,5% a 7%, em nosso estudo esteve presente em 22% dos pacientes, demonstrando uma ampliação das descrições de oligogenicidade quando comparados aos estudos prévios utilizando somente a técnica de Sanger. A nova técnica de sequenciamento genético (SPLE), utilizada em nosso estudo, foi capaz de ampliar de 22% para 31,5% (41 em 130 pacientes) a porcentagem de pacientes com diagnóstico molecular definido, quando comparado aos dados prévios utilizando a técnica de Sanger, mostrando-se rápida, confiável e eficaz / Congenital isolated hypogonadotropic hypogonadism (IHH) is a rare condition caused by GnRH deficiency, due to defective hypothalamic gonadotropin-releasing hormone (GnRH) production or secretion, or by pituitary resistance to the GnRH action. Congenital IHH is more prevalent in men and about 50% to 60% of affected individuals present with associated anosmia or hyposmia, characterizing Kallmann\'s syndrome. Several genes have already been associated with the pathogenesis of congenital IHH, but most cases still remain without a molecular diagnosis. Until recently, identification of the genetic causes of IHH was performed by sequencing candidate genes using the Sanger technique. However, with the growing number of genes and the genetic complexity of IHH, it has become almost impossible to keep the screening of all candidate genes updated using the traditional techniques. The advent of next-generation sequencing (NGS) has allowed the simultaneous genotyping of several regions, faster and with lower relative cost. The present project was developed with the objective of tracking candidate genes in patients with congenital IHH using large-scale parallel sequencing, in aiming to increase the genetic knowledge of this rare condition. A total of 130 unrelated patients with IHH was studied by targeted NGS, using a panel containing 36 IHH associated genes. Initially, 104 potentially pathogenic variants were identified in 77 patients (59.2%). However, after an individualized analysis of each variant, the number of patients considered to carry pathogenic or probably pathogenic variants dropped to 41 (31.5%). The genes KAL1, FGFR1, CHD7 and GNRHR were the most frequently affected and these results confirm the importance of genes classically associated with IHH. It is noteworthy the high prevalence of variants in CHD7 (10.8%), a rather extensive gene, leading to technical difficulty of sequencing by traditional methods, which had not been studied in this cohort. CHD7 is the causative gene of CHARGE syndrome, however it has been recently identified in a growing number of congenital IHH patients with or without additional features of the syndrome. Among the results, we emphasize a novel mutation in the GNRH1 gene, a rare cause of IHH, and the identification of deleterious variants in the IGSF10 gene, recently associated with pubertal delay but without a clear role in the IHH phenotype, in two patients with reversible hypogonadism. Probably pathogenic variants in genes with few descriptions or even no reports of association with the IHH phenotype (SPRY4, FLRT3, IGSF1, NSMF, SOX10 and OTX2) were also identified in this cohort, increasing the genetic knowledge of IHH. Oligogenicity, previously described with a prevalence of 2.5% to 7%, was observed in 22% of our patients, demonstrating an increase in oligogenicity cases when compared to previous studies using only the Sanger sequencing. In conclusion, targeted NGS was able to increase the percentage of patients with molecular diagnosis from 22% to 31.5% in our cohort when compared to the previous data using the Sanger sequencing, and has been shown to be a fast, reliable and effective tool in the molecular diagnosis of congenital IHH

O objetivo deste trabalho foi aperfeiçoar o filtro lento em escala domiciliar (FLD) à realidade das comunidades isoladas brasileiras com o uso conjunto de pré-tratamento e pós-tratamento acessíveis. As espécies cactáceas Opuntia cochenillifera e Opuntia ficus indica foram avaliadas em ensaios de tratabilidade como coagulantes naturais em pó. A melhor condição para remoção de turbidez e cor aparente foi com 30 mg.L-1 de O. cochenillifera em pH sem a necessidade de correção (± 6,8). O pré-tratamento adaptado às comunidades isoladas utilizando o coagulante natural apresentou valores compatíveis de turbidez (7,83 ± 2,32 uT) para inserção aos filtros. Os FLDs em fluxo contínuo (taxa de filtração de 1,22 m³/m².dia) e em fluxo intermitente (taxa de filtração máxima de 2,79 m³/m².dia) demonstraram redução da turbidez, da cor aparente, dos coliformes totais e da Escherichia coli e produziram água filtrada com ausência de cistos de Giardia spp. e oocistos de Cryptosporidium spp. O FLD em fluxo contínuo teve a maior eficiência em relação à turbidez (± 81,2%); enquanto que o FLD operado em fluxo intermitente apresentou melhores eficiências para a remoção da cor aparente (48,4%), dos coliformes totais (2,60log ± 0,99) e da E. coli (2,86log ± 0,79). Os principais microrganismos atuantes no schmutzdecke dos FLDs foram ciliados (como, por exemplo, a Vorticella sp.), nematódeos e rotíferos. A esponja-vegetal (Luffa cylindrica) e o quiabo (Hibiscus esculentus) foram ponderados como desinfetantes alternativos para o pós-tratamento aos FLDs. As sementes da L. cylindrica e o fruto da H. esculentus foram avaliados através do preparo do extrato e as folhas da L. cylindrica através do preparo em pó. A melhor condição para inativação de E. coli foi com 1 g.L-1 do extrato de H. esculentus em pH sem a necessidade de correção (± 6,8) durante um tempo de contato de 30 min. Essa condição forneceu 1,12log (± 0,16) para a inativação de E. coli, contudo não afetou a viabilidade dos (oo)cistos de Giardia spp. e Cryptosporidium spp. Em ensaios toxicológicos com Chironomus xanthus, a água filtrada não apresentou toxicidade. / The aim of this study was to improve the household-scale slow sand filter (HSSF) to the reality of isolated Brazilian communities with combined use of pre-treatment and post-treatment approachable. Cacti species Opuntia cochenillifera e Opuntia ficus indica were evaluated in Jar tests as powder natural coagulants. The best condition for turbidity and apparent color removal was with 30 mg.L-1 of O. cochenillifera at pH without correction (± 6,8). Pre-treatment adapted to isolated communities using natural coagulant showed compatible turbidity values (7.83 ± 2.32 uT) for insertion into the filters. The HSSFs with continuous (filtration rate of 1,22 m³/m².day) and intermittent operation (maximum filtration rate of 2,79 m³/m².day) demonstrated reductions in turbidity, apparent color, total coliforms and Escherichia coli and produced filtered water without Giardia spp. cysts and Cryptosporidium spp. oocysts. The HSSF in continuous operation had the highest turbidity efficiency (± 81.2%); while the HSSF in intermittent operation had showed better efficiencies for the apparent color (48,4%), total coliforms (2,60log ± 0,99) and E. coli (2,86log ± 0,79). The main microorganisms active in the schmutzdecke were ciliates (for example, Vorticella spp.) nematodes and rotifers. Luffa cylindrica and Hibiscus esculentus were evaluated as alternative disinfectant for post-treatment to HSSF. L. cylindrica seed and H. esculentus fruit were tested as extract and L. cylindrica leave was tested powder. The best condition for E. coli inactivation was with 1 g.L-1 of H. esculentus extract at pH without correction (± 6,8) during 30 min. This condition provided 1,12log (± 0,16) for E. coli inactivation, however did not affect Giardia spp. cysts and Cryptosporidium spp. oocysts viability. In toxicological tests with Chironomus xanthus, the filtered water showed no toxicity.

Orientador: Mara Martins Jacomeli / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Educação / Made available in DSpace on 2018-08-22T14:27:45Z (GMT). No. of bitstreams: 1 Cardoso_MariaAngelica_D.pdf: 3012362 bytes, checksum: abe1b7b90e84ccee1e1bbbcafcc0b9cb (MD5) Previous issue date: 2013 / Resumo: Esta pesquisa tem por objeto a organização do trabalho didático nas escolas isoladas paulistas. Fundamentando-se no método histórico-crítico, estabeleceu a organização do trabalho didático como categoria de análise, objetivando analisar a história e a forma como as escolas isoladas se organizaram didaticamente, no período de 1893 a 1932. Ao partir da tese de que as escolas isoladas carregaram vestígios do método lancasteriano (ou mútuo), tornou-se necessário um recuo ao Período Imperial, aprofundando o estudo sobre as escolas de primeiras letras para, em seguida, investigar as escolas isoladas. Para tanto, foi realizada uma revisão bibliográfica tendo como referência teórica os trabalhos de Saviani (2008); Alves (2001, 2005); Neves (2003); Marcílio (2005); Bastos e Faria Filho (1999); Souza (1998, 2006, 2009); Reis Filho (1995); Infantosi (1983); Nagle (1976); Schelbauer (1998). Os documentos constantes no Arquivo do Estado de São Paulo (AESP) foram as fontes primárias consultadas. No primeiro período republicano, a necessidade de universalização da instrução pública levou à criação dos grupos escolares, que atendiam, inicialmente, os grandes centros urbanos; para os centros menores e bairros populosos, criaram-se as escolas reunidas. Nos bairros periféricos, nas vilas e áreas rurais foram adotadas as escolas isoladas cuja função era dar uma formação básica - leitura, escrita e as operações elementares da aritmética - à população pobre, residente nesses locais. A organização do trabalho didático, nessas escolas, se aproximava da organização adotada nas escolas de primeiras letras sob o método mútuo presentes no Período Imperial. A história das escolas isoladas está marcada por aspectos tanto de suas antecessoras, as escolas de primeiras letras, quanto da moderna escola burguesa. Funcionando em casebres, elas herdaram das escolas de primeiras letras, principalmente, o atendimento, em uma só sala, de crianças em diferentes níveis de adiantamento, o controle da presença, os registros administrativos; da escola burguesa, dentre outros aspectos, o controle e a divisão ordenada do tempo. / Abstract: This research has as object organization of the didactic work in isolated schools in São Paulo. Basing on the historical-critical method, established the organization of the didactic work as a category of analysis, in order to study the history and analyze how schools are organized didactically isolated in the period from 1893 to 1932. When from the thesis that the isolated schools carry traces of the lancasterian system (or mutual), it became necessary to a retreat Imperial Period, deepening the study of the schools of first letters to then to investigate the isolated schools. To this end, was conducted a bibliography review with reference to the theoretical work Saviani (2008), Alves (2001, 2005), Neves (2003); Marcílio (2005); Bastos and Faria Filho (1999); Souza (1998, 2006, 2009); Reis Filho (1995); Infantosi (1983), Nagle (1976); Schelbauer (1998). The documents in the Arquivo do Estado de São Paulo (AESP) were the primary sources consulted. In the first Republican period, the need for universal public education led to the creation of school groups, which met initially to large urban centers; for smaller centers and populous neighborhoods, schools were created together. In the suburbs, towns and rural areas were adopted isolated schools whose function was to provide a basic education - reading, writing and elementary arithmetic operations - to the poor population of these locations. The organization of the didactic work in these schools approached the organization adopted in schools of the first letters under the mutual method present in the imperial period. The history of isolated schools is marked by both aspects of its predecessors, the schools of first letters, as the modern bourgeois school. Working in hovels, they inherited the school of first letters mainly care in one room, children at different levels of play, controlling presence, administrative records; the bourgeois school, among other things, control and orderly division of time. / Doutorado / Filosofia e História da Educação / Doutora em Educação

In canonical boundary layer transition, a uniform laminar flow perturbed by 2-d T-S waves develops downstream into 3-d waves, which eventually break down with turbulent spots appearing. Previous experimental studies have established that this kind of development is absent, is by-passed, in transition induced by free-stream turbulence or surface roughness. However a common, characteristic feature of the late, three-dimensional stage is the prevalence of streamwise vorticity and streaks. Isolated and multiple streamwise vortices are present in both, canonical transition and bypass transition. This thesis de-scribes an experimental study of the late stages of boundary layer transition after a single or a pair of streamwise vortices have formed. The present work can be considered both as a study of transition induced by surface roughness and as a study of the late stages of transition in general. The experiments were made on a zero-pressure-gradient boundary layer in a low speed wind tunnel. Various hill configurations, mounted on a flat plate, were used to create isolated and multiple streamwise vortices. Particle image velocimetry (PIV) and hot-wire anemometry used for measurements. Numerical simulations of the initial laminar stage were carried out to understand the vortex formation at the edge of the hills. Computations have shown that the streamwise vorticity induced by the spanwise asymmetry of the hill rolls up into a single streamwise vortex. The streamwise vortex causes high speed fluid to be brought close to the wall and low speed fluid to move away. In turn, streamwise velocity profiles acquire inflections in both the spanwise and wall-normal directions. Previous studies have concluded that the inviscid instability of inflectional profiles are essential, or at least common, precursors to transition. Another view of the structure of bypass transition induced involves a secondary instability of streaks that can be sinuous or varicose. These two types follow from instabilities of the inflectional spanwise and wall-normal profiles of the streamwise velocity, respectively. However the present study confirms that the occurrence of inflections is not sufficient for transition. The first series of experiments were with smooth Gaussian shaped hills that spanned one-half of the tunnel. Two hill shapes were taken, steep and shallow. Isolated streamwise vortices formed by the side of the hill. Hill heights were less than that of the incoming boundary layer, and they were mounted within the subcritical part of the boundary layer. At low free stream speeds, streaks formed, with inflectional wall-normal and spanwise velocity profiles, but without effecting transition. The necessary conditions for inviscid instability Rayleigh’s inflexion-point theorem and Fjortoft’s theorem are satisfied for these low-speed non-transitional cases. Transition observed at higher speeds show two kinds of development. With the steep hill, the streamwise vortex is not too close to the plate and it exhibits oscillations over some distance before flow breaks down to turbulence; streamwise velocity signals exhibited the passage of a wave packet which intensified before break-down to turbulence. This dominant mode persists far downstream from the hill even while the flow breaks down and frequency content grows over a range of scales. With the shallow hill, the breakdown develops continuously without such a precursor stage; there was a broad range of frequencies present immediately downstream of the hill. For the steep hill the maximum fluctuation is observed on the upwash side of the vortex. With the shallow hill, the fluctuation level is maximum at the location between low and high speed streak. Features of breakdown to transition caused by these single streamwise vortices are found to be similar to those in transition by other causes such as surface roughness, freestream turbulence etc. With the steep hill, the growth of fluctuations(urms, the peak levels of streamwise velocity component fluctuations) is remarkably similar to that in the K-type transition. Unlike in freestream induced transition, the initial growth of u2 rms,max with downstream distance was not linear. Profiles of urms/urms,max vs. y/δ∗where δ∗,is the displacement thickness is partially matching with the optimal disturbances, for the steep hill case. The phase velocity matches as in previous measurements of roughness induced transition. The flow exhibits the breakup of a shear layer near the outer edge of the boundary layer into successive vortices. This breakdown pattern resembles to those in the recent numerical simulations. The passing frequency of these vortices scales with freestream velocity, similar to that in single-roughness induced transition. Quadrant analysis of streamwise and wall-normal velocity fluctuations show large ejection events in the outer layer. The difference in the route to transition between the steep and shallow hills was considered to the relative proximity of the initial streamwise vortex to the flat plate and its interaction with the wall. To examine this conjecture, two configurations were prepared to produce two types of counter-rotating streamwise vortices. One is a hill that span the tunnel except for a short gap, and the other, its complement, is a short span hill. The short-gap hill produce a pair of vortices with the common flow directed away from the wall. This resulted in a separation bubble that formed a short distance downstream and breakdown to turbulence. The short-span hill configuration seems to have a stabilizing effect. With the short gap hill, transition occurs for lower freestream speeds than with the isolated vortices considered before. Also, the breakdown location is further downstream when the gap is larger. The initial velocity profiles look similar for transitional and non-transitional flow cases, and are inflectional, which clearly indicates that inflectional instabilities are not effective here. A separation index was computed to identify unsteadiness of the separated flow region. The separation is itself steady, where as the reattachment is unsteady. Fluctuations grow near this reattachment zone. The unsteadiness of the reattachment coexists with the appearance of strong fluctuations and transition. It is likely that the this last stage of breakdown results when the shear layer, lifted up by the separation bubble, breaks down near the edge of the boundary layer and the consequent unsteadiness is in the reattachment also. Coherent cat’s-eye-like patterns were observed in a longitudinal, plane normal to the wall. With isolated vortices sinuous oscillations and with stream-wise vortex pairs varicose oscillations were observed in wall-parallel planes. In both cases passing frequency of these vortices scales with freestream velocity. Λ-type vortices were identified in spanwise plane with counter-rotating legs. These experiments have identified some possible roles of streamwise vortices in the last stages of boundary layer transition. Vortices may undergo their own instability in the background shear layer, evident as oscillations, if they are not too close to the wall. Otherwise the breakdown is without such a stage. Wall interaction of these vortices seems to be a necessary last stage. Inflectional instability is not indicated. Wall interaction that results in separation results in break-down in the unsteady reattachment zone. Breakdown coexists with the reattachment and not at separation, even though it may be the separating shear layer that breaks down.

Creative ways of utilising renewable energy sources in electricity generation especially in remote areas and particularly in countries depending on imported energy, while increasing energy security and reducing cost of such isolated off-grid systems, is becoming an urgently needed necessity for the effective strategic planning of Energy Systems. The aim of this research project was to design and implement a new decision support framework for the optimal design of hybrid micro grids considering different types of different technologies, where the design objective is to minimize the total cost of the hybrid micro grid while at the same time satisfying the required electric demand. Results of a comprehensive literature review, of existing analytical, decision support tools and literature on HPS, has identified the gaps and the necessary conceptual parts of an analytical decision support framework. As a result this research proposes and reports an Iterative Analytical Design Framework (IADF) and its implementation for the optimal design of an Off-grid renewable energy based hybrid smart micro-grid (OGREH-SμG) with intra and inter-grid (μG2μG & μG2G) synchronization capabilities and a novel storage technique. The modelling design and simulations were based on simulations conducted using HOMER Energy and MatLab/SIMULINK, Energy Planning and Design software platforms. The design, experimental proof of concept, verification and simulation of a new storage concept incorporating Hydrogen Peroxide (H2O2) fuel cell is also reported. The implementation of the smart components consisting Raspberry Pi that is devised and programmed for the semi-smart energy management framework (a novel control strategy, including synchronization capabilities) of the OGREH-SμG are also detailed and reported. The hybrid μG was designed and implemented as a case study for the Bayir/Jordan area. This research has provided an alternative decision support tool to solve Renewable Energy Integration for the optimal number, type and size of components to configure the hybrid μG. In addition this research has formulated and reported a linear cost function to mathematically verify computer based simulations and fine tune the solutions in the iterative framework and concluded that such solutions converge to a correct optimal approximation when considering the properties of the problem. As a result of this investigation it has been demonstrated that, the implemented and reported OGREH-SμG design incorporates wind and sun powered generation complemented with batteries, two fuel cell units and a diesel generator is a unique approach to Utilizing indigenous renewable energy with a capability of being able to synchronize with other μ-grids is the most effective and optimal way of electrifying developing countries with fewer resources in a sustainable way, with minimum impact on the environment while also achieving reductions in GHG. The dissertation concludes with suggested extensions to this work in the future.

While the formalism of isolated horizons is known for some time, only quite recently the near horizon solution of Einstein's equations has been found in the Bondi-like coordinates by Krishnan in 2012. In this framework, the space-time is regarded as the characteristic initial value problem with the initial data given on the horizon and another null hypersurface. It is not clear, however, what ini- tial data reproduce the simplest physically relevant black hole solution, namely that of Kerr-Newman which describes stationary, axisymmetric black hole with charge. Moreover, Krishnan's construction employs the non-twisting null geodesic congruence and the tetrad which is parallelly propagated along this congruence. While the existence of such tetrad can be easily established in general, its explicit form can be very difficult to find and, in fact it has not been provided for the Kerr-Newman metric. The goal of this thesis was to fill this gap and provide a full description of the Kerr-Newman metric in the framework of isolated horizons. In the theoretical part of the thesis we review the spinor and Newman-Penrose formalism, basic geometry of isolated horizons and then present our results. Thesis is complemented by several appendices.

La production de photons de grande impulsion transverse lors des collisions proton-proton (p-p), est décrite par la chromodynamique quantique perturbative (pQCD). Parmi ces photons, ceux issus directement d’un processus partonique énergétique (appelés photons directs) sont particulièrement intéressants car leur mesure permet de tester précisément les prédictions de la pQCD et offre la possibilité de mieux contraindre les fonctions de structure du proton. Ce travail de thèse a pour objectif l’étude et la mesure des photons directs produits dans les collisions p-p à 7 TeV avec le détecteur ALICE. Le calorimètre électromagnétique d’ALICE (EMCal), est utilisé pour réaliser cette mesure qui s’appuie notamment sur l’utilisation d’une procédure d’isolement permettant de réduire le bruit de fond provenant des autres modes de production (fragmentation, décroissance). Les aspects relatifs à la qualité des données dans EMCal, à l’identification des photons, mais aussi ceux liés à la correction du spectre ou encore à sa normalisation sont mis en avant. Finalement, la première section efficace de photons isolés mesurée dans les collisions p-p à 7 TeV avec le détecteur ALICE est présentée et comparée avec les prédictions théoriques avant d’être mise en regard avec les résultats issus des autres grandes expériences du LHC. / The high transverse momentum photon production inproton-proton collisions (p-p) is described by perturbativequantum chromodynamics (pQCD). Among thesephotons, those produced directly by an energetic partonicinteraction (called direct photons) are of great interestsince their measurement allows to test pQCDpredictions and it allows also the constraint of protonstructure functions. The work of this thesis aims atstudying and measuring direct photons produced in p-pcollisions at 7 TeV with the ALICE detector. The ALICEelectromagnetic calorimeter (EMCal) is used to achievethis measurement which is based on an isolation procedurethat allows to reduce background coming fromother photon production modes (fragmentation, decay).Multiple aspects like EMCal data quality, photon identificationas well as spectrum correction and its normalizationare highlighted. Finally, the first isolated promptphoton cross-section measured with ALICE detector ispresented, compared to theoretical predictions and tothe last results from other LHC experiments.

Introduction : La perfusion pulmonaire isolée (PPI) est une technique expérimentale dont l’objectif est d’administrer de fortes doses de chimiothérapie dans le poumon sans effets secondaires systémiques. Cette thèse s’est déroulée en trois étapes : première étape, déterminer in vitro la chimiothérapie la plus efficace en 30 minutes sur les cellules de cancers colo-rectaux (CCR). Deuxième étape, mettre au point la technique de PPI chez le porc. Troisième étape, mener une étude d’escalade de dose de chimiothérapie chez le porc en PPI.Méthodes : Première étape, des tests de cytotoxicité in vitro ont été menés sur un panel de cellules d’adénocarcinome colique humain avec les drogues de chimiothérapie les plus efficaces dans les essais cliniques. Deuxième étape, des porcs étaient traités par perfusion de chimiothérapie dans le poumon gauche isolé de la circulation générale pendant 30 minutes puis maintenus en vie pendant un mois. Troisième étape, les doses de chimiothérapie injectés étaient augmentées par palier jusqu’à obtenir une toxicité aigüe ou le décès des animaux.Résultats : La gemcitabine (GEM) fut la drogue ayant la plus grande efficacité anti-tumorale pour un traitement de 30 min. La PPI de GEM permit d’obtenir des concentrations élevées de GEM dans le parenchyme pulmonaire et la survie des animaux pendant un mois. Il n’existait pas de fuites systémiques de GEM. L’augmentation des doses de GEM permis de déterminer la dose maximale toxique à 320 mg et la toxicité limitant la dose à 640 mg. Conclusions : La technique de PPI avec la GEM est une technique sûre et reproductible permettant d’obtenir de fortes concentrations de GEM dans le parenchyme pulmonaire. / Introduction: The isolated lung perfusion (ILP) is an experimental technique which main objective is to deliver high dose of cytotoxic agent to the lung tissue without systemic exposure. The thesis took place in three stages: first stage, setting in vitro the chemotherapy the most efficient against colorectal cancer (CCR) cells in 30 min. Second stage, develop the ILP technique in a pig model. Third stage, lead a dose escalation study with chemotherapy by ILP.Methods: First stage, efficacy of various cytotoxic molecules against a panel of human CCR cell lines was tested in vitro after a 30-minute exposure. Second stage, pigs were treated with chemotherapy delivered by ILP during 30 minutes and kept alive during a month. Third stage, chemotherapy doses were increase in order to obtain acute toxicity or death of animals.Results: Gemcitabine (GEM) was the most efficient drug against CCR cells in 30 minutes. ILP with GEM permit to maintain high concentration in the lung parenchyma and pigs survival during one month. No systemic leaks were detected. Dose increase of GEM conduct to determine the maximal tolerated dose of GEM by ILP to 320 mg. Conclusions: ILP with GEM is a safety and reproducible technique allowing high GEM concentrations in the lung tissue.

Orientador: Carlos Raimundo Ferreira Grosso / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T10:13:53Z (GMT). No. of bitstreams: 1 Costa_BiancaSouzada_M.pdf: 3645103 bytes, checksum: ee9884c2d54163da7814b0ea032a53c5 (MD5) Previous issue date: 2014 / Resumo: O objetivo deste trabalho foi produzir partículas de pectina e alginato por gelificação iônica, com posterior recobrimento com isolado proteico de soja (IPS), gelatina de pele de tilápia (GPT) e com a mistura dessas duas proteínas (IPS:GPT), avaliando suas características físico-químicas e seu comportamento frente a diferentes tratamentos. No estudo preliminar foram analisadas as condições que promovessem a carga elétrica livre que otimizassem a interação entre os polissacarídeos e proteínas, formando complexos insolúveis. A partir desses ensaios foram estabelecidas as seguintes proporções de polissacarídeo: proteína e valores de pH: 1:2 em pH4 para recobrimento com gelatina de pele de tilápia, e 1:0,75 em pH3 para interação com o isolado proteico de soja e para a mistura das duas proteínas. Foram testados 4 níveis de proteína em solução ( 1, 2, 4 e 8 %) para recobrimento das partículas de pectina e alginato. As partículas obtidas foram caracterizadas pelo teor de umidade, conteúdo proteico adsorvido e pela sua morfologia. A partir deste estudo preliminar foi selecionada a concentração de 8% de proteína em solução, devido à produção de partículas com alto teor proteico. Estas partículas foram avaliadas com relação à sua estabilidade frente a variações de pH, diferentes concentrações de NaCl e sob simulação das condições gastrointestinais in vitro. Utilizando a concentração de 8% de proteína em solução foram obtidos altos valores de adsorção proteica, resultando no percentual de proteína de 61,87%, 47,61% e 52,06% para as partículas recobertas com GPT, IPS e IPS:GPT, respectivamente. A variação de pH e das concentrações de sal influenciaram na solubilidade da camada proteica, apresentando uma maior solubilidade em condições de extrema acidez (pH 1) e a medida em que aumentava a concentração de sal. Nas faixas de pH (1 a 7) e nas concentrações de NaCl (0 a 584 mM) estudadas, independente do valor da solubilidade proteica obtida, todas as partículas permaneceram íntegras. No ensaio gastroentérico in vitro, as partículas de pectina e alginato (PEC:ALGPart) sem recobrimento e as recobertas com gelatina de pele de tilápia (GPTPart) foram resistentes as condições gástricas e entéricas, permanecendo íntegras. As partículas recobertas com isolado proteico de soja (IPSPart) e com a mistura de proteínas (IPS:GPTPart), foram resistentes às condições gástricas, porém desintegraram-se em meio intestinal, liberando o material encapsulado / Abstract: The aim of this work was to produce particles of pectin and alginate by ionic gelation with subsequent coating with isolated soy protein (IPS), tilapia skin gelatin (GPT), and a mixture of these two proteins (IPS:GPT), evaluating their physico-chemical characteristics and behavior to different treatments. In the preliminary study, the conditions that promote the balance of free electrical charge due the interaction between polysaccharides and proteins were analyzed. From these tests the following proportions of polysaccharide:protein and pH values were established: 1:2 at pH 4 for covering with tilapia skin gelatin, and 1:0.75 at pH3 for interaction with the isolated soybean protein and mixing of the two proteins. Four protein levels were tested (1, 2, 4 and 8%) in solutions for coating the particles of pectin and alginate. The particles obtained were characterized by moisture content, adsorbed protein content and their morphology. From this preliminary study it was selected the concentration of 8% protein solution, due to the production of particles with high protein content. These particles were evaluated for their stability against pH changes, different concentrations of NaCl and simulation under the simulated gastrointestinal conditions. Using a concentration of 8% protein solution high levels of protein adsorption were obtained, resulting in percentage of protein adsorbed of 61.87%, 47.61% and 52.06% for the coated particles GPT, IPS and IPS: GPT, respectively. The variation of pH and salt concentrations influence the solubility of the protein layer having a higher solubility in conditions of extreme acidity (pH 1) and the extent to which increased salt concentration. In the pH ranges and concentrations of NaCl studied, regardless of the amount of protein solubility obtained, all the particles remained intact. In the gastrointestinal assay, pectin and alginate particles (PEC: ALGPart) uncoated and coated particles with tilapia skin gelatin (GPTPart) were resistant to the gastric and enteric conditions, remaining intact. The coated particles with isolated soy protein (IPSPart) and the protein mixture (IPS:GPTPart) were resistant to gastric conditions, but disintegrated in the intestinal environment, releasing the encapsulated material / Mestrado / Nutrição Experimental e de Alimentos / Mestra em Alimentos e Nutrição