O objetivo deste trabalho é estudar o nível de organização dos cristalitos de surfactantes aniônico, catiônico e zwiteriônico com o aumento na densidade destas moléculas sobre um cristal de germânio. As análises foram realizadas por espectroscopia de infravermelho com transformada de Fourier acoplada à técnica de reflexão total atenuada (FTIR-ATR). Este estudo apresenta importantes aspectos na organização do dodecilsulfato de sódio (SDS), do N-hexadecil-N,N-dimetil-3-amônio-1-propanosulfato (HPS), do brometo de hexadeciltrimetilamônio (CTAB) e do brometo de dodeciltrimetilamônio (DTAB). No SDS é observado um deslocamento de 1.7 cm-1 para valores de maior frequência na banda de estiramento assimétrico do CH2, vass (CH2), enquanto que é observado um deslocamento de 0.9 cm-1 na banda de estiramento simétrico, vsim (CH2). Este deslocamento para valores de maior frequência nas bandas de estiramento está associado com um aumento na desorganização da cadeia alifática com o aumento na densidade de moléculas sobre o elemento de ATR. A banda de deformação angular do CH2, δ(CH2), apresenta um valor em 1468 cm-1 que é também um indicativo de desorganização. No CTAB não é observado variações acentuadas nos valores das frequências vibracionais. Na banda vass (CH2) do DTAB é observado um deslocamento de 4.5 cm-1 para valores de menor frequência. Embora seja observado valores próximos de 2920 cm-1 para a banda vass (CH2), que é um indicativo do estado líquido de surfactantes, o que é observado nesse estudo são cristalitos de DTAB. O deslocamento da banda vsim (CH2) do DTAB é da ordem de 2 cm-1. Estas mudanças nas bandas vass (CH2) e vsim (CH2) são um indicativo da diminuição nas conformações gauche e um aumento nas conformações trans ao longo da cadeia alifática. O valor da freqüência em torno de 1472 cm-1 para a banda δ(CH2) é também um indicativo de uma maior organização na cadeia de CH2 do DTAB. Para o HPS é observado um deslocamento de 2.6 e 2.7 cm-1 para valores de maior frequência nas bandas vass (CH2) e vsim (CH2), respectivamente. A banda δ(CH2) do HPS apresenta um deslocamento de 4 cm-1 para valores de maior frequência. A variação nas bandas vass (CH2), vsim (CH2), e δ(CH2) ressalta o aumento na desorganização da cadeia alifática com o aumento na densidade de moléculas de HPS sobre o germânio. / The objective of this work is study the order level of anionic, cationic and zwitterionic surfactants with the increase of their density packing on the surface of a germanium element. The analyses were performed by a Fourier transform infrared-attenuated total reflection spectroscopy. This study shows important insights on the conformational order of sodium dodecyl sulfate (SDS), N-hexadecyl-N-N -dimethyl-3-ammonio-1-propane-sulfonate (HPS), hexadecyl-trimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB). It is observed a shift of 1.7 and 0.9 cm-1 to higher frequency values of the CH2 asymmetric (vass (CH2)) and symmetric (vsim(CH2)) stretching bands for the SDS molecules, respectively. The latter shift to higher frequency values is associated with the disorder of the aliphatic chain due to the increase of density packing of SDS molecules on the germanium element. The CH2 scissoring band [δ (CH2)] shows a value in 1468 cm-1, which is also an indicative of conformational disorder. It is not observed any accentuated change on the vibrational frequency values of the CTAB molecules. The vass (CH2) band of the DTAB molecules is shifted 4.5 cm-1 to lower frequency values. Although it is observed values near 2920 cm-1 for the vass(CH2) band, indicating a surfactant liquid phase, it is shown in this study that there are indeed DTAB crystallites. The shift of DTAB vsim(CH2) band is around 2 cm-1. These changes in vass(CH2) and vsim(CH2) band are an indicative of a decrease in gauche conformations and an increase in all-trans conformations over the aliphatic chain. The frequency value around 1472 cm-1 for the δ(CH2) band is also an indicative of the order in CH2 chain of DTAB. It is observed a shift of 2.6 and 2.7 cm-1 to higher frequency values of vass (CH2) and vsim(CH2) bands of the HPS molecule, respectively. The δ(CH2) band of the HPS molecule presents a shift of 4 cm-1 to higher frequency values. These variations in vass (CH2), vsim(CH2), and δ(CH2) bands stand out the disorder of the aliphatic chain due to the increase of the density packing for the HPS molecules on the germanium surface.

Submitted by Michele Mologni (mologni@unoeste.br) on 2018-08-08T17:41:50Z No. of bitstreams: 1 Luís Fernando Xavier Gasqui.pdf: 694924 bytes, checksum: 34ee156d2e9833414ddbea664e5394bb (MD5) / Made available in DSpace on 2018-08-08T17:41:50Z (GMT). No. of bitstreams: 1 Luís Fernando Xavier Gasqui.pdf: 694924 bytes, checksum: 34ee156d2e9833414ddbea664e5394bb (MD5) Previous issue date: 2018-06-28 / The present research aims to stablish DRIS guidelines for sugarcane plantation based on Total Recoverable Sugars (ATR), through the study of the relation between nutrients and quality as well as their interactions that influence ATR production at sugarcane plantation, like some of the variable able to affect them. Were used foliar analysis results for the nutrients N, P, K, Ca, Mg, S, B, Cu, Fe, Mn and Zn with the productivity of 80 stands which belonged to Maracaí database, an area in the São Paulo State countryside, comprising a total area of approximately 1.020 hectares. These stands presented 4 different genetic materials, RB 72454, RB 845210, SP 813250 and RB 935744. The foliar samples were collected in the months of April to June 2012.It was used, for the formation of the database, the division in 7 ATR productive levels ≥150, ≥152.5, ≥155, ≥157.5, ≥160, ≥162.5, ≥165 Kg.ton-1, for high productivity, with the establishment of all the binary relations among the nutrients studied, then it was averaged the rate, the standard deviation and the variation of each nutrient concentration, as well as the relation among them, in each sub-population, being kept 55 relations that presented the biggest ratio in the variation among the sub-population. It was calculated the index for each nutrient and each population IBN. So, it was defined the productivity of 160 Kg.ton-1 as an index to be used because it has been the one which has presented the best relationship between IBN and the analyzed productivities. It was verified that, among all the analyzed samples, the scarcest were Ca, S, B and Cu. Referring to the nutrients P, K, Fe, Mn, the levels haven’t been different to the existing standards, however, with regard to the others, it was observed in this study, that they have been below existing standards. / O presente trabalho têm por objetivo estabelecer normas DRIS para a cultura da cana-de-açúcar fundamentado nos Açúcares Totais Recuperáveis (ATR), através do estudo da relação existente entre nutrientes e a qualidade bem como interações entre eles que influenciam a produção de ATR na cultura da cana-de-açúcar, bem como algumas das variáveis capazes de afetá-los. Utilizou-se resultados das análises foliares para os nutrientes N, P, K, Ca, Mg, S, B, Cu, Fe, Mn e Zn com as produtividades de 80 talhões pertencentes ao banco de dados referente a região de Maracaí-SP, compreendendo uma área total de aproximadamente 1.020 ha. Estes talhões expressaram 4 diferentes materiais genéticos, RB 72454, RB 845210, SP 813250 e RB 935744. As amostras foliares foram coletadas nos meses de abril a junho do ano de 2012. Utilizou-se para a formação do banco de dados a divisão em 7 níveis produtivos de ATR ≥150, ≥152.5, ≥155, ≥157.5, ≥160, ≥162.5, ≥165 Kg.ton-1, para altas produtividades, com o estabelecimento de todas as relações binárias entre os nutrientes estudados, então calculou-se a média, o desvio padrão e a variância para cada concentração de nutriente, como também para as relações entre estes, em cada sub-população, sendo mantidas 55 relações, que apresentaram a maior razão na variância entre as sub-populações. Calculou-se o índice de cada nutriente e o IBN de cada população. Definiu-se assim a produtividade de 160 Kg.ton-1 como índice a ser usado por ser o que apresentou melhor correlação entre o IBN e as produtividades analisadas. Verificou-se que dentre as amostras analisadas os elementos mais escassos foram Ca, S, B e Cu. Para os nutrientes P, K, Fe, Mn os teores não diferem dos padrões existentes, porém para os demais, observou-se no estudo, que ficaram abaixo dos padrões existentes.

Ataxia telangiectasia and Rad3-related protein (ATR) is a serine/threonine-protein kinase of the PI3K family and is well known for its key role in regulating DNA damage responses in the nucleus. In addition to its nuclear functions, ATR also was found to be a substrate of the prolyl isomerase Pin1 in the cytoplasm where Pin1 isomerizes cis ATR at the Ser428-Pro429 motif, leading to formation of trans ATR. Cis ATR is an antiapoptotic protein at mitochondria upon UV damage. Here we report that Pin1’s activity on cis ATR requires the phosphorylation of the S428 residue of ATR and describe the molecular mechanism by which Pin1-mediated ATR isomerization in the cytoplasm is regulated. We identified protein phosphatase 2A (PP2A) as the phosphatase that dephosphorylates Ser428 following DNA damage. The dephosphorylation led to an increased level of the antiapoptotic cis ATR (ATR-H) in the cytoplasm and, thus, its accumulation at mitochondria via binding with tBid. Inhibition or depletion of PP2A promoted the isomerization by Pin1, resulting in a reduction of cis ATR with an increased level of trans ATR. We conclude that PP2A plays an important role in regulating ATR’s anti-apoptotic activity at mitochondria in response to DNA damage. Our results also imply a potential strategy in enhancing cancer therapies via selective moderation of cis ATR levels.

Peptidyl-prolyl isomerization is an important post-translational modification of protein because proline is the only amino acid that can stably exist as cis and trans, while other amino acids are in the trans conformation in protein backbones. This makes prolyl isomerization a unique mechanism for cells to control many cellular processes. Isomerization is a rate-limiting process that requires a peptidyl-prolyl cis/trans isomerase (PPIase) to overcome the energy barrier between cis and trans isomeric forms. Pin1, a key PPIase in the cell, recognizes a phosphorylated Ser/Thr-Pro motif to catalyze peptidyl-prolyl isomerization in proteins. The significance of the phosphorylation-dependent Pin1 activity was recently highlighted for isomerization of ATR (ataxia telangiectasia- and Rad3-related). ATR, a PIKK protein kinase, plays a crucial role in DNA damage responses (DDR) by phosphorylating hundreds of proteins. ATR can form cis or trans isomers in the cytoplasm depending on Pin1 which isomerizes cis-ATR to trans-ATR. Trans-ATR functions primarily in the nucleus. The cis-ATR, containing an exposed BH3 domain, is anti-apoptotic at mitochondria by binding to tBid, preventing activation of pro-apoptotic Bax. Given the roles of apoptosis in many human diseases, particularly cancer, we propose that cytoplasmic cis-ATR enables cells to evade apoptosis, thus addicting cancer cells to cis-ATR formation for survival. But in normal DDR, a predominance of trans-ATR in the nucleus coordinates with a minimal level of cytoplasmic cis-ATR to promote DNA repair while preventing cell death; however, cells can die when DNA repair fails. Therefore, a delicate balance/equilibrium of the levels of cis- and trans-ATR is required to ensure the cellular homeostasis. In this review, we make a case that this anti-apoptotic role of cis-ATR supports oncogenesis, while Pin1 that drives the formation of trans-ATR suppresses tumor growth. We offer a potential, novel target that can be specifically targeted in cancer cells, without killing normal cells, to significantly reduce the adverse effects usually seen in cancer treatment. We also raise important issues regarding the roles of phosphorylation-dependent Pin1 isomerization of ATR in diseases and propose areas of future studies that would shed more understanding on this important cellular mechanism.

Les ondes térahertz s’étendent de 0.1 à 10x1012 Hz, à la frontière entre les domaines de l’optique et des radiofréquences. Cette position intermédiaire originale en a longtemps rendu l’accès difficile : les technologies térahertz n’ont pris leur essor qu’au cours des années 90. Le domaine n’a pas encore atteint la maturité des domaines des microondes ou de l’infrarouge qui le jouxtent. Cependant, les motivations exploratoires sont fortes, de par la sensibilité spectroscopique du térahertz aux états moléculaires (rotationnels, vibrationnels..) et aux liaisons faibles établies dans et entre les molécules. Dans le cas des objets biologiques, le térahertz est particulièrement sensible à l’eau : sa quantité, son état physico-chimique et ses solutés.Nous avons mis en œuvre un montage d’imagerie en réflexion interne totale atténuée (ATR) pour pouvoir distinguer des cellules vivantes de leur milieu physiologique. Au cours de ce travail, le montage d’imagerie ATR a été caractérisé théoriquement, puis expérimentalement. La première démonstration de l’origine du contraste sur ces images térahertz a été réalisée. Il provient du contenu intracellulaire, plus spécifiquement des protéines et peptides dissouts dans le cytoplasme.Une analyse fine des mécanismes sous-jacents à la nature protéique du contraste térahertz a également été développée. Elle donne accès à des informations spectroscopiques inédites sur l’eau, les protéines dissoutes et la couche de solvatation les entourant.Mettant à profit cette compréhension de notre montage térahertz, nous l’avons proposé comme outil non invasif de suivi quantitatif de la perméabilisation de cellules en conditions physiologiques. Lors de la perméabilisation, augmentation des transferts moléculaires à travers la membrane, notre outil permet de quantifier le passage des peptides et protéines. La perméabilisation de cellules vivantes a une gamme d’application vaste, de l’entrée de fluorochromes pour l’imagerie ou de médicaments à la thérapie génique. Afin d’assurer ces passages à travers la membrane des cellules, il est nécessaire d’altérer ses propriétés, sans pour autant compromettre la viabilité cellulaire. L’étude de deux types de perméabilisation avec notre outil térahertz est proposée : la perméabilisation chimique et l’électroporation. Dans les deux cas, des mécanismes d’effet dose ont été caractérisés quantitativement. Notre outil térahertz a démontré de grands avantages devant les méthodes actuellement utilisées pour quantifier ces dynamiques de perméabilisation et en caractériser la réversibilité. / Lying between 0.1 to 10x1012 Hz, the terahertz radiation occupies a middle ground between microwaves and infrared light waves, sometimes named “the terahertz gap” for technologies relevant to generation and detection have only risen at the beginning of the 90’s and aren’t fully developed yet. Nevertheless, there are strong exploratory incentives because of terahertz spectroscopic sensitivity to molecular states (rotational, vibrational…) and weak bounds in and between molecules. In the case of biological object, terahertz waves are especially sensitive to water: its quantity, physico-chemical state and solutes. We implemented an Attenuated Total internal Reflection (ATR) imaging setup in order to distinguish live cells from their physiological bathing medium. Throughout this work, we characterized both experimentally and experimentally the ATR setup. The first demonstration of the contrast origin in the terahertz images obtained was done. It arises from the intracellular content, more specifically the proteins and peptides dissolved in the cytoplasm.A precise analysis of the underlying mechanism of this proteinaceous terahertz contrast has also been developed. It gives access to original spectroscopic information about water, dissolved proteins and the hydration shell around them.Taking advantage of our whole setup comprehension, we proposed it as a non-invasive tool for quantitative live-cell permeabilization assessment in physiological conditions. During permeabilization, aka increased molecular transfers through the cell membrane, our tool allows to quantify the transfer of peptides and proteins. Live-cell permeabilization has a large application range, from fluorochrome entry in imaging, to drugs or gene therapy. In order to ensure molecules crossing the cell membrane, it’s necessary to alter its properties without compromising cell viability.A study of two permeabilization methods is proposed: chemical permeabilization and electroporation. In both cases dose effect mechanisms were quantitatively characterized. Our terahertz tool demonstrated great advantages over classical permeabilization quantification methods and permeabilization reversibility assessment methods.

High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB). This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells. We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dovitinib (DOV), to increase GB cell death. We show that DOV downregulates key BER proteins, attenuates pSTAT3-coordinated Lin28A and HMGA2 expression. Our results suggest that a sequential therapeutic strategy of pretreating GB cells with DOV followed by a sequence of TMZ and DOV diminishes TMZ resistance and enhances the ability of TMZ to induce GB cell death. Overall, we identified HMGA2 as a multifunctional survival factor in human cancer cells and showed that targeting HMGA2 is a valid strategy to combat HMGA2+ cancer cells. / February 2016

Demonstrou-se pela primeira vez que é possível determinar surfactantes e água em formulações de Sabonetes Líquidos (SL) e Shampoos (SH), direta e simultaneamente pela técnica de infravermelho por transformada de Fourier (FTIR) acoplada à uma cela de reflectância total atenuada (ATR). Tradicionalmente, a principal aplicação do infravermelho médio (2,50 - 15,0 µm) é a identificação de compostos orgânicos. O desenvolvimento de novos acessórios, principalmente de ATR, os avanços na área de microinformática e de métodos quimiométricos, vem viabilizando as análises quantitativas rápidas com excelentes resultados mesmo em meio aquoso. A determinação simultânea de surfactantes em formulações por FTIR-ATR é investigada em detalhes nesta tese. Dentre os surfactantes utilizados, o Lauril Éter Sulfato de Sódio (LESS) e a Cocoamidopropil Betaína (CAPB) são comuns em ambas as formulações, sendo a Coco Dietanolamida (CDEA) empregada em shampoo e o Alquilpoliglicosídeo (APG) em sabonete líquido. Espectros de absorbância de amostras padrão e de verificação foram adquiridos na região do infravermelho médio (800-1600 e 1900-3000 cm-I). Para a regressão de mínimos quadrados clássicos (CLSR), selecionou-se 200 números de onda, enquanto que para a regressão de mínimos quadrados inversos (ILSR), apenas 10. Nas regressões de componentes principais (PCR) e de mínimos quadrados parciais (PLSRI e PLSR2), utilizaram-se de 300 à 1100 números de onda. Dois conjuntos de amostras padrão foram preparados, o primeiro, contendo 27 misturas padrão, foi estudado somente pelos métodos CLSR e ILSR, enquanto que o segundo conjunto, contendo 48 amostras padrão, foi avaliado por todos os métodos mencionados acima. A seleção das regiões de quantificação favoreceu números de onda dos componentes minoritários CAPB, APG e CDEA e resultados satisfatórios foram encontrados para 18 amostras de shampoo e sabonete líquido. Interferentes como NaCl e perfume foram incluídos no segundo conjunto e os métodos PCR e PLSR proporcionaram melhores resultados. Os erros relativos (RSEP%) para água (correspondendo a 84-88% do produto) e LESS (6-10%) não excederam 1%; para CAPB (<3%) e CDEA (<2%), o RSEP% situou-se entre de 2-4% e para APG (<3%), não excedeu 5%. Avaliações do processo de normalização, repetibilidade, vulnerabilidade a interferentes (perfumes), redução no número de padrões de calibração foram conduzidos, encontrando-se resultados satisfatórios para todos os casos, com erro relativo inferior à 5,0%. Um dispositivo simples para injeção direta da amostras no acessório de ATR foi construído, permitindo aumentar a freqüência analítica de 20 para 60 análises por hora. / It is demonstrated for the first time that the principal constituents of a shampoo as well as of a liquid soap -three surfactants and water- can be determined directly, simultaneously and quickly in undiluted samples by Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy in the middle infrared region, despite the broad absorption bands ofthe solvent. The main application of the middle infrared (2,50 - 15,0 µm) was the identification of organic compounds. The development of new accessories, such as ATR, and the advance of computers and chemometrics, extended the technique to quantitative analysis, with excellent results. The simultaneous determination of surfactants in shampoos and liquid soap formulations by FTIRA TR is investigated in detail in this thesis. Two of the surfactants, sodium lauryl ether sulfate (LESS) and cocoamidopropyl betaine (CAPB) are common to both formulations; alkylpolyglucoside (APG) is the third surfactant of the liquid soap and cocodiethanolamide (CDEA), the corresponding ingredient of the shampoo. Absorbance data of the undiluted verification samples and calibration standards was collected in the middle infrared region of the spectrum (800-1600 and 1900-3000 cm-1). Five methods of multivariate quantification were compared: Classical Least Squares Regression (CLSR), where absorbance data measured at 200 wavenumbers was processed, Inverse Least Squares Regression (ILSR), were data at 10 selected wavenumbers was analyzed, Principal Components Regression (PCR), and Partial Least Squares Regression, which has two different approaches, PLSR1 and PLSR2. Two sets of standard samples were prepared for the method calibration. The first one, consisting of 27 standard mixtures, was evaluated by the CLSR and ILSR methods; a second set, with 48 standards, was evaluated by all the methods mentioned above. Potential interfering, such as NaCI and perfume, were inc\\uded in this second set. By favoring wavenumbers where absorption bands of the minor components (CAPB in both formulations, APG in the liquid soap and CDEA in the shampoo) are more intense, good results were obtained for 18 simulated samples of shampoo and 18 samples of liquid soap. For the second set, PCR and PLSR methods were most favorable; relative errors (RSEP%) for water (major component, 84-88%) and LESS (6-10%) did not exceed 1%; for CAPB (<3%) and CDEA(<2%), RSEP% of 2-4% were observed, and for APG(<3%), 5% was not exceeded. Tests for repeatability, normalization performance evaluation, effect of interferents presented favorable results. A simple device for direct sample injection was designed and evaluated. It permitted an increase ofthe analytical frequency from 20 to 60 samples per hour.

Demonstrou-se pela primeira vez que é possível determinar surfactantes e água em formulações de Sabonetes Líquidos (SL) e Shampoos (SH), direta e simultaneamente pela técnica de infravermelho por transformada de Fourier (FTIR) acoplada à uma cela de reflectância total atenuada (ATR). Tradicionalmente, a principal aplicação do infravermelho médio (2,50 - 15,0 µm) é a identificação de compostos orgânicos. O desenvolvimento de novos acessórios, principalmente de ATR, os avanços na área de microinformática e de métodos quimiométricos, vem viabilizando as análises quantitativas rápidas com excelentes resultados mesmo em meio aquoso. A determinação simultânea de surfactantes em formulações por FTIR-ATR é investigada em detalhes nesta tese. Dentre os surfactantes utilizados, o Lauril Éter Sulfato de Sódio (LESS) e a Cocoamidopropil Betaína (CAPB) são comuns em ambas as formulações, sendo a Coco Dietanolamida (CDEA) empregada em shampoo e o Alquilpoliglicosídeo (APG) em sabonete líquido. Espectros de absorbância de amostras padrão e de verificação foram adquiridos na região do infravermelho médio (800-1600 e 1900-3000 cm-I). Para a regressão de mínimos quadrados clássicos (CLSR), selecionou-se 200 números de onda, enquanto que para a regressão de mínimos quadrados inversos (ILSR), apenas 10. Nas regressões de componentes principais (PCR) e de mínimos quadrados parciais (PLSRI e PLSR2), utilizaram-se de 300 à 1100 números de onda. Dois conjuntos de amostras padrão foram preparados, o primeiro, contendo 27 misturas padrão, foi estudado somente pelos métodos CLSR e ILSR, enquanto que o segundo conjunto, contendo 48 amostras padrão, foi avaliado por todos os métodos mencionados acima. A seleção das regiões de quantificação favoreceu números de onda dos componentes minoritários CAPB, APG e CDEA e resultados satisfatórios foram encontrados para 18 amostras de shampoo e sabonete líquido. Interferentes como NaCl e perfume foram incluídos no segundo conjunto e os métodos PCR e PLSR proporcionaram melhores resultados. Os erros relativos (RSEP%) para água (correspondendo a 84-88% do produto) e LESS (6-10%) não excederam 1%; para CAPB (<3%) e CDEA (<2%), o RSEP% situou-se entre de 2-4% e para APG (<3%), não excedeu 5%. Avaliações do processo de normalização, repetibilidade, vulnerabilidade a interferentes (perfumes), redução no número de padrões de calibração foram conduzidos, encontrando-se resultados satisfatórios para todos os casos, com erro relativo inferior à 5,0%. Um dispositivo simples para injeção direta da amostras no acessório de ATR foi construído, permitindo aumentar a freqüência analítica de 20 para 60 análises por hora. / It is demonstrated for the first time that the principal constituents of a shampoo as well as of a liquid soap -three surfactants and water- can be determined directly, simultaneously and quickly in undiluted samples by Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy in the middle infrared region, despite the broad absorption bands ofthe solvent. The main application of the middle infrared (2,50 - 15,0 µm) was the identification of organic compounds. The development of new accessories, such as ATR, and the advance of computers and chemometrics, extended the technique to quantitative analysis, with excellent results. The simultaneous determination of surfactants in shampoos and liquid soap formulations by FTIRA TR is investigated in detail in this thesis. Two of the surfactants, sodium lauryl ether sulfate (LESS) and cocoamidopropyl betaine (CAPB) are common to both formulations; alkylpolyglucoside (APG) is the third surfactant of the liquid soap and cocodiethanolamide (CDEA), the corresponding ingredient of the shampoo. Absorbance data of the undiluted verification samples and calibration standards was collected in the middle infrared region of the spectrum (800-1600 and 1900-3000 cm-1). Five methods of multivariate quantification were compared: Classical Least Squares Regression (CLSR), where absorbance data measured at 200 wavenumbers was processed, Inverse Least Squares Regression (ILSR), were data at 10 selected wavenumbers was analyzed, Principal Components Regression (PCR), and Partial Least Squares Regression, which has two different approaches, PLSR1 and PLSR2. Two sets of standard samples were prepared for the method calibration. The first one, consisting of 27 standard mixtures, was evaluated by the CLSR and ILSR methods; a second set, with 48 standards, was evaluated by all the methods mentioned above. Potential interfering, such as NaCI and perfume, were inc\\uded in this second set. By favoring wavenumbers where absorption bands of the minor components (CAPB in both formulations, APG in the liquid soap and CDEA in the shampoo) are more intense, good results were obtained for 18 simulated samples of shampoo and 18 samples of liquid soap. For the second set, PCR and PLSR methods were most favorable; relative errors (RSEP%) for water (major component, 84-88%) and LESS (6-10%) did not exceed 1%; for CAPB (<3%) and CDEA(<2%), RSEP% of 2-4% were observed, and for APG(<3%), 5% was not exceeded. Tests for repeatability, normalization performance evaluation, effect of interferents presented favorable results. A simple device for direct sample injection was designed and evaluated. It permitted an increase ofthe analytical frequency from 20 to 60 samples per hour.

Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD+) to produce poly (ADP-ribose) (PAR) polymers on target proteins or itself. As a result, hyper-activity of PARP1 may lead to necrosis by excessively depleting ATP pool which results in mitochondrial energetic collapse. On the other hand, it is known that Ca2+ overload induces necrosis, but much still remains unknown about how Ca2+ overload-induced necrosis is regulated in cells. In this study, we show that ATR, besides its hallmark regulatory role in DDR, also plays a role in NDDR by suppressing ionomycin-induced necrosis. Ionomycin as a Ca2+ ionophore can dramatically raise the intracellular level of Ca2+, leading to necrosis. We found that this Ca2+ overload-induced necrosis occurs without inducing DDR in cells. Instead, the hyper-poly(ADP-ribosyl)ation (PARylation) activity of activated PARP1 could be a reason leading to necrosis, as NAD+ supplied to media can rescue ionomycin-induced necrosis. In vitro PARylation assay also demonstrates that PARP1 hyper-activation is Ca2+ dependent. In cells, ATR-PARP1 interaction happened after ionomycin treatment. Furthermore, ionomycin treatment induces more full-length PAR polymers formed in ATR-deficient cells than in ATR-proficient cells. The interaction of kinase-dead ATR and PARP1 dramatically decreased as compared to wild-type ATR. Therefore, ATR plays a novel role in NDDR wherein it is able to suppress Ca2+ overload-induced PARP1-mediated necrosis. Ca2+ overload-induced cell death is a major cause of many human medical conditions and diseases, such as brain injury, stroke and ischemia et al. Our ongoing studies will help to define the molecular mechanisms of the anti-necrosis activities of ATR, which may support ATR as a new clinical target for therapeutic treatment of those diseases.

This dissertation focuses on vowel harmony in Maasai, an Eastern Nilotic language spoken in Kenya and Tanzania. The main goal of this dissertation is to determine whether an adequate account of the Maasai pattern of Advanced Tongue Root (ATR) harmony can be formulated in Optimality Theory. Ultimately it is seen that it can, relying on directional Maximal Licensing constraints Walker (2011). Maasai is a language with dominant-recessive harmony. There are two sets of vowels--ATR and non-ATR. A word can only include members of one vowel set; if there is an AT R vowel anywhere in a word, all vowels will be ATR in the output. The only exception to this is the non-ATR low vowel, which lacks an ATR counterpart. It is opaque--it does not harmonize and it blocks the spread of harmony if it is followed by an ATR vowel, but it harmonizes to [o] when preceded by an ATR vowel. All earlier analyses have been based on mainly one source, Tucker and Mpaayei (1955). To avoid using inaccurate or inaccurate data, the data analyzed in this thesis were collected from native speakers in Arusha, Tanzania. Earlier accounts have been based on impressionistic transcriptions. Acoustic analysis of the data were performed to explore the properties of the vowels. The height of the first formant was found to be the most robust acoustical cue to differentiate ATR and non-ATR vowels, though the height of the second formant has some use as a secondary cue. Like many previous studies of languages with an ATR contrast, in this study, it was found that the ATR vowels in Maasai have lower F1s than their non-ATR counterparts (Ladefoged 1964, Lindau et al. 1972, Lindau 1976, Jacobson 1980, Hess 1992, Maddieson and Gordon 1996, Fulop et al. 1998, Anderson 1999, 2007, Przezdziecki 2005, Gick et al. 2006, Starwalk 2008, Kang and Ko 2012). Guion (2004)'s acoustic analysis of Maasai, which showed that ATR and non-ATR vowels in minimal pairs or near minimal pairs differ in F1 was confirmed. Unlike previous research, vowels that have undergone harmony were also investigated. It was observed that not only does Maasai show an ATR/ non-ATR distinction, but that the harmony process is neutralizing. An ATR suffix will force a non-ATR root to harmonize, and an ATR root will force a non-ATR prefix to harmonize. The vowel that has undergone harmony to become ATR is not distinguishable from one is always ATR. It was also found that distance from the trigger (the ATR vowel that causes harmony) does not affect the harmony process. Maasai has been described as having one lexically ATR prefix which causes only less peripheral prefixes to harmonize (Tucker and Mpaayei 1955, Mol 1995, 1996). This claim was investigated, but no acoustic evidence was found to support the claim that there is an ATR prefix. Instead, it is suggested that the perception of the prefix as ATR arises from coarticulatory effects that are the result of the unique environment of the prefix. Acoustic analysis of prefixes preceding the putative ATR prefix were found to be non-ATR. Although previous OT analyses of Maasai have been either unduly complex, incapable of accounting for all the data or have dismissed elements of the harmony as morphological, the harmony system can be accounted for rather simply with two directional harmony constraints. Walker (2011) suggests that languages which appear to demonstrate one bidirectional harmony process might actually be the result of two unidirectional harmony processes. The analysis of Maasai presented her supports this suggestion. There are two directional Maximal Licensing constraints which are high-ranked there is another constraint that must be ranked between them to account for the asymmetric behavior of the low vowel.