Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

MOLECULAR INVESTIGATION OF THE EFFECTS OF ANTIOXIDANTS ON RAT BRAIN TISSUES

In this study, structural and functional aspects of adenovirus type 5 (Ad5) E4orf3 protein were analyzed by biophysical and biochemical methods. Ad5 is one of the mostly used gene therapy vectors to date. However, some of its proteins possess oncogenic potential and their presence comprises safety risks. E4orf3 is one of the oncoproteins of Ad5. It also takes important roles in viral infection, and is beneficial for therapy vectors. Therefore, understanding the functions of E4orf3 is very important for developing efficient and safe adenovirus vectors. Most of the present knowledge about the functions of E4orf3 comes from its mutational analysis. It has never been expressed or purified successfully due to its extreme insolubility. Therefore, this study focused on the optimization of expression of E4orf3 protein. As a result, full-length E4orf3 was obtained in soluble form as a Glutathione-S-transferase (GST) fusion protein and purified by GST affinity chromatography for the first time. Subsequently, the interaction of E4orf3 with four different proteins, DNA-PK, Aup1, E1B-55 kDa and E4orf6 was analyzed in detail by GST-pulldown technique. In these experiments, E4orf3 was shown to associate with Aup1, E1B-55 kDa and E4orf6 in vitro, and the C-terminal of E4orf3 was determined to be responsible for these interactions. Finally, basic structural information about E4orf3 protein was also obtained for the first time by the direct analysis of the fusion protein in glutathione beads with Fourier Transform Infrared (FTIR) spectroscopy. Since the purified E4orf3 protein could not be separated from the glutathione beads due to its hydrophobic regions, the secondary structures in this protein were determined after subtracting glutathione and H2O absorption bands, and the GST moiety.

The main objective of this study was to investigate the effects of high hydrostatic pressure on the stability of blood constituents for the purpose of an effective reduction of viral and bacterial count. The effect of HHP treatment on the several blood constituents were analyzed at different HHP levels at 25 0C for 5 minutes. The bovine blood as the model material was separated into two major parts / namely, serum and blood cells by centrifugation. Erythrocytes were found to be mostly stable up to 220 MPa pressure treatment displaying only surface modifications, but the cells lose their morphology at 350 MPa. White Blood Cells and platelets were found to be more sensitive, being degraded at around 110 MPa pressures putting an upper limit for the HHP treatment for the whole blood. But serum components and parameters studied showed much higher stability up to 220 MPa pressure level. The HHP treated blood cells were analyzed by FTIR spectroscopic technique and found to be stable in the macromolecular level. HHP treated proteins display only minimal changes in their secondary structures shown by the artificial neural network and curve fitting studies. Changes in the lipid bands indicated the changes in the membranes of the blood cells. In the microbiologic part of the study, Listeria innocua was found to be more stable than Bovine Herpes Virus type 1 as the model bacterium and virus respectively and their inactivation levels were compared with that of blood constituents.

Cleaning validation, in which cleaned surfaces are analysed for residual material, is an important process in pharmaceutical manufacturing and research facilities. Current procedures usually consist of either swab or rinse-water sampling followed by analysis of the samples. The analysis step is typically either rapid but unselective (conductivity, pH, total organic carbon, etc.), or selective but time-consuming (HPLC). This thesis describes the development of an in situ surface-spectroscopic analysis that removes the need for swab sampling and is both rapid and selective. This method has the potential to complement existing analyses to increase the efficiency of cleaning-validation protocols. The spectrometric system consists of a Fourier-transform infrared (FTIR) spectrometer coupled to a fibre-optic grazing-angle reflectance probe, and allows the measurement of infrared reflection-absorbance spectra (IRRAS) from flat surfaces in ~10 s. Multivariate chemometric methods, such as partial least squares (PLS) regression, are used to exploit the high information content of infrared spectra to obtain selective analyses without physical separation of the analyte or analytes from whatever interfering species may be present. Multivariate chemometric models require considerably more effort for calibration and validation than do traditional univariate techniques. This thesis details suitable methods for preparing calibration standards by aerosol deposition, optimising and validating the model by cross- and test-set validation, and estimating the uncertainty by resampling and formula-based approaches. Successful calibration models were demonstrated for residues of acetaminophen, a model active pharmaceutical ingredient (API), on glass surfaces. The root-mean-square error of prediction (RMSEP) was ~0.07 µg cm⁻². Simultaneous calibration for acetaminophen and aspirin, another API, gave a similar RMSEP of 0.06 µg cm⁻² for both compounds, demonstrating the selectivity of the method. These values correspond to detection limits of ~0.2 µg cm⁻², well below the accepted visual detection limit of ~1-4 µg cm⁻². The sensitivity of the method with a stainless steel substrate was found to depend strongly on the surface finish, with highly polished surfaces giving more intense IRRAS. RMSEP values of 0.04- 0.05 µg cm⁻² were obtained for acetaminophen on stainless steel with three different finishes. For this system, severe nonlinearity was encountered for loadings 1.0 µg cm⁻². From the results presented in this thesis, it is clear that IRRAS has potential utility in cleaning validation as a complement to traditional techniques.

In this dissertation,the membrane protein channelrhodopsin-1 from the green flagellate algae Chlamydomonas agustae (CaChR1) is studied using a variety of spectroscopic techniques developed in the Rothschild Molecular Biophysics Laboratory at Boston University. Over the last decade, channelrhodopsins have proven to be effective optogenetic tools due to their ability to function as light-gated ion channels when expressed in neurons. This ability allows neuroscientists to optically activate an inward directed photocurrent which depolarizes the neuronal membranes and triggers an action potential. Although a variety of channelrhodopsins with different properties have been used, the underlying mechanisms of channelrhodopsin functionality is not yet fully understood. The protein studied here has several advantageous properties compared to the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii including a red shifted visible absorption and slower light inactivation despite having a lower channel current. Elucidating the internal molecular mechanisms underlying the function of CaChR1 provides critical insight into the large class of channelrhodopsin proteins leading toward improved bioengineering for specific optogenetic applications. Here near-IR pre-resonance Raman spectroscopy of CaChR1 provides information on the structure of the unphotolyzed (P0) retinal chromophore, the Schiff base protonation state, and presence of carboxylic acid residues interacting with the Schiff base. Low-temperature FTIR difference spectroscopy combined with site-directed mutagenesis and isotope labeling provide information on changes occurring in the retinal chromophore and protein during the primary phototransition (P0 to P1). This includes information about changes involving protonation state of binding-pocket residues, protein backbone structure, and internal water molecules. Further experiments combining low-temperature and time-resolved FTIR-difference spectroscopy reveal additional information about structural changes during the transition from the unphotolyzed state to the active (open channel) state of the protein (P0 to P2). This work has resulted in an initial model that describes key proton transfer events which occur between the Schiff base and carboxylic acid residues inside the active site of CaChR1. The model raises the possibility that ion channel gating and ion specificity is regulated by the protonation changes of two key residues (Glu 169 and Asp299) located near the Schiff base.

Made available in DSpace on 2014-06-11T19:27:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-18Bitstream added on 2014-06-13T18:56:08Z : No. of bitstreams: 1 lopes_vcp_me_rcla.pdf: 959048 bytes, checksum: 8ef5fde75931039770927a47c11ea7f8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Recentemente o mundo começou a se preocupar com o consumo exacerbado de embalagens e a sua rápida descartabilidade. Por conseguinte, alternativas de amenização deste problema têm sido propostas tais como a utilização de plásticos biodegradáveis, o uso de blendas poliméricas e o emprego de aditivos que facilitem a biodegradação. Sendo assim, este trabalho teve por objetivo avaliar os diferentes tratamentos: foto, bio e foto/biotratamento, aplicados na degradação de filmes finos de PEBD, PHB e de suas blendas, empregando o fungo P. chrysosporium CCB 478 no processo biodegradativo. As blendas de PEBD/PHB foram preparadas nas composições de 90:10, 80:20 e 70:30 (m/m) juntamente com os filmes de homopolímeros pelo processo de prensagem sob aquecimento. As amostras foram submetidas à radiação UV e em seguida foram aplicados os testes de biodegradação em meio mineral líquido com o fungo P. chrysosporium CCB 478 pelo período de 120 dias. Análises de perda de massa, ângulo de contato, FTIR, análises visuais, microscopia óptica, MEV, foram técnicas utilizadas para avaliar a biodegradação. O fototratamento realizado com todas as amostras dos filmes foi relevante para o processo de adesão e colonização da superfície dos filmes. As blendas de composição 70/30 e 80/20 apresentaram alterações morfológicas e estruturais mais significativas, por MEV e FTIR, além de apresentaram melhores resultados de biodegradação, após a fotoirradiação. / Recently the world began to worry about the high consumption and rapid disposability of packaging. Therefore, alternatives to improve this problem have been proposed such as use of biodegradable plastics, polymer blends and additivies which facilitates the biodegradation. Thus, this study aimed to evaluate the results of different treatments: biotreatment, phototreatment, and photo/biotreatment, applied on the degradation of thin films of LDPE, PHB and its blends, using as fungus Phanerochaete chrysosporium CCB 478 in biodegradation process. Blends of LDPE/PHB were prepared in the compositions of 90:10, 80:20 and 70:30 (m/m) along with homopolymers films by pressing process under heating. Samples were exposed to UV radiation and then were submitted to the biodegradation tests in liquid mineral medium with the P. chrysosporium CCB 478 fungus for 120 days. Analysis of weight loss, contact angle, FTIR, visual analysis, optical microscopy and SEM, were employed to estimate biodegradation. Phototreatment applied to all films was relevant to the adhesion process and colonization the surface films. The blends of composition 70/30 and 80/20 showed significant morphological and structural changes, verified by SEM and FTIR techniques, besides showed better results of biodegradation after photoirradiation.

Made available in DSpace on 2014-06-11T19:27:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-04-26Bitstream added on 2014-06-13T18:56:11Z : No. of bitstreams: 1 campos_a_me_rcla.pdf: 4152569 bytes, checksum: 41a529d2c0be5a0a79eb44e0ec1448ea (MD5) / Os plasticos constituem um dos materiais mais utilizados em nosso cotidiano. Assim, os residuos plasticos tem aumentado bastante e hoje representam 20% do total do volume de residuos, em lixoes municipais. O PVC (policloreto de vinila) e um polimero sintetico, instavel em relacao ao calor e luz, que degrada a temperatura relativamente baixa (aproximadamente 130oC), e libera HCl. A desidrocloracao gera novas especies na cadeia polimerica, ou seja, sequencias de polienos que podem ser verificadas atraves da espectroscopia de absorcao na faixa do UV-Visivel. A reciclagem do PVC apresenta algumas dificuldades intrinsecas ao comportamento do polimero, durante o processo tecnologico e, alem disso, sua biodegradacao e dificil. O PCL (poli α-caprolactona), um poliester sintetico, e sensivel a acao de enzimas microbianas, e pode sofrer hidrolise e degradacao. A mistura desses polimeros, isto e, a blenda de PVC e PCL pode constituir um meio para facilitar o ataque microbiano ao PVC, alem de melhorar ou manter as propriedades mecanicas da blenda. Processos termo e fotodegradativos podem facilitar o ataque microbiano a matriz polimerica do PVC. O presente trabalho pretende investigar a termo, foto e biotransformacao de filmes de blendas de PVC/PCL 1:1 e 3:1 com fungos de solo (Phanerochaete chrysosporium e Aspergillus fumigatus). As mudancas estruturais foram analisadas atraves da espectroscopia de absorcao no infravermelho (FTIR) e no UV-Visivel e as mudancas morfologicas, atraves de Microscopia Eletronica de Varredura (MEV) / The plastics are one of used the most materials daily. Thus, the plastics wastes have increased considerably and today represent 20% of the waste total volume in the municipal landfills. PVC (poly vinyl chloride) is a synthetic polymer, unstable in the presence of heat and light, that degrades on relative low temperature (approximately 130ºC), and releases HCl. The dehydrochlorination produces new species on polymeric chain, thus, polyenes sequences, that can be checked by the UV-Visible absorption spectroscopy. The recycling of PVC presents some difficulties due its behaviour during the technological processing and its biodegradation is difficult. PCL (polycaprolactone), a synthetic polyester is susceptible to enzymes action, and undergo hydrolysis and degradation. The blend of PVC and PCL can be one way to facilitate the microorganisms attack to the PVC polymer and to improve the mechanical properties of the blend. Thermal and photodegradation process can to facilite the microorganisms attack on PVC polymeric chain. The present work intends to investigate the thermal, photo and biotransformation of polymer films and blends of PVC/PCL 1:1 and 3:1 blends, with soil fungi (Phanerochaete chrysosporium and Aspergillus fumigatus). The structural changes were analysed by infrared absorption spectroscopy (FTIR) and the UV-Visible. The morphologics changes were investigated by scanning electron microscopy (SEM)

Introdução: Nos últimos anos, procedimentos cirúrgicos envolvendo a glândula tireoide têm aumentado em todo o mundo, bem como a incidência de neoplasias malignas sem, no entanto, se observar aumento da taxa de mortalidade. Muitas técnicas foram propostas para alcançar um diagnóstico pré-operatório acurado em doenças da tireoide. As técnicas de espectroscopia de FTIV (Infravermelho por transformada de Fourier) já apresentam evidências na caracterização de múltiplos tecidos, entre eles a glândula tireoide, tendo como vantagem sua rapidez e preservação do tecido analisado. O estudo de novas técnicas na diferenciação dos nódulos tireoidianos pode auxiliar na seleção de pacientes que serão submetidos ao tratamento cirúrgico adequado, evitando o sobretratamento. Objetivos: Caracterizar os tecidos tireoidianos sadios e patológicos à luz da espectroscopia de infravermelho (IV), testar a viabilidade do método de espectroscopia no IV em diferenciar tecidos patologicamente alterados da glândula tireoide comparativamente aos achados espectroscópicos encontrados em tecidos sadios. Casuística e Métodos: Os pacientes foram selecionados no Serviço de Cirurgia de Cabeça e Pescoço do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP-USP), no período de 2014 a 2015. A amostra foi composta por 44 pacientes de ambos os sexos, com idade acima de 18 anos, com indicação de tireoidectomia. Quarenta e quatro amostras de nódulos em tireoide e 44 de tecidos normais foram analisadas por espectroscopia no IV utilizando-se um espectrômetro Nicolet 380 - Nicolet USA® em tecidos a fresco. A análise foi realizada definindo-se as áreas de cada banda, utilizando-se para os cálculos o programa OriginPro 8.6.0 (OriginLab Corporation Northrampton, MA 01060 USA). A seguir, efetuou-se a normalização pela banda a 1240 cm-1. A comparação das áreas foi calculada por meio do teste t- Student com p<0,05. Após o cálculo das médias realizou-se avaliação de derivadas de segunda ordem do espectro para se evidenciarem as posições de cada banda de absorção. Realizou-se, também, a análise de cada banda aplicando o teste t-Student para amostras pareadas e amostras independentes; e o teste de Wilcoxon para amostras pareadas e para comparação de duas amostras independentes. Resultados: O espectro IV de cada peça foi obtido, sendo expresso em função da absorbância e dos números de onda no IV médio (4000 - 900 cm-1). O presente estudo demonstrou que na análise do tecido tireoidiano pela espectroscopia no IV é possível diferenciar os nódulos benignos do tecido sadio, com diferença significativa na área da banda B entre tecido sadio e bócio, que corresponde a 1452,90 cm-1 no tecido sadio (Proteínas e Lipídeos) e 1069,80 cm-1 no bócio (DNA); e também diferença estatisticamente significativa entre os tecidos normal e carcinoma para largura na banda C, onde a largura foi maior no tecido com carcinoma do que no tecido normal. Conclusões: A espectroscopia no IV é capaz de diferenciar os tecidos tireoidianos patologicamente alterados da glândula tireoide comparativamente aos achados em tecidos tireoidianos sadios. Concluiu-se que nos pacientes com doença nodular benigna da glândula tireoide é possível diferenciar o tecido sadio do bócio com significância estatística, bem como também diferenciar nódulos malignos do tecido sadio por meio da espectroscopia no IV. / Introduction: In recent years, surgical procedures involving the thyroid gland have increased worldwide, as well as the incidence of malignant neoplasia without, however, observing an increase in the mortality rate. Many techniques have been proposed to achieve an accurate preoperative diagnosis in thyroid diseases. The FTIR spectroscopy techniques already present evidence in the characterization of multiple tissues, among them the thyroid gland, having as an advantage its rapidity and preservation of the tissue analyzed. The study of new techniques in the differentiation of thyroid nodules can help in the selection of patients who will undergo the appropriate surgical treatment avoiding overtreatment. Objectives: To characterize healthy and pathological thyroid tissues in the light of infrared spectroscopy, to test the viability of the infrared spectroscopy method in differentiating pathologically altered tissues from the thyroid gland compared to the spectroscopic findings found in healthy tissues. Casuistic and methods: Patients were selected at the Head and Neck Surgery Service of the Hospital of Clinics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto-SP, Brazil, from 2014 to 2015. The sample consisted of 44 patients of both sexes, aged over 18 years, oriented about their participation in the study and with indication of thyroidectomy. The analysis was performed by defining the areas of each band, using the program OriginPro 8.6.0 (OriginLab Corporation Northrampton, MA 01060 USA). The band was then normalized to 1240 cm -1. The mean area was calculated using the Student t-test with p<0.05. After the calculation of the averages, the second-order derivative of the spectrum was evaluated to show the positions of each absorption band. We also performed the analysis of each band by applying the t-Student test for paired samples, Wilcoxon test for paired samples, Student\'s t-test for independent samples and the Wilcoxon test for comparison of two independent samples. Results: The infrared spectrum of each piece was obtained, being expressed as a function of absorbance and wave numbers in the mean IR (4000 - 900 cm -1). The present study demonstrated that in the analysis of thyroid tissue by infrared spectroscopy, we can differentiate benign nodules from healthy tissue, with a significant difference in the area of the B-band between healthy tissue and goiter, which corresponds to 1452.90 cm -1 in healthy tissue (Proteins and Lipids) and 1069.80 cm -1 in goiter (DNA) and also a significant difference in width between normal thyroid tissue and carcinoma of the C band. Conclusions: Infrared spectroscopy is able to differentiate pathologically altered thyroid tissues from the thyroid gland compared to findings in healthy thyroid tissues. After the analysis of the results it was possible to conclude that in patients with benign nodular disease of the thyroid gland it is possible to differentiate healthy goiter tissue with statistical significance, as well as it is possible to differentiate malignant nodules from healthy tissue through infrared spectroscopy