Made available in DSpace on 2014-06-12T15:48:50Z (GMT). No. of bitstreams: 2 arquivo2047_1.pdf: 2722890 bytes, checksum: 408df99108380d5e3dc79885dccc69e4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente trabalho descreve a preparação e caracterização de sais derivados do ácido N-(3,5-dinitrobenzoil)-α-fenilglicina, utilizando como contra-íon as seguintes aminas protonadas: NH4 + (DNBAm), (CH3-CH2-)2NH2 + (DNBD), (CH3-CH2-CH2-)NH3 + (DNBP), (CH3-CH2-CH2-CH2-)NH3 + (DNBBu) e (CH3-CH2-)3NH+ (DNBT). Estes sais apresentaram efeito fotocrômico quando submetidos à exposição de radiação ultravioleta (UV) ou radiação solar, tanto no estado sólido como em solução de DMSO. Os sais foram preparados seguindo-se uma mesma rota de síntese a partir da N-(3,5-dinitrobenzoil)-α-fenilglicina com bons rendimentos. Estes sais foram analisados e caracterizados estruturalmente, através das técnicas: RMN-1H, RMN- 13C, infra-vermelho (I.V.), análise elementar (AE), análise térmica gravimétrica (TGA) e UV-Vis. Testes espectroscópicos revelaram a formação de um complexo de transferência de carga intramolecular após exposição à radiação ultravioleta. O estudo de eficiência fotocrômica em meio de DMSO revelou também que o contraíon do sal de N-(3,5-dinitrobenzoil)-α-fenilglicinato tem uma baixa influência na eficiência da reação fotoquímica para formação do complexo de transferência de carga, estes resultados foram correlacionados a valores de pKBH+.tanto em meio aquoso como em meio de DMSO. Foram efetuadas também algumas medidas de condutividade para o DNBAm e DNBT, indicando que ocorre dissociação de ambos os sais em meio de DMSO, sendo a solução de DNBAm a mais condutora. A avaliação inicial dos sais preparados em meio sólido foi efetuada através da irradiação de luz UVC controlada. O teste visual para mudança de coloração (branco rosa) aponta para a seguinte ordem de intensidade de cor obtida: DNBT > DNBD > DNBP > DNBBu > DNBAm

Despite the decades of intensive research, nucleic acids represent still a permanent object of structural studies. Within the framework of the doctoral work, the apparatus for measurement of UV excited resonance Raman spectra (UV RRS) was built up and optimized. A realistic and complex interpretation table was prepared based on analysis of published data and extensive series of UV RRS measurements on NA model structures, mononucleotides, and polynucleotides. The established methodology was verified when applied in several structural studies of nucleic acids, mainly the study of the influence of magnesium ions on the equilibrium between duplexes and triplexes formed by PolyA and PolyU homopolynucleotides, a study of temperature-induced structural changes in DNA double helix and DNA hairpin, and investigation of slow structural transitions of guanine quadruplexes induced by the presence of potassium ions. The results of the test measurements and the above-mentioned studies have shown that the created methodology for studying UV RRS of nucleic acids brings most of the expected benefits of the resonance excitation: the possibility of Raman scattering measurements at the same concentrations as in the case of UV absorption, high sensitivity to fine temperature-induced structural changes and good interpretability...

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of lorazepam in oral solution. The chromatographic conditions include an isocratic separation (25% water / 75% methanol at a flow rate of 0.500 ml/min) on a Waters X=Bridge C18 column (150 x 4.6 mm; 3.5-micron particle size). The method was validated using guidance from the United States Pharmacopeia (USP) chapter, including investigation of system suitability, precision, accuracy, linearity, and specificity. The calibration curves on three non-consecutive days met the linearity criteria R2 >0.99. Each chromatogram for 200 mcg/mL calibration samples, designated as 100% assay level, met system suitability criteria of resolution ≥2.0, tailing factor ≤2.0, column efficiency (theoretical plates) ≥2000, and precision of prior metrics % RSD ≤1.0. Three replicates of each concentration, 150 mcg/mL at 75% assay, 200 mcg/mL at 100% assay, and 250 mcg/mL at 125% assay were assessed for precision and accuracy over 3 days. Precision and accuracy were evaluated and met the inter-day (repeatability) criteria % RSD and % Error ≤ 2% and intra-day (intermediate) criteria % RSD and % Error ≤ 5% at the 75%, 100%, and 125% assay levels. To assess for specificity, 200 mcg/mL samples were assessed for degradation after being subjected to heat (>60°C), oxidation (3% H2O2), acidic (0.1M HCl), and basic (0.1M NaOH) environments. Samples from each condition were evaluated for lorazepam recovery at 0, 24, and 48 hours. Most drug loss was observed with the samples subjected to acidic and oxidative environments, with 14.71% and 13.16% drug loss, respectively after 48 hours. This method was developed to support the 30-day stability investigation of lorazepam oral solution when stored in oral syringes at room and refrigerated temperatures.

We have used a non-replicating recombinant adenovirus, AdCA17𝘭𝘢𝘤𝘡, which expresses the β-galactosidase (β-gal) reporter gene under the control of the human cytomegalovirus immediate early (HCMV-IE) promoter, to examine host cell reactivation (HCR) of a UVC-damaged reporter gene in human fibroblasts. Since β-gal expression in mammalian cells is expected to occur following transcription of a lesion-free 𝘭𝘢𝘤𝘡 reporter gene, the removal of UV-induced DNA lesions and subsequent β-gal expression is a measure of the DNA repair capacity of the infected cell. In the present work, we have examined HCR and UV-enhanced HCR of the UV-damaged reporter construct, AdCA17𝘭𝘢𝘤𝘡, in normal and nucleotide excision repair (NER) deficient fibroblasts. Xeroderma pigmentosum (XP) group C and E fibroblasts are deficient in the global genome pathway (GGR) of NER, while Cockayne syndrome (CS) fibroblasts are deficient in the transcription-coupled repair pathway (TCR) of NER and XP group A fibroblasts are deficient in both TCR and GGR. HCR of the UV-damaged reporter gene activity was significantly reduced in XP-A, XP-C, XP-E and CS-B cell strains relative to that in normal human fibroblasts, indicating that both TCR and GGR contribute to expression of the UV-damaged reporter gene in human cells. Pre-treatment of human fibroblasts with UVC (12 J/m²) immediately prior to infection results in enhanced HCR of the UV-damaged reporter gene. UV-enhanced HCR of the UV-damaged reporter gene was detected in normal fibroblasts at 12 hr post-infection and in CS-B, XP-C and XP-E fibroblasts, but not XP-A fibroblasts at 24 and 40 hr post-infection. These results indicate that both TCR and GGR are UV-inducible in human cells. We have examined the colony survival of various human cells following exposure to UVC and UVA radiation. HaCAT, an immortalized human keratinocyte cell line, demonstrated increased resistance to UVC exposme compared to glioma (U373, T98G) and colon carcinoma (HT29) cell lines, while exhibiting increased sensitivity to UVA radiation and H₂O₂ treatment. This increased sensitivity towards both UVA and H₂O₂ treatment suggests that HaCAT cells have a reduced capacity to repair oxidative DNA damage. In addition, we show that p53-null keratinocytes (HPV-G) have reduced clonogenic survival compared to p53-mutant keratinocytes (HaCAT) following H₂O₂ treatment, consistent with an involvement of p53 in the survival of keratinocytes following H₂O₂ treatment. We also examined whether the interaction of H₂O₂ with growth media resulted in chemical by-products that were toxic to cells. Even though the H₂O₂ levels in the media were reduced following 24 hr incubation, similar clonogenic survival curves of HaCAT cells were observed following treatment of cells with immediately-prepared or 24hr-incubated H₂O₂-containing media solutions. Thus, extended incubation (24 hr) of H₂O₂ with media did not alter its cytotoxicity towards HaCAT cells, indicating that no detectable levels of toxic chemical by-products were produced. Ultraviolet irradiation of cells can have both cytotoxic and/or mutagenic consequences, resulting in increased levels of cell death or the induction of a state of genomic instability that persists for several cell generations after irradiation. However, the extent of the effects induced in non-irradiated cells by UV -irradiated cells has not been fully investigated. Using the medium transfer technique, we have tried to address whether UV irradiation of cells can induce biological effects in non-irradiated cells. Medium obtained from UVA, but not UVC, irradiation of various human cell lines in phosphate-buffered saline (PBS) solution was capable of reducing the relative clonogenic survival and colony size of non-irradiated HaCAT cells. In the absence of cells, UVA-irradiation of PBS reduced the clonogenic survival, but not the colony size, of various non-irradiated human cells. These results indicate a cytotoxicity of UVA-irradiated PBS towards non-irradiated cells. Hydrogen peroxide, a reactive oxygen species (ROS) generated following UV A irradiation, was measured following UV A treatment (or mock-treatment) of PBS (in the presence or absence of cells) either immediately or 24 hr post-irradiation. Hydrogen peroxide levels increased immediately following UV A irradiation, suggesting that it may contribute to the reduced survival of non-irradiated human cells. However, human glioma T98G and U373 cells produced elevated H₂O₂ levels in mock-irradiated conditions at 24 hr post-incubation, while demonstrating different sensitivities towards treatment with medium containing UVA-irradiated PBS. These results suggest that UVA-induced H₂O₂ is not responsible for the reduction in clonogenic survival of non-irradiated human cells. We also examined colony number and size at different times following the addition of media containing UVA-irradiated PBS and media from UVA-irradiated cell cultures. Increasing the time before scoring for colonies resulted in an increased clonogenic survival and a decrease in relative colony size for HaCAT cells. These preliminary results suggest that the relative survival and relative colony size of non-irradiated cells are inter-related, and indicate that treating non-irradiated HaCAT cells with medium from UVA-irradiated cells or medium containing UVA-irradiated PBS reduced the growth rate of HaCAT colonies. Therefore, in order to properly evaluate clonogenic survival, a time-dependent examination of relative survival and relative colony size should be conducted in order to address whether the treatment results in an inhibition of cell growth and/or true cell killing. / Thesis / Master of Science (MS)

We consider a modified non-relativistic quantum mechanics where the position and momentum operators satisfy a non-standard commutation relation of the form [X_i, P_j] = 𝑖ℏ({1 + βP²) + β′P_iP_j}. Such a theory incorporates an absolute minimal length, UV/IR mixing and non-commutative position space. The possible representations in terms of differential operators are analyzed and their equivalence to first order is established. Simple quantum systems, namely the harmonic oscillator, the Coulomb potential and the gravitational well are studied in one of these representations, the pseudo-position one, and results are compared to previously published results. The Coulomb potential is also analyzed by an alternative analytical/numerical method. A constraint of ~ 3 GeV on the scale of the parameters β, β′ is obtained from precision experimental data on the atomic hydrogen energy levels. / Ph. D.

The benefits of low UV dose treatment of horticultural produce – also known as hormetic treatment - have been attested to in numerous studies conducted over the last 15 years. However, commercial growers have not adopted the concept of hormesis. With increasingly stringent controls on the use of fungicides and other chemical agents the time has come to examine how hormetic treatment might be applied in the horticulture sector. The objectives of this work were firstly, to confirm UV-induced hormetic effects applied postharvest for a number of different types of produce, namely, tomatoes, broccoli, strawberries and mangoes. Secondly, to evaluate the use of rollers to ensure full surface treatment of produce, and thirdly to evaluate the possibility of treating produce preharvest. In order to investigate surface UV dose distributions, a polystyrene sphere (Diameter 70 mm) was used to simulate fruits such as tomatoes, apples, peaches etc., that have an approximately spherical form. Biodosimetry based on spores of Bacillus subtilis was employed to experimentally determine UV doses and to compare the results obtained with theoretical predictions. Good agreement was obtained and the modelling approach was extended to other types of produce. This showed the amenability of mechanical rollers to ensure full surface treatment of produce. Postharvest treatment of produce was carried using conventional low intensity UV sources principally emitting at 254 nm and also a commercially available high energy pulsed UV source. Treatment using the conventional UV source was carried out on mechanical rollers within a UV cabinet designed for this work at a fixed distance from the source and at an intensity of 1000 μW/cm2. A 5 minute conventional UV treatment of tomatoes was approximately comparable to fruit given a 3-pulsed treatment using the pulsed source (507 J/pulse of polychromatic light). The colour and texture of both groups of fruit were significantly maintained as compared with controls. The treated tomatoes also showed a significant increase in the ascorbic acid levels during storage. Similarly, a 15 minute conventional UV treatment of broccoli heads was comparable to heads given a 10-pulsed treatment using the pulsed source. Where both treatments gave rise to a statistically significant retention of green colour of treated broccoli. In addition, mangoes given a 10 minute conventional UV treatment were comparable to fruit given a 20-pulsed treatment using the pulsed source with both treatments leading to maintenance of texture as compared to control fruit. This confirmed the UV-hormetic effects. The effects of conventional and pulsed treatments are compared and discussed. Preharvest treatment of tomatoes and strawberries was carried out in commercial glasshouses. Doses of either 3 or 8 kJ/m2 were delivered to the fruits using a treatment device designed for the work, which delivered a combined intensity of 2000 μW/cm2 from two low pressure UV sources. The treated tomatoes showed a delay in development of colour as measured on the vine and after picking. Picked tomatoes were inoculated with P. digitatum and C. gloeosporioides and the results obtained showed a significant inhibition of the development of the fungi in the treated fruit during the storage period. These results suggest that the beneficial response shown by the preharvest treatment is not a localised one but a systematically induced resistance observable throughout the treated plant. This was shown by monitoring tomato fruits on treated plants which themselves where not directly exposed to the UV light. The two doses elicited different responses in the treated strawberries, with the 8 kJ/m2 dose causing the fruit to redden significantly faster than the 3 kJ/m2 treated fruits and controls. This could have significant nutritional benefit as the red colour of strawberries has been correlated with anthocyanin levels. On the other hand, treatment at the lower UV dose led to a lag in colour development. The amenability of the equipment utilised for commercial application is discussed.

O aumento do conhecimento em relação aos danos provocados pela radiação solar, tanto na faixa do UVB quanto na faixa do UVA, o avanço nas tecnologias relacionadas ao produto e às formas de avaliação bem como a disponibilização de novas moléculas no mercado levaram ao aumento da qualidade da proteção proporcionada pelos fotoprotetores. Entretanto, ainda há preocupações em relação à segurança de alguns filtros solares devido a sua fotoinstabilidade e penetração cutânea. Dessa forma, torna-se necessário o desenvolvimento de novos filtros solares mais efetivos e seguros, que apresentem relevância científica e potencial de inovação. Assim, a presente pesquisa teve como objetivo desenvolver novos análogos ao filtro solar benzofenona-3 e determinar o seu potencial fotoprotetor por meio da avaliação da absorção UV e avaliação da fotoestabilidade, fototoxicidade e atividade antioxidante. Para tal, inicialmente seis análogos ao filtro solar químico benzofenona-3 foram sintetizados visando aumentar o tamanho da molécula e aumentar a sua absorção no UVA longo (340-400nm). Foram avaliados os espectros de absorção no UV e a fotodegradação dessas substâncias. A fototoxicidade das substâncias selecionadas foi avaliada por meio do uso de cultura de fibroblastos 3T3, para a determinação da viabilidade celular na presença e ausência da radiação. A atividade antioxidante das substâncias foi avaliada por meio da quimioluminescência gerada pela reação HRP-H2O2-luminol. Os resultados permitiram demonstrar a importância da relação entre a estrutura molecular dos compostos e sua absorção no UV. As moléculas benzofenona-3 fenil amino (B5) e o carbazol da benzofenona-3 fenil amino (B6) apresentaram maior absorção no UVA, longo e curto, e no UVB, quando comparadas com a molécula de partida (benzofenona-3). Apenas as substâncias que apresentavam ponte de hidrogênio intramolecular (B5 e B6) foram consideradas fotoestáveis. Somente a molécula B5 não apresentou potencial para fototoxicidade aguda. Além disso, essa molécula apresentou atividade antioxidante, o que sugere o seu grande potencial para utilização com filtro solar. / The increase of our knowledge of not only UVB but also UVA-induced damages, the advances on product and testing technologies as well as new sunscreen molecules leaded to and enhancement of quality of UV protection provided by topical sunscreens. However there are some safety concerns involving some UV-filters due to their photoinstability and skin penetration. Therefore it is necessary to develop new safer and more effective UV-filters, which also presents scientific relevance and innovation potential. Thus, the aim of the present research was to develop new analogues based on benzophenone-3, and to evaluate their photoprotective potential through their photostability, phototoxicity and antioxidant activity. For this purpose, firstly six new synthetic analogues based on benzophenone-3 were prepared in order to promote molecular weight enhancement as well as improve long-wave UVA absorption (340-400 nm). The UV absorption spectra and photodegradation of these compounds were also analyzed. Phototoxicity of selected compounds was evaluated by using 3T3 monolayer fibroblast culture to determine cell viability in the presence and absence of UVA radiation. The antioxidant activity was evaluated by HRP-H2O2-luminol induced chemiluminescence. The results showed that relationship between the molecular structure and UV absorption. Me molecules phenylamine benzophenone-3 (B5) and phenylamine benzophenone-3 carbazol (B6) showed higher short and long-wave UVA and UVB absorption, when compared to benzophenone-3. Only B5 and B6, which presented intermolecular hydrogen bond, were considered photostable. B5 did not present any acute phototoxicity potential; in addition it has antioxidant activity, which suggests its high UVfilter potential.

O aumento do conhecimento em relação aos danos provocados pela radiação solar, tanto na faixa do UVB quanto na faixa do UVA, o avanço nas tecnologias relacionadas ao produto e às formas de avaliação bem como a disponibilização de novas moléculas no mercado levaram ao aumento da qualidade da proteção proporcionada pelos fotoprotetores. Entretanto, ainda há preocupações em relação à segurança de alguns filtros solares devido a sua fotoinstabilidade e penetração cutânea. Dessa forma, torna-se necessário o desenvolvimento de novos filtros solares mais efetivos e seguros, que apresentem relevância científica e potencial de inovação. Assim, a presente pesquisa teve como objetivo desenvolver novos análogos ao filtro solar benzofenona-3 e determinar o seu potencial fotoprotetor por meio da avaliação da absorção UV e avaliação da fotoestabilidade, fototoxicidade e atividade antioxidante. Para tal, inicialmente seis análogos ao filtro solar químico benzofenona-3 foram sintetizados visando aumentar o tamanho da molécula e aumentar a sua absorção no UVA longo (340-400nm). Foram avaliados os espectros de absorção no UV e a fotodegradação dessas substâncias. A fototoxicidade das substâncias selecionadas foi avaliada por meio do uso de cultura de fibroblastos 3T3, para a determinação da viabilidade celular na presença e ausência da radiação. A atividade antioxidante das substâncias foi avaliada por meio da quimioluminescência gerada pela reação HRP-H2O2-luminol. Os resultados permitiram demonstrar a importância da relação entre a estrutura molecular dos compostos e sua absorção no UV. As moléculas benzofenona-3 fenil amino (B5) e o carbazol da benzofenona-3 fenil amino (B6) apresentaram maior absorção no UVA, longo e curto, e no UVB, quando comparadas com a molécula de partida (benzofenona-3). Apenas as substâncias que apresentavam ponte de hidrogênio intramolecular (B5 e B6) foram consideradas fotoestáveis. Somente a molécula B5 não apresentou potencial para fototoxicidade aguda. Além disso, essa molécula apresentou atividade antioxidante, o que sugere o seu grande potencial para utilização com filtro solar. / The increase of our knowledge of not only UVB but also UVA-induced damages, the advances on product and testing technologies as well as new sunscreen molecules leaded to and enhancement of quality of UV protection provided by topical sunscreens. However there are some safety concerns involving some UV-filters due to their photoinstability and skin penetration. Therefore it is necessary to develop new safer and more effective UV-filters, which also presents scientific relevance and innovation potential. Thus, the aim of the present research was to develop new analogues based on benzophenone-3, and to evaluate their photoprotective potential through their photostability, phototoxicity and antioxidant activity. For this purpose, firstly six new synthetic analogues based on benzophenone-3 were prepared in order to promote molecular weight enhancement as well as improve long-wave UVA absorption (340-400 nm). The UV absorption spectra and photodegradation of these compounds were also analyzed. Phototoxicity of selected compounds was evaluated by using 3T3 monolayer fibroblast culture to determine cell viability in the presence and absence of UVA radiation. The antioxidant activity was evaluated by HRP-H2O2-luminol induced chemiluminescence. The results showed that relationship between the molecular structure and UV absorption. Me molecules phenylamine benzophenone-3 (B5) and phenylamine benzophenone-3 carbazol (B6) showed higher short and long-wave UVA and UVB absorption, when compared to benzophenone-3. Only B5 and B6, which presented intermolecular hydrogen bond, were considered photostable. B5 did not present any acute phototoxicity potential; in addition it has antioxidant activity, which suggests its high UVfilter potential.

Thiol-ene chemistry is versatile and efficient and can be used as a powerful tool in polymer synthesis. In this thesis, the concept of thiol-ene chemistry has been central, where it has been explored as a tool for the synthesis of well-defined hydrogels and dopa-functional materials towards biomedical applications; such as hydrogels, primers for adhesive fixation of bone fractures, self-healing gels, and micelles for drug-delivery. Using thiol-ene chemistry, well-defined hydrogels were realized in order to study how the structure influences properties such as swelling, stiffness and hydrolytic degradation. It was found that all these characteristics are related to each other, as a more loosely crosslinked hydrogel experiences higher swelling, lower stiffness and higher degradation rates. Dopa-functional materials have gained a lot of interest throughout the years due to the remarkable adhesive properties they possess in wet environments. In the pursuit of new primers towards thiol-ene functional crosslinked bone adhesives, compounds with dopa moieties were proposed. Primers derived from dopamine were found to enhance the adhesion towards bone, and it was concluded that addition of NaOH was essential to achieve good adhesion. The strongest adhesion was achieved when thiol and ene-functional primers were used in combination. Most synthetic routes to dopa-functional polymers involve several protection and deprotection steps and a more simplistic synthetic route is therefore desired. The possibility of using UV-initiated thiol-ene chemistry to produce dopa-functional polymers was therefore investigated. The resulting polymers were shown to exhibit self-healing properties upon complexation with Fe3+ ions. Finally, the developed synthetic route was used to produce dopa and allyl-functional triblock-co-polymers. These triblock-co-polymers were then used to form micelles and evaluated as drug-delivery vehicles for the cancer-drug doxorubicin. The micelles were found to have high drug-loading capacities and slow release profiles and showed promising results when evaluated against breast-cancer cells. / Reaktioner mellan tioler och omättade kemiska föreningar utgör ett mångsidigt och effektivt redskap inom polymersyntes. I denna avhandling har begreppet tiol-en kemi varit centralt och kemin har använts för syntes av såväl väldefinierade hydrogeler som dopa-funktionella material. Dessa material har sedan utvärderats mot biomedicinska tillämpningar såsom hydrogeler, primers för fixering av benfrakturer, självläkande geler och kontrollerad läkemedelsleverans. Tiol-en-kemi har i denna avhandling använts för att framställa väldefinierade hydrogeler som sedan utvärderats med avseende på hur strukturen påverkar egenskaper såsom svällningsgrad, styvhet och nedbrytningshastighet. Det visade sig att alla dessa egenskaper är relaterade till varandra och att lösare tvärbundna hydrogeler uppvisar högre svällning, lägre styvhet och högre nedbrytningshastigheter. Marina musslor har en exceptionell förmåga att fästa mot olika ytor och på grund av detta har det visats en hel del intresse för dopa-funktionella material genom åren. På jakt efter en primer för att öka vidhäftningen hos benlim proponerades därför föreningar med dopafunktionella grupper. Det visade sig att dopaminderivat kunde förbättra vidhäftningen mot ben och det visade sig även att tillsats av natriumhydroxid var viktigt för att uppnå god vidhäftningsförmåga. Den starkaste vidhäftning uppnåddes när derivat med tiol och omättade bindningar användes i kombination. Syntes av dopafunktionella material involverar ofta flera reaktionssteg och en förenklad syntesväg är därför att eftersträva. UV-initierad tiol-en-kemi undersöktes därför som en möjlig syntesväg för att framställa dopafunktionella polymerer. Polymererna visade sig ha självläkande egenskaper vid komplexbildning med järnjoner. Slutligen användes denna syntesväg för att framställa blocksampolymerer. Dessa blocksampolymerer användes sedan för att bilda miceller med lovande resultat vid utvärdering för leverans av läkemedel mot bröstcancer. / <p>QC 20160125</p>

The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.