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abstract: In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment. / Dissertation/Thesis / Ph.D. Chemistry 2011

Orientadores: Jorge Humberto Nicola, Ester Maria Danielli Nicola / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T06:03:03Z (GMT). No. of bitstreams: 1 Hernandez_JoaoWagnerRodrigues_D.pdf: 4481265 bytes, checksum: 5351d4ae13e0c0baa8fd480610047a9b (MD5) Previous issue date: 2009 / Resumo: A pesquisa de fluorescência nativa em tecidos biológicos tem despertado grande interesse na Biologia e Medicina. A fluorescência óptica tornou-se instrumento indispensável aos diagnósticos, sendo um método eficiente e não invasivo. Neste trabalho investigamos a presença de fluorescência nativa em áreas corpóreas de ratos Wistar, subdivididos em dois grupos: um com alimentação normal e outro com dieta de glicose 5%. Em áreas internas (experimento 1), não ficou evidenciada a fluorescência nativa em animais com alimentação normal de água e ração. Nos animais submetidos à dieta de glicose, observamos a presença de fluorescência nativa vermelha, captada por espectroscopia, alcançando um pico máximo de emissão luminosa em 120 horas de dieta e retomando valores iniciais, quando sua dieta voltou ao normal. O conteúdo do lúmen intestinal foi analisado espectroscopicamente e por prova bioquímica. O gráfico obtido foi semelhante ao da PpIX e o teste bioquímico (Eales modificado ) resultou positivo à presença de substância porfirínica. A fluorescência em áreas externas, bolsa escrotal, focinho, pata e cauda, também foi pesquisada nas mesmas condições de dieta (normal e glicosada), evidenciando que animais com dieta normal, não apresentaram fluorescência e nos de dieta de glicose, deparamos com pico máximo de fluorescência em 120 horas de dieta e retomando valores iniciais, quando sua dieta voltou ao normal. A fluorescência nativa encontrada nos animais submetidos à dieta glicosada, evidencia uma nova condição fisiopatológica e que se assemelha a um quadro de porfiria reversível, bem identificada e documentada pelo experimento 2. Os resultados mostram a importância da espectroscopia óptica como um método de diagnóstico precoce, simples e confiável, para uso em diversos tipos de doenças ou disfunções com acúmulo de pigmentos fotossensíveis. Chama, ainda, a atenção para situações em que fluorescências nativas possam ser tomadas como diagnóstico falso-positivo de alterações patológicas ou não. / Abstract: The research of native fluorescence in biological tissues has aroused great interest in Biology and Medicine. The optic fluorescence became an important instrument for the diagnosis, being an efficient and non-invasive method. In this work we investigated the presence of native fluorescence in corporal areas of Wistar rats, subdivided in two groups: one with normal feeding and another one with glucose diet 5%. In the intestinal tract (Experiment 1), there was no evidence of fluorescence in animals with normal feeding of water and ration. In the animals submitted to the glucose diet, we observed the presence of red native fluorescence, caught by spectroscopy, reaching a maximum peak of luminous emission in 120 hours of diet with the return to normal values after the re-introduction of normal diet. The content of the intestinal lumen was analyzed spectroscopically and by biochemical test. The obtained graph was similar to the one of the PpIX and the test modified by Eales resulted positive to the presence of porfirinic substance. For Experiment 2, a group of 30 animals were kept at the same diet conditions (normal and glucose 5%) up to 120 hours. Four external areas were selected for the study (paw, tail, nose and scrotum). The group os animals under glucose diet presented a marked fluorescence on the selected areas with a maximum pick at 120hours. After 24 and 48 hours of reestablishment of the regular solid diet the fluorescence decreased disappearing totally at 48 hour. The native fluorescence found in the animals submitted to the glucose diet, evidences a physiopathological condition and it is similar to a condition of reversible porfiria, well identified and registered by Experiment 2. The results show the importance of the optic spectroscopy as a method of precocious, simple and trustworthy diagnosis, for use in diverse types of illnesses or dysfunctions with accumulation of photosensitive pigments. It also calls attention, for situations where native fluorescence can be taken as false-positive diagnosis of malignant alterations. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Climate change has given rise to higher demand of renewable energy. Conventional non-organic solar cells are still seen the most on the market but the last 10-20 years there has been an exponential increase in research about organic solar cells. The research demands measurements done on increasing fast time scales as insights about the topic and technology develops. Here is presented configurations done at the Isaak spectrometer at Freie Universit\"{a}t Berlin. The spectrometer is used for studying charge transport in organic solar cells as well as hybrid solar cells made from organic and inorganic semiconductors. One important feature for the time response is the Q factor property of the resonator in which the sample is placed. The Q factor describes the rate of energy loss relative to the stored energy of the resonator. The Q factor dependency over different temperature was studied in order to gain vital knowledge about the time response of the setup. Further new equipment was been installed at the setup and programmed in FSC2. This in order to increase the sensitivity of the optical setup, secure the personnel from possible dangerous excitation laser and in order to do more automated measurements. As the optical path was modified by installing a new optical fiber a new calibration had to be done. Finally, in order to try the functionality of the new equipment an automated energy dependency measurement was done on the polymer MEH-PPV and the polymer:fullerene blend MEH-PPV:PCBM. The automatic process of measuring was successful.

Cette thèse porte sur le développement d'un appareil couplant la spectrométrie de masse avec la spectrométrie de mobilité ionique et la spectroscopie laser, dans le but d'effectuer des analyses conformationnelles sur des édifices biomoléculaires. La construction, la mise au point et l'optimisation de l'appareil font l'objet de la première partie de ce mémoire. En particulier, il s'est agi de déterminer les caractéristiques de l'appareil (résolution, fréquence de fonctionnement…) par une série de tests sur des systèmes modèles. Dans un deuxième temps, nous avons effectué des mesures de spectroscopie d'action sur des conformères sélectionnés en mobilité. Nous avons étudié la relation entre la conformation et les propriétés optiques d'un système en mesurant le photo-détachement d'électron de divers conformères sélectionnés. Dans le cadre de la spectroscopie d'action, nous avons utilisé les possibilités nouvelles offertes par l'appareil pour réaliser des expériences de photo-isomérisation cis-trans sur des complexes non-covalents. Sur cet exemple, nous avons montré l'intérêt de cet appareil pour mesurer des spectres d'action de photo-isomérisation. Et enfin, nous avons montré la possibilité de réaliser des mesures de spectroscopie d'action basée sur le Transfert d'Energie par Résonance de Förster (FRET), en phase gazeuse, résolu en conformation / This thesis deals with the development of a new instrument coupling mass spectrometry, ion mobility spectrometry and laser spectroscopy. The aim is to perform structural analysis on biomolecular systems.The first part of this thesis focuses on the construction, the development and the optimization of the set-up. The main point was to determine the features of the set-up (resolution, working frequency) by series of tests with model systems.In a second phase, we did conformer resolved action spectroscopy. We studied the relation between the conformation and the optical properties of one system by measuring photo- electron detachment on different selected conformers. In the framework of action spectroscopy we used the new capacity of the set-up to perform cis-trans photo-isomerization on non-covalent complexes. We showed with this example interest of the use of this instrument to measure photo-isomerization action spectra. We finally showed the possibility to perform conformer resolved action spectroscopy measurements based on Förster Resonance Energy Transfer (FRET) in the gas phase

One of the aims of forensic sciences is to determine the identities of victims of crime. In some cases the investigators may have ideas as to the identities of the victims and in these situations, ante mortem photographs of the victims could be used and identities established through skull-photo superimposition. The aim of this study was to evaluate the accuracy of a newly developed digital photographic superimposition technique on a South African sample of cadaver photographs and skulls, from the Pretoria Bone Collection. Forty facial photographs were selected and for each photograph, 10 skulls (including the skull corresponding to the photograph) were used for superimposition. The investigator did not know which of the 10 skulls corresponded to the photograph in question. The skulls were scanned 3-dimensionally, using a Cyberware™ Model 3030 Colour-3D Scanhead scanner. Once scanned, the raw data for the skulls were ‘cleaned’ using Cysurf™ programme. The photographs were also scanned for superimposition in the 3D Studio Max programme. Superimposition in 3D Studio Max involves a morphological superimposition, whereby a skull is superimposed over the photograph and assessed for a morphological match. Superimposition using selected anatomical landmarks was also performed to assess the match. A total of 400 skull-photo superimpositions were carried out using the morphological assessment and another 400 using the anatomical landmarks. In 85% of cases the correct skull was included in the possible matches for a particular photograph using morphological assessment. However, in all of these cases, between zero and three other skulls out of 10 possibilities could also match a specific photograph. In the landmark based assessment, the correct skull was included in 80% of cases. Once again, however, between one and seven other skulls out of 10 possibilities also matched the photograph. When using the morphological and landmark assessments combined, 97.5% of correct skulls were included in the list of possibilities, but between one and seven false positives per case were found. This study indicates that skull-photo superimposition has limited use in the identification of human skeletal remains, but may be useful as an initial screening tool. Corroborative techniques should also be used in the identification process. / Thesis (PhD)--University of Pretoria, 2011. / Anatomy / unrestricted

Nitrate esters of aromatic alcohols were synthesized by esterification which involved competition between 0-nitration and aromatic C-nitration. TLC analysis gave a pattern of adsorption affinities for the nitroxy group and other substituents consistent with the molecular conformations. The NMR frequency of the α-protons showed a linear correlation with the accepted group electronegativities of the substituents in molecules with rigid carbon skeletons and gave a value of 4.18 kcal/mole for the nitroxy group. The symmetric and asymmetric IR stretching frequencies of nitroxy groups in dilute cyclohexane solution were shifted to higher values by steric interaction between contiguous groups when the C-ONO₂ bonds were constrained to coplanarity. The UV spectra showed benzenoid, π→π*, and n→π* bands and a solvent perturbation effect assigned to a solvent → solute charge-transfer interaction. The nitrate esters reacted with the solvent when irradiated in solution in the wavelength range of the n→π* excitation. Product analysis indicated that C-C bond cleavage occurred via intermediate alkoxyl radicals. Rate studies showed the following order of reactivity: benzyl nitrate < dl-hydrobenzoin dinitrate < meso-hydrobenzoin dinitrate, < trans-1, 2-acenaphthenediol nitrate < cis-1, 2-acenaphthenediol dinitrate. The rate measurements and ESR spectra gave evidence of intramolecular energy transfer from the naphthalene moiety to the nitroxy groups in the 1, 2-acenaphthenediol dinitrates assigned as a singlet→singlet transfer. Calculations from the apparent first-order rate constants and spectra showed that benzyl nitrate, and meso- and dl-hydrobenzoin dinitrates photolysed with a quantum yield of about 2 in benzene solution. A solvent effect caused k[subscript Et₂O] > k[subscript EtOH] >k[subscript PhH]. On the basis of product analysis, rate measurements, estimated quantum yields and ESR spectra a mechanism for the nitrate ester photolysis was proposed. / Science, Faculty of / Chemistry, Department of / Graduate

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

Magister Artium - MA / This project was born after the researcher, a practicing social worker at a psychiatric facility, observed the presence of high rates of anxiety and depressive disorders among transgender patients. These patients were often also abandoned by their family or primary caregivers. This research was part of a larger National Research Foundation (NRF) project in the Western Cape and Gauteng, which explored LGBT older persons’ care needs. It differed from the main project in that it focused on the mental health care challenges experienced by older transgender people. The project was funded by the NRF and the researcher was allocated funding from that project to explore LGBT aging and care in the marginalised areas. LGBT discrimination has been indicated as a key factor in the onset of mental health issues later in adulthood. Older adults are generally at a higher risk of developing mental disorders. The older transgender community with mental health care needs thus often suffers multiple forms of oppression within a heteronormative society. The aim of the research was to determine the mental health care challenges experienced by older transgender people in the Cape Metropole, Western Cape. Objectives to reach this aim included exploring and describing the unique challenges faced by older transgender people, their experiences when accessing mental health care, and describing strategies of addressing their mental health care needs. The research methodology entailed a qualitative approach. Snowball sampling was applied for selecting five older transgender participants and five key informants. Photo voice, a Participatory Action Research (PAR) design, was used. Data collection consisted of in-depth interviewing, focus groups, and photo journaling. Themes were developed from the data utilising Thematic Analysis, aided by Atlas.ti software. Ethics and trustworthiness were certified through guidance by the research supervisor. This research was classified as high risk, since it involved marginalised individuals from the aged LGBT community. Anxiety in the group was anticipated and dealt with by providing further counselling where needed. The findings indicate that older transgender people experience minority stress across all racial and age cohorts. They suffer heightened anxiety when accessing healthcare services, as they anticipate transphobia and oppression. In addition, the intersectional socio-economic status of age and gender identity seems to contribute to building resilience within the participants. Lastly, substance use and social and professional support were identified as coping strategies in the face of on-going discrimination.

Passive acoustic monitoring has been successfully used on many elasmobranch species, but no such study has yet been published for the whale shark (Rhincodon typus). In some ways this is surprising as the known whale shark aggregation sites would seem to be ideal targets for this method. For this dissertation, two acoustic studies were carried out in Saudi Arabia and Tanzania. Each was performed in parallel with visual surveys and the Saudi population was also studied using satellite telemetry. Sighting and acoustic data were compared at both sites, and the results were mixed. The acoustic monitoring largely confirmed the results of visual surveys for the Saudi Arabian sharks, including seasonality, residency and a degree of parity and integration between the sexes that is unique to this site. Satellite tracks of tagged Saudi sharks were used to confirm that some animals migrated away from the aggregation site before returning in subsequent seasons, confirming philopatric behavior in this species. In contrast, the acoustic results in Tanzania demonstrated year-round residency of whale sharks in the area, despite seasonal declines in visually estimated abundance. Seasonal changes in habitat selection render the sharks at this site temporarily cryptic to visual sampling. The differing results are compelling because both the philopatric behavior demonstrated in Saudi Arabia and the cryptic residency of the Tanzanian sharks could explain the seasonal patterns in whale shark abundances reported at other aggregation sites. Despite their differences, both sites in this study can be classified as secondary whale shark nurseries and each may be a vital feeding ground for its respective population.