Dynamics and interactions of nuclear proteins revealed by quantitative photobleaching microscopy

Tese de doutoramento em Biofísica (Biofísica), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007The nucleus is a complex cellular organelle, exhibiting a high degree of organization and also a highly dynamic nature. Live cell imaging using fluorescent proteins (FPs) as molecular tags and photobleaching techniques have been essential in revealing the dynamic nature of the cell nucleus. In this thesis, these tools were used to study molecular dynamics and interactions inside this cellular compartment. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) were used to analyze the kinetic behavior of spliceosome components SmE, U2AF65, U2AF35, SF1 and SC35 in the nucleus of living cells. The recruitment mechanism of splicing factors (SFs) to the sites of transcription is still poorly understood. Our results rule out the hypothesis that a transcription specific signal recruits SFs from the speckles. They also suggest the formation of multi-protein complexes distinct from the spliceosome. The existence of these complexes was confirmed by Fluorescence Resonance Energy Transfer (FRET) techniques, which revealed that SFs could interact with each other even in the absence of active splicing. A novel U2AF65 self-interaction was also detected, suggesting altogether that levels of SFs in speckles are consistent with self-organization mechanisms. The intranuclear mobility of mRNPs was studied using two GFP-tagged mRNA-binding proteins, PABPN1 and TAP, as mRNA markers. A novel FLIP method was devised to quantify the mobility of the RNA-bound and unbound pools of molecules and used to test whether myosin motors were implicated in mRNP movement. We show that this is not the case and that myosin inhibition appears to affect transcription instead. A novel FLIP after Photoactivation method was developed to study the nucleocytoplasmic exchange dynamics of nuclear proteins, yielding the permanence times of molecules inside the nucleus. The method was used to study the role of the structural domains of TAP in its shuttling activity.O núcleo celular é um organito complexo, dotado de um elevado grau de organização mas também uma natureza extremamente dinâmica. A utilização de proteínas fluorescentes como marcadores moleculares para visualização em células vivas, bem como as técnicas de photobleaching, têm sido essenciais na descoberta da natureza dinâmica do núcleo. Neste trabalho, estas ferramentas foram aplicadas no estudo da dinâmica e interacções moleculares dentro deste compartimento celular. As técnicas de Fluorescence Recovery After Photobleaching (FRAP) e Fluorescence Loss In Photobleaching (FLIP) foram utilizadas na análise do comportamento cinético dos componentes do spliceosoma SmE, U2AF65, U2AF35, SF1 e SC35 no interior do núcleo de células vivas. O mecanismo de recrutamento dos factores de splicing (SFs) para os locais de transcrição é ainda pouco conhecido. Os nossos resultados excluem a hipótese de haver um sinal associado à transcrição que seja responsável por este recrutamento. Sugerem ainda a formação de complexos multi-proteicos distintos do spliceosoma. A existência destes complexos foi confirmada por técnicas de Fluorescence Resonance Energy Transfer (FRET), que mostraram que os SFs podiam interagir uns com os outros mesmo na ausência de splicing activo. Foi ainda descoberta uma nova auto-interacção para o factor U2AF65, sugerindo os resultados no seu conjunto que a distribuição de SFs no núcleo é compatível com mecanismos de auto-organização. A mobilidade de mRNPs no núcleo foi estudada utilizando como marcadores moleculares duas proteínas que se ligam ao mRNA marcadas com GFP, PABPN1 e TAP. Foi desenvolvido um método de FLIP para quantificação da mobilidade das fracções ligadas e não ligadas ao mRNA e usado para testar a possibilidade de motores de miosina estarem envolvidos no movimento de mRNPs. Mostramos que tal não acontece e que a inibição de miosina parece antes afectar a transcrição. Um novo método de FLIP após foto-activação foi desenvolvido para estudar a dinâmica de trocas entre o núcleo e o citoplasma de proteínas nucleares, permitindo a estimação do tempo de permanência de moléculas dentro do núcleo. O método foi utilizado para investigar o papel dos diferentes domínios estruturais da proteína TAP na sua actividade de exportação nuclear.Fundação para a Ciência e Tecnologia (BD/21518/99); European Commission (“RNOMICS” QLG2-CT-2001-01554 and “Integrated Technologies for in vivo Molecular Imaging” LSHG-CT-2003-503259

Angle’s classification among Brazilian racial biotypes: a university based observational study

Background: The purpose of this study is to determine demographic profile and the pattern of malocclusion from the initial clinical records of patients who sought the dental clinics for orthodontic treatment and to determine difference in Angle’s classification among racial biotypes.Materials and Methods: Between the years 2011 and 2014, 1576 clinical records were selected and reviewed by one examiner to identify epidemiological characteristics. Angle’s classification, age, dentition, and others were scored, and after tabulation, data were grouped to find percentiles.Results: Class I was found at 57.9%; Class II, 31.4%, and Class III, 10.8%; there was no significant difference in gender distribution (49.6% of men and 50.4% of women); the orthodontic treatment plan indicated was comprehensive (77.4%) and the sample was composed, mainly, by white and mulatto biotypes (40.8% and 41.1%, respectively) and by youth aged 5–10 y.o. (32.1%) and aged 11–15 y.o. (29.4%). Conclusion: There was a high incidence of Class I and indication for comprehensive treatment. There were differences in the incidence of malocclusion for the Afro-Brazilian ethnic group

Live-Cell Visualization of Pre-mRNA Splicing with Single-Molecule Sensitivity

SummaryRemoval of introns from pre-messenger RNAs (pre-mRNAs) via splicing provides a versatile means of genetic regulation that is often disrupted in human diseases. To decipher how splicing occurs in real time, we directly examined with single-molecule sensitivity the kinetics of intron excision from pre-mRNA in the nucleus of living human cells. By using two different RNA labeling methods, MS2 and λN, we show that β-globin introns are transcribed and excised in 20–30 s. Furthermore, we show that replacing the weak polypyrimidine (Py) tract in mouse immunoglobulin μ (IgM) pre-mRNA by a U-rich Py decreases the intron lifetime, thus providing direct evidence that splice-site strength influences splicing kinetics. We also found that RNA polymerase II transcribes at elongation rates ranging between 3 and 6 kb min−1 and that transcription can be rate limiting for splicing. These results have important implications for a mechanistic understanding of cotranscriptional splicing regulation in the live-cell context

Facial harmony in orthodontic diagnosis and planning

Facial Harmony is one of the main goals of orthodontic treatment, and it is not always correlated with the attainment of cephalometric objectives. The purpose of this study was to evaluate two groups of subjects presenting a clinically balanced soft tissue profile using cephalometric radiographs. Thirty lateral cephalometric radiographs of white females, divided in two groups, one with excellent facial profile (Group 1), and the other with good facial profile (Group 2) were used. Student's t-test (P?< .05) was used to compare the cephalometric parameters of the 2 groups. Linear regression analysis was also performed between 1.NB and SnV-Pog and between AB horizontal and SnV-Pog'. Group 2 showed higher mean values than group 1 for ANB (p = 0.002), AB horizontal (p < 0.001), 1.NB (p < 0.001), and a lower mean value for SnV-Pog (p = 0.003). The higher the SnV-Pog value, the lower the 1.NB value, no matter what group was evaluated. For each 1 mm increase in SnV-Pog, a 0.61º decrease could be expected in 1.NB (p = 0.003). The higher the SnV-Pog value, the lower the AB horizontal value, although group 2 presented greater AB horizontal values. For each 1 mm increase in SnV-Pog, a 0.24 mm decrease could be expected in AB horizontal (p = 0.019). We concluded that women with good facial profile do not necessarily present the same cephalometric values, and facial analysis should be the main reference in planning orthodontic treatments and should be considered together with cephalometric analysis in an individualized way

A Stochastic View of Spliceosome Assembly and Recycling in the Nucleus

How splicing factors are recruited to nascent transcripts in the nucleus in order to assemble spliceosomes on newly synthesised pre-mRNAs is unknown. To address this question, we compared the intranuclear trafficking kinetics of small nuclear ribonucleoprotein particles (snRNP) and non-snRNP proteins in the presence and absence of splicing activity. Photobleaching experiments clearly show that spliceosomal proteins move continuously throughout the entire nucleus independently of ongoing transcription or splicing. Using quantitative experimental data, a mathematical model was applied for spliceosome assembly and recycling in the nucleus. The model assumes that splicing proteins move by Brownian diffusion and interact stochastically with binding sites located at different subnuclear compartments. Inhibition of splicing, which reduces the number of pre-mRNA binding sites available for spliceosome assembly, was modeled as a decrease in the on-rate binding constant in the nucleoplasm. Simulation of microscopy experiments before and after splicing inhibition yielded results consistent with the experimental observations. Taken together, our data argue against the view that spliceosomal components are stored in nuclear speckles until a signal triggers their recruitment to nascent transcripts. Rather, the results suggest that splicing proteins are constantly diffusing throughout the entire nucleus and collide randomly and transiently with pre-mRNAs

Opto-mechanical eye models, a review on human vision applications and perspectives for use in industry

The purpose of this review is to aggregate technical information on existent optomechanical eye models (OME) described in the literature, for image quality assessment in different applications. Several physical eye models have been reviewed from peer-reviewed papers and patent applications. A typical eye model includes an artificial cornea, an intraocular lens or other lens to simulate the crystalline lens, an aperture as the pupil, and a posterior retinal surface, which may be connected to a light sensor. The interior of the eye model may be filled with a fluid to better emulate physiological conditions. The main focus of this review is the materials and physical characteristics used and the dimensional aspects of the main components including lenses, apertures, chambers, imaging sensors and filling medium. Various devices are described with their applications and technical details, which are systematically tabulated highlighting their main characteristics and applications. The models presented are detailed and discussed individually, and the features of different models are compared when applicable, highlighting strengths and limitations. In the end there is a brief discussion about the potential use of artificial eye models for industrial applications.This work is supported by European Structural and Investment Funds in the FEDER component, through the Operational Competitiveness and Internationalization Programme (COMPETE 2020) [Project nº 39479; Funding Reference: POCI-01-0247-FEDER-39479]

Biodanza cómo proceso de renovación existencial para los ancianos

Neste estudo, buscamos, a partir do método etnográfico, identificar os efeitos da Biodança nos idosos que a vivenciam. O estudo foi realizado com oito idosos que integram o grupo de Biodança do SESC de Fortaleza, Ceará. Os dados coletados por meio da entrevista semi-estruturada e da observação participante foram analisados pelo método de análise de narrativa. Na busca do significado da Biodança para os idosos, evidenciou-se que ela constitui um mecanismo de enfrentamento das dificuldades com a saúde, estimulando a mudança de comportamento em relação às condições de saúde, por aumentar o ímpeto vital e a vontade de viver. Neste sentido, a Biodança promove o resgate da saúde de pessoas que envelhecem.In this study, we searched, from the ethnographic method, to identify the effects of Biodance in elders that lived it. The study was done with eight elderly that integrate the group of Biodance of SESC from Fortaleza, Ceará. The data were collected through semi-structured interview and participant observation, analyzed by method of narrative analysis. In the search of meaning of Biodance to elders, it highlighted that it constitutes a mechanism of facing of difficulties with health, stimulating the change of behavior in relation to health conditions, due increase the vital impetus and will to live. In this sense, the Biodance promotes the rescue of health that grew older.En este estudio se busca, a partir del método etnográfico, identificar los efectos de la biodanza en los adultos mayores que la experimentan. El estudio fué realizado con 8 adultos mayores que integran el grupo de biodanza de la SESC de la ciudad de Fortaleza, Ceará, Brasil. Los datos recolectados a traves de la entrevista semiestructurada y de la observación participante, fueron analizados por el método del análisis narrativo. En la busqueda del segnificado de la biodanza para las personas mayores de edad, se evidenció que ella constituye un mecanismo de enfrentamiento hacia las dificultades en su salud, estimulandolos a los cambios de comportamiento en relación a las condiciones de salud, aumentar su impetu vital y la voluntad de vivir. En este sentido, la biodanza promueve el rescate de la salud de las personas que envejecen

¡Que siga la función! Aprender teatro haciendo teatro

La presente propuesta recoge un nuevo enfoque docente en torno al género dramático y su proceso de enseñanza-aprendizaje en la Educación Secundaria. El teatro, así como los elementos que lo componen, se presentan como un recurso didáctico y pedagógico para su inclusión en el aula, de manera que el género nos sirva de herramienta para que los alumnos aprendan todo lo relativo al teatro mediante la práctica. Así, a través del trabajo cooperativo y la concepción artística del género, provocaremos una renovación metodológica, de forma que el género teatral adquiera una dimensión más cercana al alumnado, rompiendo con la imagen denostada que le persigue y que lo sitúa en el último eslabón de los géneros literarios. Para ello, se ofrecerá una visión renovada del género en la que serán los alumnos quienes guíen su aprendizaje a través de la escritura creativa, la lectura dramatizada y la dramatización; recursos que no solo servirán como vehículo de conocimientos, sino también como potenciadores de sus habilidades comunicativas, creativas y sociales y que conllevarán al desarrollo personal de los estudiantes. Valores como el respeto, la empatía o la cooperación se ponen de manifiesto en este proyecto de la mano del género dramático y dan paso a una educación inclusiva que atiende a la diversidad del aula.<br /

Rhinometric evaluation of nasal cavity geometry and its relation to the upper arch transverse distance

The objective of this study was to evaluate children's respiratory patterns in the mixed dentition, by means of acoustic rhinometry, and its relation to the upper arch width development. Fifty patients were examined, 25 females and 25 males with mean age of eight years and seven months. All of them were submitted to acoustic rhinometry and upper and lower arch impressions to obtain plaster models. The upper arch analysis was accomplished by measuring the interdental transverse distance of the upper teeth, deciduous canines (measurement 1), deciduous first molars (measurement 2), deciduous second molars (measurement 3) and the first molars (measurement 4). The results showed that an increased left nasal cavity area in females means an increased interdental distance of the deciduous first molars and deciduous second molars and an increased interdental distance of the deciduous canines, deciduous first and second molars in males. It was concluded that there is a correlation between the nasal cavity area and the upper arch transverse distance in the anterior and mid maxillary regions for both genders