Orientador: Idomeo Bonetti Filho / Resumo: O objetivo deste estudo foi avaliar a eficiência de três métodos de remoção da medicação intracanal contendo hidróxido de cálcio(CH) com propilenoglicol(PG) e meticelulose(MC) e sua repercussão sobre a resistência de união e penetração de um cimento endodôntico (AH Plus) à dentina radicular. Inicialmente sessenta dentes unirradiculares foram preparados até o instrumento F5, em seguida foram divididos em 2 grupos(n=30) de acordo com o veículo(PG) e (MC). Após 7 dias, foram realizadas microtomografias dos espécimes, e mensurados a área total de preenchimento da medicação contendo hidróxido de cálcio no canal radicular. Em seguida os espécimes foram submetidos a três protocolos de remoção n=10: irrigação convencional(CI), irrigação ultrassônica passiva(PUI) e irrigação ultrassônica contínua(CUI) e uma nova análise microtomográfica foi realizada. A persistência dos resíduos HC (em mm3) foi avaliada após o uso dos protocolos de remoção. Em seguida os mesmos espécimes foram obturados com cimento endodôntico à base de resina epóxi associados com rodamina, e após a divisão em terços cervical, médio e apical, foram submetidos aos testes de push-out e posteriormente análise dos tipos de fratura e em seguida análise da penetração dentinária com microscopia a laser confocal. Houve similar persistência de resíduos de HC, independentemente do segmento analisado (P>0.05). Os valores obtidos no teste de push-out e penetração dentinária foram similar nos terços cervical e médio radicular (P >... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

[pt] Devido à diminuição do número de descobertas de novas reservas de óleo e gás nas últimas décadas, as companhias de petróleo têm demonstrado um interesse cada vez maior em melhorar a eficiência dos processos de recuperação de óleo. Geralmente, após as fases de recuperação primário e secundário, uma grande quantidade de óleo permanece dentro do reservatório, pois a extração se torna não rentável. Assim, cresce o número de estudos voltados para a recuperação avançada de petróleo, com o objetivo de obter uma melhor fração de recuperação. O foco deste trabalho é estudar os fundamentos do deslocamento de óleo em meios porosos usando um microscópio confocal de varredura a laser, que possibilita visualizações 3D com boa resolução. A análise foi no deslocamento de óleo resultante da injeção de uma suspensão de capsulas de goma gelana em água após a injeção de água. Estas capsulas, movendo com a água, bloqueiam alguns dos caminhos preferenciais e forçam a água a deslocar uma parte do óleo preso. O resultado alcançado foi uma coleção de imagens 3D de meios porosos artificiais, nas quais foi possível distinguir a distribuição das fases (microcápsulas, fase aquosa e oleosa) dentro dos meios porosos, antes e após a injeção das microcápsulas. Essas imagens mostraram que as microcápsulas de goma gelana bloqueiam os caminhos preferenciais da água e que, após o bloqueio, alguns gânglios de óleo foram deslocados de suas posições originais. Esta tese aplica técnicas modernas de microscopia para examinar o conceito por trás da recuperação avançada de óleo usando microcápsulas. / [en] Thanks to decay of new discoveries of oil and gas reserves in the past decades, oil companies have a growing interest in the increase of oil recovery efficiency. Commonly, after primary and secondary recovery phases, a largeamount of oil remains inside the reservoir, as it becomes unprofitable to continue the extraction. Thus, the number of studies focused on enhanced oil recovery is growing, aiming to obtain a better recovery fraction. The focus of this work is to study the fundamentals of oil displacement in porous media using a confocal laser scanning microscope, which enables 3D visualization with a good resolution. The analysis was on oil displacement that results from the use of a suspension of gellan gum microcapsules in water injected after water injection. These microcapsules, moving along with the water, blocked some of the preferential paths and forced the water to displaces parcels of the trapped oil. The result achieved was a collection of 3D images from artificial porous media, in which it was possible to distinguish the distribution of phases (microcapsules, oil, and aqueous phases) inside the porous media, before and after the microcapsules injection. These images showed that indeed the gellan gum microcapsules blocked preferential water paths and that, after the blockage, some oil ganglia were displaced from their original positions. This thesis applies modern techniques of microscopy to investigate the concept behind enhanced oil recovery using microcapsules.

La microscopía confocal de reflectancia in vivo (MCR) es una técnica no invasiva que permite obtener un diagnóstico de forma inmediata y en tiempo real de patología cutánea tumoral con una precisión diagnóstica que se acerca al diagnóstico histológico convencional. La MCR puede permitir un seguimiento clínico de respuesta a tratamientos no invasivos de patología tumoral, como la terapia fotodinámica, sin necesidad de realizar biopsias. El presente trabajo reúne cuatro estudios realizados con el fin de demostrar la utilidad de la técnica de MCR en el estudio y manejo clínico de las neoplasias cutáneas. En el primer estudio se incluyeron de forma prospectiva 154 tumores cutáneos para establecer qué parámetros de microscopía confocal se relacionaban con cada tumor, analizar si estos criterios eran reproducibles y finalmente evaluar la correlación de estos parámetros con la dermatoscopia y la histología convencional. Según los resultados de este primer trabajo, cuatro características observables mediante MCR permitían diferenciar las lesiones melanocíticas (LM) de las no melanocíticas (LNM): patrón epidérmico en empedrado, crecimiento pagetoide, presencia de nidos celulares en la dermis y presencia de papilas dérmicas bien definidas en toda la lesión. Dentro de las LM, la presencia de células redondas en estratos suprabasales y de células nucleadas atípicas en la dermis se asociaban al diagnóstico de melanoma; mientras la presencia de papilas dérmicas con contorno reflectante (anillos basales) así como la observación de células basales típicas, se asociaban a nevus. Basándonos en la reproducibilidad y correlación histológica y dermatoscópica de los criterios de MCR, se desarrolló un algoritmo en dos etapas para el diagnóstico de melanoma consiguiendo una sensibilidad del 86,1% y una especificidad del 95.3% (J Am Acad Dermatol. 2009;61:216-29). En el segundo trabajo, se estudiaron los parámetros de microscopía confocal en melanomas de extensión superficial (MES) en fase de crecimiento vertical y en melanomas nodulares (MNs). Para ello se incluyeron de forma prospectiva 10 MNs, 10 MES con área nodular y 10 MES con área palpable. Se realizó un análisis sistemático y estudio estadístico de las características dermatoscópicas, confocales e histológicas de los 3 grupos de lesiones. Mientras los MNs mostraban patrones de dermatoscopia inespecíficos, los MES exhibían patrones multicomponente y puntuaciones más altas en los algoritmos de dermatoscopia. Por MCR, los MNs tenían poco crecimiento pagetoide y presentan a menudo un patrón normal de la epidermis, a diferencia de los MES que se caracterizaban por un patrón desestructurado de la epidermis y la presencia de abundantes células en estratos suprabasales Tanto en los MNs como en las áreas nodulares de los MES, en la unión dermo-epidérmica no se visualizaban las papilas dérmicas, en su lugar se observa una proliferación de células reflectantes atípicas no agregadas. En la dermis, los MNs exhibían a menudo unos agregados celulares característicos de aspecto cerebriforme, que podían observarse también en las áreas nodulares de algunos MES (Arch Dermatol. 2008;144:1311-20). En un tercer artículo estudiamos las características del carcinoma basocelular pigmentado (CBC) mediante MCR, histología e inmunohistoquímica (S-100, MelanA, HMB-45 y CD1a).Mediante MCR demostramos la presencia de estructuras reflectantes muy características de aspecto dendrítico dentro de los nidos tumorales situados en la dermis de los CBC estudiados, que correspondían por estudio inmunohistoquímico a melanocitos no neoplásicos que poblaban los nidos (Arch Dermatol. 2007;143:883-6). Finalmente, en un cuarto trabajo estudiamos la aplicabilidad de la MCR en el diagnóstico y seguimiento de pacientes con genodermatosis de alto riesgo de desarrollar cáncer cutáneo (síndrome de Gorlin -SG- y Xeroderma pigmentoso -XP-) afectos de CBCs múltiples y tratados con terapia fotodinámica con metil-aminolevulinato (MAL). Se incluyeron 4 pacientes con SG y 2 hermanos con XP. Se trataron lesiones únicas o múltiples en zonas localizadas con 1 a 3 ciclos de TFD-MAL. La exploración con MCR se realizó antes y 3 meses después del tratamiento en las lesiones diana. Se trataron 13 CBCs pigmentados faciales en los pacientes afectos de XP y múltiples lesiones en cara o tronco (hasta 200) en los afectos de SG. Globalmente se obtuvo una remisión clínica completa en un 25- 67% de las lesiones. La respuesta al tratamiento puedo ser correctamente evaluada mediante MCR (J Eur Acad Dermatol Venereol. 2011;25:819-27). En conclusión, la MCR parece ser útil en el diagnóstico diferencial de las lesiones pigmentadas y complementa a la dermatoscopia en el diagnóstico del melanoma. Esta técnica permite caracterizar subtipos de melanoma como el melanoma nodular. También sirve de ayuda en el diagnóstico y caracterización del carcinoma basocelular pigmentado y en su monitorización tras tratamiento con terapias no invasivas como la terapia fotodinámica. / This work brings together four studies to demonstrate the clinical utility of reflectance confocal microscopy (RCM) in the clinical management of cutaneous neoplasms. In the first study we prospectively included 154 skin tumors in order to develop an algorithm for the in vivo diagnosis of skin tumors. We analyzed RCM features on stored images before excision and performed statistical analyses to determine the association of RCM features with tumor types. Four confocal features differentiated melanocytic lesions (ML) from non-melanocytic lesions (NML): cobblestone pattern of epidermal layers, pagetoid spread, mesh appearance of the dermoepidermal junction, and the presence of dermal nests. Within ML, the presence of roundish suprabasal cells and atypical nucleated cells in the dermis was associated with melanoma, and the presence of edged papillae and typical basal cells was associated with nevi. Based on the reproducibility and histological and dermoscopic correlation of MCR criteria, we developed a two-step algorithm for the diagnosis of melanoma, achieving a sensitivity of 86.1% and specificity of 95.3% (J Am Acad Dermatol. 2009; 61: 216-29). In the second work we characterized nodular melanoma (NM) by dermoscopy, RCM and histopathology. We included 10NMs, 10 superficial spreading melanomas (SSMs) with a nodular area, and 10 SSMs with a blue palpable area but not yet nodular. Whereas NMs had predominantly nonspecific global dermoscopic patterns, SSMs exhibited a multicomponent pattern and higher dermoscopic scores. By RCM, distinctive confocal features were observed at all cutaneous levels in NMs compared with SSMs, suggesting different biological behavior. (Arch Dermatol. 2008, 144:1311-20). In the third article we explored the confocal features of pigmented basal cell carcinoma (BCC) and correlated the findings with histology and immunohistochemistry. RCM revealed highly refractive dendritic structures within tumor nests that corresponded to the presence of melanocytes within the tumor. RCM allowed the study of pigmented BCC and the identification of specific criteria. (Arch Dermatol. 2007; 143:883-6). Finally, in the fourth paper we aimed to evaluate the efficacy of methyl-aminolevulinate (MAL) photodynamic therapy (PDT) in basal cell carcinomas (BCCs) from patients with Gorlin syndrome (GS) and Xeroderma pigmentosum (XP), and to determine the utility of RCM in the diagnosis and the evaluation of therapeutic response. We included 4 patients with GS and 2 siblings with XP. Single or multiple lesions in localized areas were treated with 1 to 3 cycles of MAL PDT. RCM was performed before and 3 months after the treatment in target lesions. Overall, we obtained a complete clinical remission in 25 to 67% of the lesions. RCM could identify confocal features for BCC and assess tumor remissions after treatment (J Eur Acad Dermatol Venereol. 2011; 25:819-27).

A reproducible in vitro model of mechanically injured (scalpel cut) articular cartilage was developed in this work utilising bovine and human osteochondral tissue. Using fluorescence-mode confocal laser scanning microscopy (CLSM), the model allowed (1) spatial and temporal quantification of in situ (within the matrix) chondrocyte viability following a full thickness cartilage injury and (2) serial evaluation of three chondroprotective strategies in injured bovine and human articular cartilage: (a) medium osmolarity (b) medium calcium concentration and, (c) subchondral bone attachment to articular cartilage. Medium osmolarity significantly influenced superficial zone chondrocyte death in injured (scalpel cut) bovine and human articular cartilage. Greatest percentage cell death occurred at 0 mOsm (distilled water). Conversely, a raised medium osmolarity (600 mOsm) was chondroprotective. The majority of in situ cell death occurred within 2.5 hours of the experimental injury, with no further increase over 7 days. Exposure of articular cartilage to calcium-free media significantly decreased superficial zone chondrocyte death in injured (scalpel cut) articular cartilage compared with exposure to calcium-rich media (2-20 mM). In calcium-rich media, the extent of percentage cell death increased with increasing medium calcium concentration but remained localised to the superficial zone of injured articular cartilage over 7 days. However, in calcium-free media, there was an increase in percentage cell death within deeper zones of injured articular cartilage over 7 days. Excision of subchondral bone from injured (scalpel cut) articular cartilage resulted in an increase in chondrocyte death at 7 days that occurred in the superficial zone of injured as well as the adjacent uninjured regions of articular cartilage. However, the presence of subchondral bone in the culture medium prevented this increase in chondrocyte death within the superficial zone. Subchondral bone may have interacted with articular cartilage via soluble mediator(s) that influenced chondrocyte survival. In human articular cartilage, healthy subchondral bone also interacted with articular cartilage in explant culture and promoted in situ chondrocyte survival, while sclerotic subchondral bone was detrimental to chondrocyte viability. These findings are of translational relevance to fluid management systems used during open and arthroscopic articular surgery, clinical and experimental research into cartilage injury, repair and degeneration as well as current techniques of tissue engineering.

The aim of this study was to investigate in vitro transdermal diffusion of a small peptide namely arginine vasopressin (AVP) with the aid of the novel PheroidTM drug delivery system. Generally, peptides seem unfit for transdermal permeation, but it was thought prudent to explore the suitability of this lipid-based system after success was achieved with entrapment of tuberculostatics, bacteria and viruses. Bestatin (a selective aminopeptidase inhibitor) was employed to circumvent any skin-related degradation of the active. Therefore, the effect of bestatin on the preservation of AVP during diffusion was investigated. Vertical Franz cell diffusion studies were conducted with female abdominal skin, with AVP at a concentration of 150 pglml in the donor phase and Hepes buffer as the receptor phase over a twelve-hour period. To prove entrapment of AVP within the lipid structures of the PheroidsTM, fluorescentlylabelled samples were monitored by means of confocal laser scanning microscopy (CLSM), which revealed definite entrapment. In vitro permeation profiles for AVP exhibited a biphasic character, with the majority of permeation occurring during the first two hours. The PheroidTM delivery system proved to be advantageous when applied as delivery medium. The inclusion of bestatin has an enhancing effect on permeation probably due to its protection of AVP. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Drosophila melanogaster and many other insects harbor intracellular bacterial symbionts that are transmitted vertically from infected host mothers to their offspring. Many of these bacteria alter host reproductive developmental processes in order to increase their transmission success. For example, Spiroplasma, a spirochete that naturally infects D. melanogaster, selectively kills males during mid-embryogenesis while sparing females. Previous studies suggested that Spiroplasma interacts genetically with the male-specific dosage compensation pathway, which causes ~2-fold up-regulation of most genes located on the male’s single X chromosome so that their expression matches the levels found in females who have two Xs. To further test this idea, I used confocal microscopy to visualize dosage compensation complex (DCC) localization and activity in infected as well as uninfected embryos. In the presence of Spiroplasma, the DCC became abnormally mis-localized across the nucleus. This pattern was accompanied by abnormal acetylation of histone H4K16, a mark induced by DCC activity and needed for proper X chromatin remodeling. My results imply that Spiroplasma directly targets the DCC by misdirecting it to uncompensated regions of the genome, an effect that leads to abnormal gene mis-regulation and consequent lethality (work from other members in our group). To further investigate this interaction, we transgenically expressed low levels of MSL-2 in both Spiroplasma infected and uninfected embryos in order to cause ectopic formation of the DCC in the female sex. I found that when infected, female embryos expressing the DCC showed significantly reduced viability in comparison to uninfected transgenic females. This result supports the notion that Spiroplasma uses the DCC in a dominant gain-of-function manner to kill embryos.

Recent studies of hoplonenemertean planuliform larvae have clarified their development and provided insight into larval evolution within the phylum. However, an assessment of viviparous development using modern techniques is lacking. To help facilitate a comprehensive comparative evaluation of developmental diversity within hoplonemerteans, we have conducted a confocal laser scanning microscopy investigation of the development in Prosorhochmus americanus, one of the few viviparous hoplonemertean species. Phalloidin staining provides evidence of a modified transitory larval epidermis, and reveals that the foregut, midgut, proboscis, central nervous system, and body wall musculature form early in development, consistent with observations for planktonic and encapsulated hoplonemertean larvae. However, invaginations characteristic of these larvae were not observed. Acetylated tubulin labeling and light microscopy shows that embryos are uniformly ciliated, and some specimens possess a caudal ciliary cirrus and/or apical tuft which are characteristic of planktonic larvae. These are interpreted as vestigial structures in the non-swimming P. americanus embryos. The findings provide additional evidence that hoplonemerteans exhibit a form of metamorphosis in their life history and thus exhibit indirect development. However, a comparative assessment of larval features in P. americanus suggests an evolutionary trend towards direct development in this species.

The anterior cingulate cortex (ACC) can influence emotional and motivational states in primates by its dense connections with many neocortical and subcortical regions. Pyramidal neurons serve as the basic building blocks of these neocortical circuits, which have been extensively studied in other brain regions, but their morphological and electrophysiological properties in the primate ACC are not well understood. In this study, we used whole-cell patch clamp and high-resolution laser scanning confocal microscopy to reveal the general electrophysiological properties and detailed morphological features of layer 2 and 3 pyramidal neurons in ACC (area 24/32) of the rhesus monkey. Neurons from both layers had similar passive membrane properties and action potential properties. Morphologically, dendrites of layer 3 ACC neurons were more complex than those of layer 2 neurons, by having dendrites with longer total dendritic lengths, more branch points and dendritic segments, spanning larger convex hull volumes. This difference in total dendritic morphology was mainly due to the apical dendrites. In contrast, the basal dendrites displayed mostly similar features between the two groups of neurons. However, while apical dendrites extend to the same layer (layer 1), the basal dendrites of layer 3 extended into deeper layers than layer 2 because of the difference in soma-pia distance. Thus, basal dendrites of the two groups of neurons receive different laminar inputs. Analysis of spines showed that more spines were found in neurons of layer 3 apical dendritic arbors than layer 2 neurons. However, the apical spine densities were similar between neurons in the two layers. Thus, while higher spine number suggests that layer 3 neurons receive more excitatory input than layer 2 neurons, the similar spine density suggests similar spatial and temporal summation of these inputs. The combined effects of increased number of excitatory input and higher dendritic complexity in layer 3 than in layer 2 ACC neurons suggest the additional information received by layer 3 neurons, especially in the apical dendrites, might undergo more complex integration.

Basic information about nail behaviour, under passive and especially iontophoretic condition, lacks in the literature. Thus, this thesis aims to fill gaps in the nail understanding by studying the potential and feasibility of the application of iontophoresis to human nail. The iontophoretic and passive delivery of Sodium Fluorescein (SF) and Nile Blue Chloride (NBC) were studied, in vitro, in order to determine their pathways as well as their depth and uniformity of penetration into the nail. The permselective properties of the nail were investigated by characterizing the contribution of electroosmosis, using mannitol as a marker, and by studying the flux of two inorganic cations, sodium and lithium, during in vitro experiments. Finally, the feasibility of transungual iontophoresis and the extraction of sodium and chloride ions from the body through the nail plate were performed on a group of human volunteers. Iontophoresis led the fluorescent markers slightly deeper into the nail plate than passive diffusion. The delivery of the bianion and of the cation was not different. Both compounds mainly penetrated the nail via the transcellular pathway. Electroosmosis resulted only in a slight enhancement of the mannitol fluxes compared to passive diffusion and the fluxes presented high variability, especially at pH 7.4 and when the current was applied in the anode-to-cathode direction. The delivery of the two inorganic cations was significantly higher at pH 7.0 than at pH 4.0 and supported that nails hold a negative charge at physiological pH. Ions were easily extractable through the nail plate during in vivo iontophoresis and all volunteers' feedbacks supported iontophoresis as an acceptable technique. This thesis demonstrated the feasibility and potential of in vivo transungual iontophoresis.

Este estudo teve como objetivo avaliar, em microscopia confocal à laser (CLSM), a ação antimicrobiana do Neo MTA Plus, do Biodentine e do MTA Angelus, utilizando um modelo de biofilme multi-espécies microcosmo, e descrever a morfologia dos microrganismos encontrados no biofilme, através da análise em microscópio eletrônico de varredura (MEV). Cinquenta e seis blocos de dentina, confeccionados a partir de raízes de dentes humanos, foram cortados em dimensões de 5 x 4 x 4 mm, limpos e esterilizados. Dois blocos foram utilizados para o controle do processo de esterilização. Os demais 54 foram fixados em placas de Hawley confeccionadas para 6 voluntários. Cada voluntário utilizou uma placa por 72 horas. Após esse período, os blocos foram mantidos em poços de cultura celular, contendo BHI, em estufa à 37ºC, por um período de 21 dias. Ao final do processo de maturação do biofilme, as amostras foram divididas aleatoriamente em 5 grupos, conforme os cimentos a serem testados e o método de análise: Neo MTA Plus (n=12), Biodentine (n=12), MTA Angelus (n=12), controle negativo (n=12) e grupo para análise em MEV (n=6). A seguir, os cimentos foram colocados em contato com o biofilme dos blocos dos grupos-teste e os conjuntos (bloco + cimento) ou os blocos do grupo controle e para análise em MEV foram recolocados em poços de cultura contendo BHI, onde permaneceram por 7 dias à 37ºC. A análise em CLSM foi realizada nos blocos de dentina e nos cimentos, separadamente. A analise em MEV foi realizada nas seis amostras do grupo correspondente. Todos os grupos apresentaram mais de 50% de bactérias viáveis. O Neo MTA Plus foi o único material a apresentar diferença significativa quando comparado ao grupo controle negativo (P=.0379). A análise em MEV mostrou um biofilme composto por bactérias de forma circular e de bastonetes, circundadas por matriz extra-celular. Em conclusão, todos os grupos apresentaram bactérias viáveis e o Neo MTA Plus teve a maior ação antimicrobiana. / This study aimed to assess, by confocal laser scanning microscopy (CLSM), the antimicrobial effect of Neo MTA Plus, Biodentine and MTA Angelus, using a microcosmo biofilm model, and to describe the microrganisms morphology, by SEM. Fifty-six dentin blocks were made from human teeth roots, cut in a size 5 x 4 x 4 mm, cleaned and sterelized. Two blocks were used to sterelization control. The others 54 blocks were fixed in Hawley retainers made for 6 volunteers that used it for 72h. After this period, they were placed in wells with BHI for 21 days, at 37ºC. At the end of the biofilm growth, the samples were randomly divided in 5 groups: Neo MTA Plus (n=12), Biodentine (n=12), MTA Angelus (n=12), negative control (n=12), and SEM analysis (n=6). The cements were placed in contact with the blocks of the tested groups. All samples were replaced in wells with BHI by 7 days at 37ºC. CLSM analysis evaluated the dentin blocks and cements isolated. SEM observation were performed in the six samples of the respective group. All groups presented a level of viable bacteria higher than 50%. Neo MTA Plus was the only one that showed significant difference from the negative control group (P=.0379). SEM analysis showed a biofilm composed of spherical and rod-shaped bacteria surrounded by extracellular matrix. All groups presented viable bacteria and Neo MTA Plus had the highest antimicrobial effect.