<p>The aims of this study were to investigate the mechanisms underlying (1) the ageing-related motor handicap at the whole muscle, cellular, contractile protein and myonuclear levels; and (2) ageing-related differences in muscle adaptability.</p><p>In vivo muscles function was studied in the knee extensors. Decreases were observed in isokinetic and isometric torque outputs in old age in the sedentary men and women and elite master sprinters. A 20-week long specific sprint and resistance training successfully improved the maximal isometric force and rate of force development in a subgroup of master sprinters.</p><p>In vitro measurements were performed in muscle biopsies from the vastus lateralis muscle. Immunocytochemical and contractile measurements in single membrane permeabilized muscle fibres demonstrated ageing- and gender-related changes at the myofibrillar level. In sedentary subjects, data showed a preferential decrease in the size of muscle fibres expressing type IIa MyHC in men, lower force generating capacity in muscle fibres expressing the type I MyHC isoform in both men and women and lower maximum velocity of unloaded shortening (V₀) in fibres expressing types I and IIa MyHC isoforms in both men and women. The master sprinters also experienced the typical ageing-related reduction in the size of fast-twitch fibres, a shift toward a slower MyHC isoform profile and a lower V₀ of type I MyHC fibres, which played a role in the decline in explosive force production capacity. The fast-twitch fibre area increased after the resistance training period. A model combining single muscle fibre confocal microscopy with a novel algorithm for 3D imaging of myonuclei in single muscle fibre segments was introduced to study the spatial organisation of myonuclei and the size of individual myonuclear domains (MNDs). Significant changes in the MND size variability and myonuclear organization were observed in old age, irrespective gender and fibre type. Those changes may influence the local quantity of specific proteins per muscle fibre volume by decreased and/or local cooperativity of myonuclei in a gender and muscle fibre specific manner.</p><p>In conclusion, the ageing-related impairments in in vivo muscle function were related to significant changes in morphology, contractile protein expression and regulation at the muscle fibre level. It is suggested that the altered myonuclear organisation observed in old age impacts on muscle fibre protein synthesis and degradation with consequences for the ageing-related changes in skeletal muscle structure and function. However, the improved muscle function in response to a 20-week intense physical training regime in highly motivated physically active old subjects demonstrates that all ageing-related in muscle function are not immutable.</p>

Our understanding of the complexity of the brain is limited by the data we can collect and analyze. Because of experimental limitations and a desire for greater detail, most investigations focus on just one aspect of the brain. For example, brain function can be studied at many levels of abstraction including, but not limited to, gene expression, protein interactions, anatomical regions, neuronal connectivity, synaptic plasticity, and the electrical activity of neurons. By focusing on each of these levels, neuroscience has built up a detailed picture of how the brain works, but each level is understood mostly in isolation from the others. It is likely that interaction between all these levels is just as important. Therefore, a key hypothesis is that functional units spanning multiple levels of biological organization exist in the brain. This project attempted to combine neuronal circuitry analysis with functional proteomics and anatomical regions of the brain to explore this hypothesis, and took an evolutionary view of the results obtained. During the process we had to solve a number of technical challenges as the tools to undertake this type of research did not exist. Two informatics challenges for this research were to develop ways to analyze neurobiological data, such as brain protein expression patterns, to extract useful information, and how to share and present this data in a way that is fast and easy for anyone to access. This project contributes towards a more wholistic understanding of the fruit fly brain in three ways. Firstly, a screen was conducted to record the expression of proteins in the brain of the fruit fly, Drosophila melanogaster. Protein expression patterns in the fruit fly brain were recorded from 535 protein trap lines using confocal microscopy. A total of 884 3D images were annotated and made available on an easy to use website database, BrainTrap, available at fruitfly.inf.ed.ac.uk/braintrap. The website allows 3D images of the protein expression to be viewed interactively in the web browser, and an ontology-based search tool allows users to search for protein expression patterns in specific areas of interest. Different expression patterns mapped to a common template can be viewed simultaneously in multiple colours. This data bridges the gap between anatomical and biomolecular levels of understanding. Secondly, protein trap expression patterns were used to investigate the properties of the fruit fly brain. Thousands of protein-protein interactions have been recorded by methods such as yeast two-hybrid, however many of these protein pairs do not express in the same regions of the fruit fly brain. Using 535 protein expression patterns it was possible to rule out 149 protein-protein interactions. Also, protein expression patterns registered against a common template brain were used to produce new anatomical breakdowns of the fruit fly brain. Clustering techniques were able to naturally segment brain regions based only on the protein expression data. This is just one example of how, by combining proteomics with anatomy, we were able to learn more about both levels of understanding. Results are analysed further in combination with networks such as genetic homology networks, and connectivity networks. We show how the wealth of biological and neuroscience data now available in public databases can be combined with the Brain- Trap data to reveal similarities between areas of the fruit fly and mammalian brain. The BrainTrap data also informs us on the process of evolution and we show that genes found in fruit fly, yeast and mouse are more likely to be generally expressed throughout the brain, whereas genes found only in fruit fly and mouse, but not yeast, are more likely to have a specific expression pattern in the fruit fly brain. Thus, by combining data from multiple sources we can gain further insight into the complexity of the brain. Neural connectivity data is also analyzed and a new technique for enhanced motifs is developed for the combined analysis of connectivity data with other information such as neuron type data and potentially protein expression data. Thirdly, I investigated techniques for imaging the protein trap lines at higher resolution using electron microscopy (EM) and developed new informatics techniques for the automated analysis of neural connectivity data collected from serial section transmission electron microscopy (ssTEM). Measurement of the connectivity between neurons requires high resolution imaging techniques, such as electron microscopy, and images produced by this method are currently annotated manually to produce very detailed maps of cell morphology and connectivity. This is an extremely time consuming process and the volume of tissue and number of neurons that can be reconstructed is severely limited by the annotation step. I developed a set of computer vision algorithms to improve the alignment between consecutive images, and to perform partial annotation automatically by detecting membrane, synapses and mitochondria present in the images. Accuracy of the automatic annotation was evaluated on a small dataset and 96% of membrane could be identified at the cost of 13% false positives. This research demonstrates that informatics technology can help us to automatically analyze biological images and bring together genetic, anatomical, and connectivity data in a meaningful way. This combination of multiple data sources reveals more detail about each individual level of understanding, and gives us a more wholistic view of the fruit fly brain.

Proliferative kidney disease (PKD) poses a high financial burden upon the freshwater salmonid aquaculture industry of Europe and North America. The alternate hosts of the causative agent, Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea), have been identified as freshwater bryozoans (Bryozoa: Phylactolaemata) within which spores capable of infecting salmonid fish develop. Currently, control of PKD relies upon complex management practices, with no licensed prophylaxis or treatment available. Assessment of the nutritional preferences of phylactolaemate bryozoans allowed development of an optimised laboratory culture system. Following laboratory maintenance, bryozoans collected from PKD-endemic sites were found to be infected with the malacosporean parasites T. bryosalmonae and Buddenbrockia plumatellae. Subsequent parasitic development was observed using light-, electron- and confocal-microscopy techniques. Methods of challenging rainbow trout with T. bryosalmonae spores were developed, with the minimum infective dose established. The presence of Thomsen-Friedenreich and Tn epitopes within the parasite was investigated, and experimental vaccine preparations based on either these specificities or T. bryosalmonae-infected bryozoans were efficacy tested in rainbow trout. In addition, salinomycin and amprolium were tested as prospective chemotherapeutants for PKD. Further insights into the development and subsequent release of malacosporean spores within their invertebrate hosts have been revealed. Long-term maintenance of T. bryosalmonae allowed controlled infection of rainbow trout previously vaccinated with experimental preparations. Findings of the project could potentially be utilised in future research into the development of control methods for PKD.

Corneal diseases are one of the major causes of visual impairment and blindness worldwide. Used for diagnoses, a laser confocal microscope provides a sequence of images, at incremental depths, of the various corneal layers and structures. From these, ophthalmologists can extract clinical information on the state of health of a patient’s cornea. However, many factors impede ophthalmologists in forming diagnoses starting with the large number and variable quality of the individual images (blurring, non-uniform illumination within images, variable illumination between images and noise), and there are also difficulties posed for automatic processing caused by eye movements in both lateral and axial directions during the scanning process. Aiding ophthalmologists working with long sequences of corneal image requires the development of new algorithms which enhance, correctly order and register the corneal images within a sequence. The novel algorithms devised for this purpose and presented in this thesis are divided into four main categories. The first is enhancement to reduce the problems within individual images. The second is automatic image classification to identify which part of the cornea each image belongs to, when they may not be in the correct sequence. The third is automatic reordering of the images to place the images in the right sequence. The fourth is automatic registration of the images with each other. A flexible application called CORNEASYS has been developed and implemented using MATLAB and the C language to provide and run all the algorithms and methods presented in this thesis. CORNEASYS offers users a collection of all the proposed approaches and algorithms in this thesis in one platform package. CORNEASYS also provides a facility to help the research team and Ophthalmologists, who are in discussions to determine future system requirements which meet clinicians’ needs.

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Dans les environnements naturels, industriels ou médicaux, les microorganismes sont majoritairement présents en étant associés aux surfaces dans des communautés hautement organisées appelées biofilms. Ces édifices biologiques constituent une stratégie de survie étonnement efficace témoignant d’une grande capacité de résistance à différent stress environnementaux tels que les traitements de nettoyage et de désinfection. L’impact des biofilms d’un point de vue sanitaire est donc considérable du fait qu’ils permettent la persistance et la transmission de germes pathogènes dans l’environnement. Dans ce contexte, ce travail de thèse avait pour objectif une meilleure compréhension des phénomènes limitant l’efficacité de désinfectants au sein des biofilms en s’appuyant notamment sur des techniques innovantes d’imagerie de fluorescence non-invasive. Le but final étant d’apporter des éléments utiles à l’optimisation des traitements de désinfection. Dans une première partie, une méthode d’investigation structurale à haut-débit par microscopie confocale a été développée et utilisée pour étudier la diversité architecturale des biofilms bactériens formés par un large panel de souches. Cette étude nous a permis d’identifier des souches d’intérêt en termes de structures de biofilms formés pour la suite du travail. Nous avons notamment pu mettre en évidence la capacité de B. subtilis à former des structures importantes et avec une architecture spécifique dans un système immergé. Dans une deuxième partie, les dynamiques d’action spatiotemporelles de désinfectants ont été visualisées dans les biofilms de souches de P. aeruginosa ou B. subtilis par des approches de microscopie confocale de fluorescence en temps réel. L’utilisation de cette technique nous a permis de mettre en évidence les difficultés de pénétration du chlorure de benzalkonium au sein des structures formées par différentes souches de P. aeruginosa. La corrélation des paramètres cinétiques d’inactivation et des données obtenues par la caractérisation biochimique de la matrice suggère un rôle majeur des substances extracellulaires dans la limitation de pénétraton du désinfectant. Nous avons également pu montrer une résistance marquée du biofilm formé par une souche de B. subtilis isolée d’un dispositif médical à l’acide péracétique, à la concentration et au temps d’utilisation du biocide dans le milieu médical. De plus, les structures tridimensionnelles formées par cette souche étaient capables de protéger le pathogène Staphylococcus aureus dans un biofilm mixte vis-à-vis du même traitement soulignant l’importance des interactions multi-espèces dans la résistance des bactéries aux désinfectants et la persistance de pathogènes dans nos environnements. / In natural, industrial or medical environments, microorganisms are present mainly in being associated with surfaces in highly organized communities called biofilms. These biological structures cosntitute a surprisingly effective survival strategy showing a large ability to withstand environmental stresses such as cleaning and disinfection treatments. Therefore, biofilms have a considerable impact on public health because they allow the persistence and transmission of pathogens. In this context, this work aimed to better understand the phenomena limiting the effectiveness of disinfectants in biofilms noticeably by using innovative imaging fluorescence non-invasive techniques. The ultimate goal was to provide data which can help to optimize disinfection treatments. In the first part, a high-throughput structural method based on confocal microscopy was developed and used to study the architectural diversity of bacterial biofilms formed by a wide range of strains. This study allowed us to identify strains of interest in terms of biofilm structure for the second part of the work. In particular, we demonstrated the ability of B. subtilis to form protruding structures with a specific architecture in a submerged system. In the second part, the spatiotemporal dynamic of the action of disinfectants were visualized in the biofilms of P. aeruginosa or B. subtilis strains by a time-lapse fluorescence confocal microscopy method. Using this technique, we showed that benzalkonium chloride encountered problems of penetration in the biofilms formed by P. aeruginosa strains. The correlation of kinetic inactivation parameters and data obtained by the characterization biochemical matrix suggested a key role of extracellular substances in the penetration limitations of the disinfectant. We also observed a pronounced resistance of the biofilm formed by a strain of B. subtilis isolated from a medical device to peracetic acid at the in-use concentration and time of biocide in medical areas. In addition, three-dimensional structures formed by this strains afforded protection to the pathogen Staphylococcus aureus in mixed biofilm against the same treatment This point highlights the importance of multi-species interactions in bacterial resistance to disinfectants and in the persistence of pathogens in our environments.

Le mélanome est un cancer agressif dont la progression est facilitée par la dégradation de la matrice extracellulaire, à la fois par les fibroblastes et les cellules tumorales. Cette dégradation génère des peptides d’élastine, notamment responsables de la prolifération des cellules saines et cancéreuses. Le peptide d’élastine (VGVAPG)3 accélère la reprise et le déroulement du cycle cellulaire de fibroblastes normaux et de cellules de mélanomes préalablement synchronisés (expression de pKi-67, détection de la phase S et quantification d’ADN). L’architecture nucléaire associée à la reprise de la synthèse des ARNm concerne les compartiments nucléaires PML-NBs et domaines SC35, partenaires indissociables de la transcription et de l’épissage, qui sont étudiés, après immunomarquages, en microscopie confocale suivie d’une reconstruction 3D. Les compartiment PML-NBs et domaines SC35 se réorganisent en fonction d’une part des phases du cycle cellulaire et d’autre part de l’activité transcriptionnelle, passant d’une séquestration du SC35 dans les PML-NBs à une interpénétration des deux compartiments. L’analyse quantitative de ces compartiments complète les résultats architecturaux en 3D. Le peptide se fixe sur le complexe récepteur de l’élastine, et induit de l’activation de la voie MEK ½ ERK ½. Un antagoniste de cette fixation (lactose) ainsi que l’inhibition de la voie ERK ½ (UO126) conduisent à l’abolition des effets dus au peptide tant pour le cycle cellulaire que pour l’organisation des PML-NBs et domaines SC35, confirmant ainsi l’implication de ces voies. / Melanoma is an aggressive cancer for which invasion is facilitated by degradation of the extracellular matrix, both by normal fibroplasts and tumor cells. This degradation generates elastin peptides, in particular responsible for the proliferation of normal and tumor cells. The elastin peptide (VGVAPG)3. accelerates recovery and progression in cell cycle of normal fibroblasts and melanoma cells previously synchronized (expression of pKi-67, S-phase detection and quantification of DNA). The nuclear architecture associated with the recovery of the synthesis of mRNA on the PML-NBs nuclear compartments and SC35 domains, inseparable partners of transcription and splicing, which are studied after immunostaining by confocal microscopy followed by 3D reconstruction. Compartments PML-NBs and SC35domains are reorganized according of the phases of the cell cycle and also the transcriptional activity, from SC35 sequestration in PML-NBs to an interpenetration of the two compartments. The quantitative analysis of these compartments consolidates the 3D architectural results. The peptide binds to the elastin receptor complex and induceds the activation of the MEK 1/2 ERK 1/2. An antagonist of the fixation (lactose) and inhibition of ERK 1/2 (UO126) lead to the abolition of effects due to the peptide for both the cell cycle and the organized of PML-NBs and SC35 domains, confirming the involvement of these pathways.

Grâce aux innovations technologiques récentes, l’exploration cutanée est devenue de plus en plus facile et précise. Le relevé topographique de la surface de peau par projection de franges ainsi que l’exploration des structures intradermiques par microscopie confocale in-vivo en sont des exemples parfaits. La mise en place de ces techniques et les développements sont présentés dans cette thèse. L’apport de l’imagerie est évident tant pour le traitement des acquisitions de ces appareils que pour l’évaluation de paramètres cutanés à partir de photographie par exemple. L’extension du modèle LIP niveaux de gris à la couleur a été réalisée pour apporter une évaluation proche de celle d’un expert grâce aux fondements logarithmiques du modèle, proches de la vision humaine. Enfin, la classification de données dans une image, sujet omniprésent dans le traitement d’images, a été abordée par les classifications hiérarchiques ascendantes, utilisant un cadre mathématique rigoureux grâce aux métriques ultramétriques / Thanks to recent developments, skin evaluation has become easier and more accurate. Topographical evaluation of skin surface by fringes projection as intra-dermal structures and exploration by in-vivo laser confocal microscopy are some examples. The use and development of these tools are developed in this thesis. Image processing contribution is obvious, as much for the treatment of these tools acquisitions, as for cutaneous parameters evaluation, based on digital camera acquisitions for example. Grey level LIP model extension to color has been realized in order to bring way of analysis near to the expert one, thanks to logarithmic bases of this model, very close to the human vision. At least, data clustering in images, a redundant topic in image analysis, has been approached by ascending hierarchical clustering, using rigorous mathematical properties thanks to the ultrametric distances

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Vývojová biologie (P1520) Studijní obor: Vývojová biologie (1501V000) Oleksandr Chernyavskiy Zdokonalené metody pro snímání obrazových dat a analýzu tkání a buněk pomocí konfokální a multifotonové mikroskopie Improved Methods of Image Acquisition and Analysis of Tissues and Cells by Confocal and Multi-Photon Microscopy Abstrakt disertační práce Školitel: RNDr. Lucie Kubínová CSc Praha, 2015 Abstract The aim of this study was to develop methods and approaches for image acquisition with subsequent image analysis of data, obtained by confocal and two- photon excitation microscopy as well as their combination, enabling new possibilities of visualization and assessment of information on biological tissues and cell structures in 3D and their measurement. We focused on methods that exploited advantages of confocal and multi-photon excitation microscopy. Our further aim was to demonstrate the applicability of non-invasive approach for in vivo applications, usefulness and the relevance of these methods in several special biological applications with emphasis on improved image acquisition, analysis and evaluation of real biological specimens. The present work was not oriented on just one specific biological problem, but rather to methodological...

A abfração, uma das causas das lesões cervicais não cariosas, caracteriza-se pela perda de tecido mineralizado, em forma de cunha, próxima à junção cementoesmalte. Hipoteticamente, é o resultado de forças oclusais excêntricas aplicadas sobre os dentes. Essas lesões são freqüentemente restauradas para impedir sua progressão, para proteger e aumentar a resistência do remanescente dental, reduzir a hiperestesia dentinária e manter a saúde periodontal. Entretanto, pouco se sabe quanto ao efeito dos fatores causadores da abfração na qualidade marginal de restaurações cervicais, quando da reabilitação dessas lesões. Este estudo in vitro propõe-se, portanto, a analisar o efeito da aplicação de carga oclusal na qualidade marginal de restaurações de cavidades cervicais em forma de cunha, utilizando microscopia confocal de varredura a laser. Para tal, foram utilizados 40 dentes prémolares superiores humanos hígidos, extraídos, nos quais foram preparadas cavidades padronizadas em forma de cunha, em sua região cervical vestibular, por meio da utilização de pontas diamantadas cilíndricas de topo plano nº 3100 (1,6 mm de diâmetro e 21 mm de comprimento), posicionadas em 45º com a superfície mais plana e próxima à junção amelo-cementária. As cavidades (1,3 mm de profundidade, 2,3 mm de abertura ocluso-cervical, 4,0 mm de abertura mésio-distal, 1,6 mm de extensão na parede oclusal e 1,9 mm de extensão na parede cervical) foram restauradas com resina composta (Z250®) em associação ao uso de um sistema adesivo (Single Bond®). Para possibilitar a análise ao microscópio confocal, o sistema adesivo foi marcado previamente com adição de rodamina B. Após imersão em água deionizada, por 7 dias e polimento das restaurações, os espécimes foram incluídos em uma matriz de resina acrílica. A porção radicular de cada dente foi revestida com uma camada de poliéter (Impregum®), de aproximadamente 1 mm, a fim de simular o ligamento periodontal. A porção coronária foi mantida totalmente exposta ao meio externo. Os dentes foram, então, submetidos à ciclagem mecânica, pela ação de uma ponta metálica em forma de torpedo, exercendo carga de 150 N, num total de 106 ciclos. Para tanto, a ponta foi posicionada de acordo com as seguintes condições experimentais: sobre a vertente triturante interna da cúspide vestibular (GI; n=10); sobre o sulco central (GII; n=10); e sobre a vertente triturante interna da cúspide lingual (GIII; n=10). O grupo controle (GIV; n=10) não foi submetido à ciclagem mecânica, permanecendo apenas armazenado em água deionizada, a 37ºC, durante todo o período experimental. Finalizada a ciclagem mecânica, as superfícies vestibulares dentárias foram, então, recobertas com duas camadas do sistema adesivo (Single Bond®), agora marcado com fluoresceína, para delimitação dos possíveis defeitos formados. Os dentes foram seccionados longitudinalmente em duas metades (mesial e distal) e submetidos à avaliação em microscopia confocal, para análise da qualidade da margem das restaurações. Foi aplicado o teste do qui-quadrado (p<0,05) para as variáveis qualitativas presença ou ausência de fendas marginais e localização das fendas em relação às paredes cavitárias (oclusal, cervical ou ambas). Para as variáveis quantitativas (amplitude e extensão das fendas) aplicou-se o teste de Kruskal-Wallis (p<0,05). Os espécimes submetidos ao carregamento oclusal, independentemente do local de aplicação da força, apresentaram maior porcentagem de fendas marginais (53,33%) quando comparados aos espécimes do grupo controle (10%; p=0,016). Não houve diferença estatisticamente significante entre o local de aplicação de força oclusal tanto para a presença ou ausência de fendas marginais (p=0,875); como para a localização das fendas em relação às paredes cavitárias (p=0,270) ou mesmo para a extensão (p=0,190) e amplitude das mesmas (p=0,070). Conclui-se, portanto, que a aplicação de carga oclusal prejudicou o comportamento marginal de restaurações cervicais, mas a variação do local de incidência de carga não interferiu no comportamento marginal das mesmas. / Tooth abfraction, one of the causes of non carious cervical lesions, is a wedgeshaped tissue loss, situated next to the cement-enamel junction, being, hypothetically, the result of eccentric occlusal loads applied to the teeth. These lesions are often restored to avoid their progression, to protect and increase remnant tooth resistance, to reduce dental hypersensitivity and to maintain periodontal health. However, there is little knowledge about the effect of abfraction etiology factors on the restoration marginal quality in the rehabilitation of these lesions. This in vitro study intends, therefore, to analyze the effect of the application of occlusal load on the marginal quality of cervical restorations in wedge-shaped cavities, by employing laser scanning confocal microscopy. Standardized wedge-shaped cavities were prepared in the cervical buccal region of 40 sound extracted upper human premolars, by the use of a cylindrical diamond bur #3100 (1.6 mm in diameter and 21 mm in length), positioned in 45º with the surface that is plainer and closer to the cementenamel junction. The cavities (1.3 mm in depth, 2.3 mm in occluso-cervical opening, 4.0 mm in mesio-distal opening, 1.6 mm in occlusal wall length and 1.9 mm in cervical wall length) were filled with a composite resin (Z250®) associated with the use of an adhesive system (Single Bond®). For the analysis at the confocal microscope to be possible, the adhesive system was previously modified by the addition of rodhamine B. After storage in deionized water for 7 days, and after the finishing and polishing of the restorations, specimens were included in acrylic resin matrixes. The radicular portion of each tooth was covered by an approximately 1 mm layer of a polyether (Impregum®), for the creation of an artificial periodontal ligament. The crown portion was kept totally exposed to the external environment. Teeth were, then, submitted to the mechanical cycling, by the action of a torpedo-shaped tip that exerted a 150 N load for a total of 106 cycles. The tip was positioned according to the following experimental conditions: over the internal triturant vertent of the buccal cusp GI; n=10); over the centre of the fissure (GII; n=10); and over the internal triturant vertent of the lingual cusp (GIII; n=10). The control group (GIV; n=10) was not submitted to the mechanical cycling, staying only stored in deionized water, at 37oC, during the whole experimental period. When the mechanical cycling was over, buccal dental surfaces were, then, covered with two layers of the adhesive system (Single Bond®), now modified by the addition of fluorescein, for delimitation of the possible formed defects. Teeth were longitudinally sectioned into two halves (mesial and distal ones), and submitted to evaluation through confocal microscopy, for analysis of the restorations marginal quality. Chi-square test (p<0.05) was applied for evaluation of qualitative variables (presence or absence of marginal defects and their location according to the cavity walls occlusal, cervical or both); and Kruskal-Wallis test (p<0.05) was applied for evaluation of quantitative variables (length and width of the defects). Specimens submitted to occlusal loading, independently of the location of the tip, presented a higher percentage of marginal defects (53.33%), when compared to the control group (10%; p=0.016). There was no significant statistical difference among the local of load placement for the presence or absence of marginal defects (p=0.875); for their localization according to the cavity walls (p=0.270); or even for their length (p=0.190) and width (p=0.070). It can be concluded, therefore, that occlusal loading was harmful for the margins of cervical restorations; but the different local of its incidence did not interfere with their marginal quality.