Master of Science / Department of Computing and Information Sciences / Mitchell L. Neilsen / ImageJ and its recent upgrade, Fiji, are image processing tools that provide extensibility via Java plug-ins and recordable macros [2]. The aim of this project is to develop a plug-in compatible with ImageJ/Fiji, which extracts length information from images for scale factor determination used in 3-D Photogrammetry for plant analysis [5]. Plant images when processed using Agisoft software, gives an image consisting of the images processed merged into a single 3-D model. The coordinate system of the 3-D image generated is a relative coordinate system. The distances in the relative coordinate system are proportional to but not numerically the same as the real world distances. To know the length of any feature represented in 3-D model in real world distance, a scale factor is required. This scale factor when multiplied by some distance in the relative coordinate system, yields the actual length of that feature in the real coordinate system. For determining the scale factor we process images consisting of unsharpened yellow colored pencils which are all the same shape, color and size. The plug-in considers each pencil as a unique region by assigning unique value and unique color to all its pixels. The distance between the end midpoints of each pencil is calculated. The date and time on which the image file gets processed, name of the image file, image file creation and modification date and time, total number of valid (complete) pencils processed, the midpoints of ends of each valid pencil, length (distance) i.e., the number of pixels between the two end midpoints are all written to the output file. The length of the pencils written to the output file is used by the researchers to calculate the scale factor. Plug-in was tested on real images and the results obtained were same as the expected result.

Bats are ecologically important around the world, partially because they eat insects. They are globally threatened by human activities. The extent of bat populations in South Asia has not been as well-monitored or researched as other parts of the world. Determining the size of a large colonial bat population is difficult, and can be aided through video footage or photographs. This study aimed to determine the population size of Tadarida plicata that inhabit the Gomantong Cave system in Borneo. Images of an evening emergence were used to determine the speed of flight, and then the number of bats to emerge from the cave through particle analysis in ImageJ. The counts, subsequent extrapolation, and comparison to previous estimates of flight speed indicated a population size approximately half the size of previous estimates, emphasizing the importance of continued monitoring. Using ImageJ particle analysis was deemed to be an effective way of estimating the number of bats in large populations.

The skin is the largest organ in the body. Its function is to protect the body from potential harm and to maintain homeostasis. The epidermis is the outermost layer of the skin. Stratum corneum is the outermost layer of the epidermis, composed of corneocytes and surrounding lipids. The lipids are produced by different enzymes that all play a role in the formation and function of the skin permeability barrier. Mutations in genes coding for these enzymes can lead to barrier dysfunction and could cause autosomal recessive congenital ichthyosis (ARCI). Nine genes have been identified as ARCI-causative and two of them are CYP4F22 and CERS3.   The purpose of this project was to study co-localization of CYP4F22 with CERS3 and also mutated CYP4F22 enzymes, by transfecting plasmids into HeLa and HaCaT cells and performing PLA on HEKn cells. Co-localization could indicate potential interactions and by studying these more in the future, novel treatment strategies can be developed for ARCI patients.   Transfection attempts showed a low transfection grade of wild type genes in both HeLa and HaCaT cells. Tendencies towards co-localization was seen in both cell types and some HeLa cells showed strong correlation after image analysis. Transfection of mutated genes failed, unfortunately. PLA showed co-localization in normal keratinocytes. The obtained results indicated a co-localization, but results need to be confirmed by immunoprecipitation and immunoblotting in the future.

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Le complexe linker of nucleoskeleton and cytoskeleton (LINC) est un complexe protéique conservé au cours de l’évolution, reliant les compartiments cytoplasmiques et nucléaires au travers la membrane nucléaire. Bien que les données récentes montrent une de ce complexe dans la régulation de la morphologie nucléaire et de la méiose, son implication dans l’organisation de la chromatine a été moins étudié chez les plantes. Le premier objectif de ce travail était de développer un plugin NucleusJ ImageJ dédié à la caractérisation de la morphologie nucléaire et de l’organisation de la chromatine en 3D. NucleusJ calcul 15 paramètres, y compris la forme et la taille des noyaux ainsi que des objets intra-nuclaires et leur position dans le noyau. Une documentation pour ce programme est disponible pour son utilisation, ainsi que des qu’un jeu données de noyaux pour tester ce programme. Plusieurs améliorations sont en cours pour développer une nouvelle version de ce plugin. Dans une deuxième partie de ce travail, des méthodes d’imagerie 3D ont été utilisées pour étudier la morphologie nucléaire et l’organisation de la chromatine dans les noyaux interphasiques chez Arabidopsis thaliana dans lequel les domaines d’htrochromatique sont groupés en régions detectable appelés chromocentres. Ces chromocentres forment un environnement répressif contribuant la rpression transcriptionnelle de séquences répétées permettant la stabilité du génome. Des mesures quantitatives de la position 3D de chromocentres dans le noyau montrent que la plupart chromocentres sont situés proximité de la périphérie du noyau, mais que cette distance peut être modifiée par le volume nucléaire ou dans certains mutants affectant le complexe LINC. Ce complexe LINC est proposé pour contribuer l’organisation de la chromatine et à son positionnement, de plus la mutation de ce complexe est associée une dérégulation l’inactivation de la transcription, ainsi qu’a une décompaction des séquences hétérochromatiques. La dernière partie de ce travail tire profit de séquences gnomiques disponibles et les données de RNA-seq pour explorer l’évolution des protines de la NE chez les plantes. Au Final, le travail réalisé durant cette thèse associe la génétique, la biologie moléculaire, la bioinformatique et de l’imagerie afin de mieux comprendre la contribution de l’enveloppe nucléaire dans l’organisation de la morphologie du noyaux et de la chromatine et suggère l’implication fonctionnelle du complexe LINC dans ces processus. / The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The first aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphology and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with different nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromocentres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC complex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes advantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional nuclear envelope. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes.

Stereology supplies image sampling rules to estimate geometric quantities such as volume, surface area, feature length and number. The method is well suited to non-invasive image acquisition methods such as Magnetic Resonance Imaging (MRI). Meanwhile, in Magnetic Resonance (MR) images analysis area, automated software packages have been continuously developed and become well-established tools especially in human brainMR images processing. The aims of the thesis are (1) to combine proper rules to sample MR images with automated or semi-automated data acquisition methods, in order to implement four different design unbiased stereological volume estimators in the study of the human brain, and (2) to compare volume estimates with those obtained from automated software packages.

Image processing is one area of signal analysis. This thesis is involved in feature extraction from image data and its implementation using Java programming language. The main contribution of this thesis lies in develop features extractors and their implementation in the program RapidMiner. The result is a robust tool for image analysis. The functionality of each operator is tested on mammogram images. A function model was developed for the removal of artifacts from the mammography images. The success rate of removal is comparable with other similar works. Furthermore, learning algorithms were compared on example detection of ventricle in ultrasound image.

The theoretical part of this master's thesis is focused on research of confocal microscopy and FLIM method. There are a principles and types of confocal microscopy and the use of broad-spectrum laser as a basic light source of these microscopes. It gives what the FLIM method and its use not only in cell biology. The practical part thesis includes the acquisition of three sets of fluorescence intensity images with use of applications tunable pulsed laser, function TimeGate and detection of hybrid detectors. For practical elaboration of this thesis is in the software Fiji created a plugin, which is the source code in the Java programming language. The types of plugins and their uses are described in the third chapter of the thesis. This plugin including the graphical user interface in the form of the dialog box, proceses the fluorescence intensity images and creates a graphical representation of data showing the fluorescence lifetime, so called pseudocolor map.

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