The aim of this semestral thesis is to compare the possibilities of available micro-CT systems. Theoretic part of this thesis there deals with possibilities of staining soft tissues and embryos because of enhancement the contrast of micro-CT images. Here follows a description of sources X-ray and detectors of available micro-CT systems. In practice there was realized the staining of embryo in cooperation with Department of histology and embryology in Brno. Then followed a measuring on FSI in Brno, ČVUT in Prague and synchrotron Elettra in Italy. In semestral thesis are described of the thesis there are compared the micro-CT systems and results of measuring embryos by means of presented systems and results are compared.The best results were obtained on micro-CT in Brno, where X-ray tube and flat panel detector were used. This images were used for a segmentation of cartilage olfactory system by means of 3D region growing. From results they were created 3D models for comparison with a manually segmented model. A less accurate results were obtain with the semi-automatic segmentation but this method isn’t too time-consuming.

The development in modern medicine has allowed to study human genome and detect predispositions to several diseases. One of very promising techniques is the analysis of human karyotype, i.e., the number and appearance of chromosomes in the cell nucleus. The most important step in the karyotype analysis is the chromosome detection and categorization. In this work, a new algorithm for detection of chromosomes from an image of microscopic DNA sample and their categorization into seven groups was developed. The algorithm was implemented in Matlab. The accuracy of segmentation and classification was tested on a set of images from two databases with 117 and 38 images, respectively. The sensitivity of the developed segmentation reached 88% while the value of positive predictivity of segmentation reached 92%. The success rate of chromosome pairing achieves 77%.

Kolorektalcancer är den tredje vanligaste cancerformen hos både män och kvinnor i Sverige där storkärls invasion, venös invasion (VI), är en viktig prognostisk indikator. Noggrann bedömning av VI är särskilt viktigt för patienter med kolorektal cancer i stadium II eftersom det kan påverka beslutet att erbjuda adjuvant behandling. Histologisk bedömning av VI kan vara utmanande på rutinmässig Hematoxylin och Eosin färgning (H&E). Elastinfärgning med syfte att identifiera elastinfibrer i kärlväggen kan underlätta bedömning av VI. I dagsläget finns inte någon bra elastinfärgning som kan användas rutinmässigt hos klinisk patologi i Värmland, Centralsjukhuset Karlstad, prover måste skickas till annat laboratorium vilket är tidskrävande. Denna studie ska finna den optimala färgningsmetoden för elastin genom att utvärdera tre olika färgningsmetoder för elastin fibrer. Färgningens resultat bedömdes rent histokemiskt av överläkare enlig kriterierna, frånvaro av avsedda kärl (skala 0–1), specifik infärgning (skala 0–1), specifik infärgnings intensitet (skala 1–3) och ospecifik infärgning (skala 0–1) och data samlades för respektive metod. Resultatet tyder på att rent histokemiskt är den manuella elastinfärgning metoden från Region Jönköpings län konsekvent och visade signifikant skillnad. Därför metoden är potentiellt användbar för den större frågeställningen om storkärlsinväxt i kolorektal cancer samt rekommenderas starkt som en rutinmässig elastinfärgning till klinisk patologi i Värmland, Centralsjukhuset Karlstad. / The third most common form of cancer in both men and women in Sweden is Colorectal cancer. Prognostic indicators of colorectal cancer include Large vessel invasion and venous invasion (VI). Careful assessment of VI is of particular interest especially with patients having stage II colorectal cancer. This is because it may influence decisions to offer adjuvant therapy. Accurate histological assessment of VI can be challenging on routine hematoxylin and eosin staining. But staining that can identify the elastin fibers in the vessel can enhance the assessment of VI. Currently, there is not any good Elastin staining that can be routinely used in clinical pathology in Värmland, Karlstad Central Hospital. Therefore, samples are required to be sent to other laboratories which in effect is time consuming. This study is aimed at finding an optimal elastin staining method through evaluations of three different staining methods for elastin fibers. Staining results were assessed histochemically by the chief physician according to the following criteria, absence of intended vessels (scale 0–1), specific staining (scale 0–1), specific staining intensity (scale 1–3) and nonspecific staining (scale 0–1), while different data were collected for each method. The results indicate that purely histochemically, elastin staining-manual method Region Jönköping County is consistent and significant. Therefore, the method is potentially useful for the detection of colorectal cancer and is strongly recommended as a routine elastin staining for clinical pathology in Värmland, Karlstad Central Hospital.

Dehydration process is an important histotechnical method of tissue samples undergoing histopathological analysis. Using different alcohols and intermedium, the samples are processed for diffusing the water out of the tissue. Impregnation with paraffin of the tissue in the later stagesis crucial for tissue stability in the remaining histotechnical methods. Sakura Tissue-Tek VIP 6 is atraditional dehydration instrument that realizes dehydrations during longer times with xylene asthe intermedium. However, LOGOS is another dehydration instrument operating with shorter times using Histolab Clear, a more environmental and cheaper intermedium.In the current study tissue samples were used from three different patient cases of each tissue:cutis, breast, intestine and prostate. Two different, new LOGOS- dehydration programs with Histolab Clear as intermedium were tested and validated in accordance with specific qualitycriteria. Parallel were tissue samples dehydrated from the same tissues in the traditional routineprogram, in order to evaluate by comparison, sections and staining quality of the new programs.Immunohistochemistry and routine staining were used for evaluation. The pathologist approved allroutine stains and disapproved some immunohistochemical analyses, depending on poorer staining and the quality of the sections.Because the amounts of the samples were insufficient in some cases, the number of the samples were determined to be expanded in order to investigate if the error occurs systematically or onlyto individual cases. Insufficient dehydration seemed to be fundamental as a cause of inferior results. Therefore, even the time of dehydration in Histolab Clear will increase by one hour, in the further validation. Optimization of dehydration time is required for approval of immunohistochemical analyzes. These analyzes are very important and definitive for the treatment of the patient, and therefore the new dehydration programs must give at least as good results as the conventional. / Dehydreringsprocessen är en viktig histoteknisk metod av vävnadsproverna som genomgår histopatologiska analyser. Med hjälp av olika alkoholer och intermedier, bearbetas proverna för att diffundera ut vattnet ur vävnaden. Impregnering av vävnaden i senare steg med paraffin, är avgörande för vävnadens stabilitet i resterande histotekniska metoder. Sakura Tissue-Tek VIP 6 är en traditionell dehydreringsmaskin som genomför dehydreringar under längre tid med Xylen som intermedium. En annan dehydreringsmaskin är LOGOS som är snabbare och som andvänder Histolab Clear, ett miljömässigare och billigare intermedium.I den aktuella studien användes vävnadsprover från tre olika patientfall vardera med vävnadstyperna cutis, bröst, intestinum och prostata. Två olika, nya LOGOS-dehydreringsprogram med Histolab Clear som intermedium utprövades samt validerades enligt bestämda kvalitetskriterier. Parallellt dehydrerades vävnadsprover från samtliga patientfall med det traditionella rutinprogrammet, med syfte att utvärdera genom jämförelse av snittens och infärgningarnas kvalité från det nya programmen.Immunhistokemiska- och rutininfärgningar användes för utvärdering. Patologerna godkände samtliga rutinfärgningar och underkände en del immunhistokemiska på grund av sämre infärgningar och dåligkvalité på snittet.Eftersom provmängden i några fall betraktades vara för få, bestämdes det att utöka antalet för att utreda om felet förekommer systematisk eller enbart vid enstaka fall. Otillräcklig dehydrering verkade vara fundamentalt vid sämre resultat. Därför kommer även dehydreringstiden i Histolab Clear att utökas meden timme till, vid den fortsatta valideringen. Optimering av dehydreringstiden krävs för godkännande av de immunhistokemiska analyserna. Analyserna är mycket viktiga och definitiva för patientens behandling och därför måste de nya dehydreringsprogrammen åtminstone ge lika bra resultat som det konventionella.

Elastinfibrer ger blodkärl och andra vävnader deras flexibilitet. Elastinfärgning är relevant när man misstänker melanom, temporalis artrit, venös invasion och efter blodkärlsoperationer. Syftet var att med hjälp av olika vävnadstyper optimera och implementera den automatiserad elastinfärgningen enligt Hart, för att ersätta den nuvarande manuella elastinfärgning enligt Verhoeff vid patologilaboratoriet på länssjukhuset Ryhov, Jönköping. Colon, njure, hud samt navelsträng färgades med den automatiska metoden enligt Hart för att hitta optimala inställningar. Vävnader från samma områden färgades med den manuella metoden enligt Verhoeff samt den automatiska metoden enligt Hart för att jämföra dem. Snitten bedömdes av en läkare så allt färgades som det skulle. Optimeringen av Harts metod resulterade i en inkubationstid på tolv minuter samt en optimal färgningsbehandling utan xylen. Resultaten av jämförelsen mellan den automatiska metoden enligt Hart och den manuella metoden enligt Verhoeff visar att den automatiska metoden enligt Hart ger bättre kontrast samt bakgrundsinfärgning. Slutsatsen blev att den automatiska metoden enligt Harts var bättre än den manuella metoden enligt Verhoeff, att i framtida studier studera möjligheten att byta ut läskningen mot till exempel ytterligare ett etanoldop samt att byta ut snitten av navelsträngen mot snitt av lever. / Elastic fibres ensure blood vessels and other tissues flexibility. Elastic staining of tissue is relevant when there is suspicion of melanoma, temporalis arteritis, venous invasion and after operations on blood vessels. The aim of the study was with the help of different tissue samples optimize and implement Hart´s elastic staining method as a substitute for Verhoeff’s at pathology lab at county hospital Ryhov, Jönköping. Colon, kidney, skin, and umbilical cord cross section got stained with Hart´s automated elastic staining method to evaluate the optimal staining procedure. Same region of the tissues was stained with Verhoeff´s manual elastic staining method and Hart´s method. All cross section were assessed and compared with the help of a pathologist doctor. Optimization of Hart´s method resulted in an incubation period of twelve minutes and optimal staining procedure without xylene. Result of comparison between Hart´s staining method and Verhoeff´s staining method showed that Hart´s staining method had a better contrast and background. Conclusions of the study was that Hart´s staining method was better than Verhoeff´s staining method, further studies could include research about a substitution of the blotting step with an extra ethanol bath as an example and liver tissue instead of the umbilical cord.

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La transplantation pulmonaire est depuis une vingtaine d’années une option thérapeutique valide pour une grande variété de pathologies pulmonaires au stade terminal. Malgré les progrès réalisés ces dernières années en matière de traitement immunosuppresseur, les rejets restent une cause majeure de la perte du greffon. Plusieurs études ont souligné l'importance du rejet aigu comme un facteur contributif important à l’évolution de la dysfonction chronique du greffon (ou CLAD) et, in fine, à la perte du greffon. Par conséquent, des outils diagnostiques fiables de rejet aigu s’imposent pour mieux prévenir le CLAD. Dans notre première étude, nous avons évalué les marqueurs tissulaires de rejet aigu humoral (RAH) pulmonaire. Nous avons montré ainsi que les lésions histologiques dont l’inflammation microvasculaire ne sont pas spécifiques et le marquage C4d est un marqueur utile pour confirmer le diagnostic de RAH. Dans un second temps, nous avons étudié en cytométrie de flux la polarisation des macrophages obtenus par lavage bronchiolo-alvéolaire (LBA) chez des patients transplantés avec et sans rejet. Nos résultats montrent les limites des marqueurs membranaires (HLA-DR et CD206) dans l’évaluation de l’état de polarisation des macrophages au cours des rejets. Ce travail montre l’intérêt des marqueurs tissulaires, en particulier le marquage C4d, dans le suivi des patients transplantés pulmonaires et souligne la nécessité d’identifier des marqueurs appropriés et utilisables en cytométrie de flux pour avancer sur l’état de polarisation des macrophages alvéolaires. / Lung transplantation is considered as a valid therapeutic option for patients with end-stage lung disease. Despite considerable progress in immunosuppressive therapy, allograft rejection remains a major cause of graft loss. Multiple studies have highlighted the importance of acute rejection as an important risk factor for the development of chronic lung allograft dysfunction (CLAD) leading to graft failure. Therefore, the improvement in the diagnosis of acute rejection represents a major challenge to prevent CLAD. In this study, we evaluated the tissue markers of acute antibody-mediated rejection (AMR) in lung transplantation. In our experience, the histopathologic findings including the microvascular inflammation in pulmonary AMR are not specific and C4d staining is a useful marker to confirm the diagnosis of AMR. Secondly, we investigated by flow cytometry the polarization of alveolar macrophage obtained by bronchoalveolar lavage (BAL) from lung transplant patients with and without acute rejection. Our results show the limits of surface markers (CD206 and HLA-DR) in the evaluation of alveolar macrophage polarization. This study shows the interest of tissue markers, especially the C4d staining, in monitoring of lung transplant patients and highlights the need to identify appropriate and available markers for future studies of alveolar macrophage polarization by flow cytometry.

Rice (Oryza sativa L.) is an important crop cultivated worldwide, and abiotic stresses limit its productivity. Different approaches were carried out to understand the mechanisms of rice defense responses against abiotic stresses, mainly drought. The NADPH-generating enzymes engaged in response to dehydration and salt stresses during the seedling stage of Nipponbare cultivar were analyzed. Enzyme activities of 6-phosphogluconate dehydrogenase (6PGDH), NADP-dependent aldehyde dehydrogenase, NADP-malic enzyme (NADP-ME) in leaves, and 6PGDH in roots were significantly increased in response to dehydration stress. NADP-ME and NADP-glutamate dehydrogenase activities in roots increased significantly in response to salt stress. These results suggest the involvement of NADPH-generating enzymes in plant responses to dehydration and salinity stresses, and the increased demands of NADPH in plants under abiotic stress can be furnished by enhanced activities of NADPH-producing enzymes. Also, a dehydration-induced protein was detected and identified as serine-hydroxymethyltransferase. This result indicates that serine-hydroxymethyltransferase can play a key role in regulating dehydration response in rice. Moreover, comparative proteomic analyses of CL163 (drought-tolerant), Cheniere (drought-sensitive), and Rex (moderately-drought-sensitive) rice varieties were performed. Drought-responsive proteome changes were profiled in leaves and roots at the seedling stage in response to drought stress imposed by polyethylene glycol (PEG-6000). Eighteen significantly differentially expressed proteins were identified by mass spectrometry. Elongate factor1 alpha and 17.9-kDa classI heat shock protein appear to have different expression patterns between CL163 and Cheniere, which may be attributable to the difference in drought response of the two rice varieties. Furthermore, a compendium of 103 drought resistance genes in rice was compiled to construct and analyze networks formed by associations between genes/proteins and to identify the most significant genes, biological processes/pathways. Genes were classified based on gene ontology and protein class into 26 groups. Forty-two genes were classified as transcription factors. Proteins encoded by the genes were localized in 8 subcellular locations and classified into three classes. Two pathways from KEGG whose genes were overrepresented in the compendium were identified. Gene expression, network presenting pairwise interactions between genes/proteins, and co-expression network were constructed. This study provides a systematic view of the crucial genes that can be contributing collectively to drought tolerance.

No description available.

No description available.