O estresse oxidativo é um dos principais responsáveis pela produção de danos espontâneos no DNA, os quais podem levar à origem de diversas neoplasias, inclusive câncer de mama (CM). Além disso, dentre os diversos fatores de risco já estabelecidos para a ocorrência do CM em mulheres, como idade, hormônios, dieta e fatores genéticos, alguns tipos de lesões benignas mamárias também aparecem como importantes elementos predisponentes. Dessa forma, a pesquisa de genes envolvidos com a produção ou reparo de danos oxidativos, bem como o estudo de elementos bioquímicos relacionados à proteção antioxidante em pacientes com lesões malignas e benignas de mama são fundamentais para o entendimento do mecanismo geral que conduz ao estresse oxidativo no CM. Os objetivos deste estudo foram determinar a frequência dos polimorfismos Ser326Cys hOGG1 e Arg38Trp AHCY pela técnica de PCR-RFLP; avaliar os níveis sanguíneos de folato, vitamina B12 e glutationa peroxidase (GSH-Px); examinar o grau de lesões oxidativas espontâneas no DNA pelo Teste do Micronúcleo (MN), Índice de Divisão Nuclear (IDN) e Ensaio Cometa em linfócitos; e por fim, verificar se os polimorfismos e os elementos bioquímicos estudados exercem influência sobre tais danos oxidativos. Foram coletadas amostras de sangue periférico de 55 voluntárias sadias, 10 pacientes com adenose esclerosante (AE) e 54 pacientes com carcinoma ductal invasivo (CDI) não tratadas. Os polimorfismos foram analisados em todas as amostras. As análises bioquímica e citogenética foram realizadas numa sub-amostra composta por 21 mulheres sadias, 10 pacientes com AE e 14 pacientes com CDI. No Ensaio Cometa foi aplicada a endonuclease OGG1 para detecção de danos oxidativos. Não foi observada relação entre os alelos Ser326Cys hOGG1 e Arg38Trp AHCY e o risco de CM. O número de indivíduos portadores do alelo Arg38Trp AHCY foi insuficiente para as demais análises. Não houve deficiência ou excesso de folato e vitamina B12 entre as voluntárias. Pacientes com CDI apresentaram níveis de GSH-Px e frequência de MNs significativamente maiores do que mulheres sadias. Não houve associação entre o grau de danos espontâneos no DNA e risco de CM. O alelo Ser326Cys hOGG1 não interfere na produção de lesões espontâneas no DNA. O folato e a vitamina B12, em níveis normais, podem provocar instabilidade genômica em pacientes com AE. / Oxidative stress is one of the most important generators of DNA damage, which can lead to carcinogenesis of many cancer types, including breast cancer (BC). Several risk factors were established for occurrence of BC in women, such as age, hormones, diet and genetic factors, however, certain types of benign breast lesions also appear as important predisposing factors. Thus, the search for genes involved in production and repair of oxidative damage, as well as the study of biochemical elements related to the antioxidant protection in patients with malignant and benign breast lesions are fundamental for understanding the general mechanism leading to oxidative stress in the BC. The aims of this study were to determine the frequency of Ser326Cys hOGG1 and Arg38Trp AHCY genetic polymorphisms by PCR-RFLP to investigate their association with BC susceptibility; evaluate folate, vitamin B-12 and glutathione peroxidase (GSH-Px) blood levels; examine the extension of spontaneous oxidative damage in DNA by Micronucleus Test (MN), Nuclear Division Index (NDI) and Comet Assay with lymphocytes; and finally, verify if polymorphisms and biochemicals investigated may influence on oxidative damage. Peripheral blood samples of 119 volunteers were collected: 55 healthy women, 10 patients with sclerosing adenosis (SA) and 54 untreated patients with invasive ductal carcinoma (IDC). Molecular analysis was performed in all samples. Biochemical and cytogenetic analysis were performed on a sub-sample of 45 individuals comprised 21 healthy women, 10 AE patients and 14 ICD patients, chosen at random from the total samples. In Comet Assay, endonuclease OGG1 was applied to detect oxidative damage. No relationship was found between Ser326Cys hOGG1 and Arg38Trp AHCY alleles and risk for BC. The number of individuals carrying the allele Arg38Trp AHCY was insufficient for further analysis. There was no deficiency or excess in folate and vitamin B12 levels among the volunteers. GSH-Px levels and frequencies of MNs were significantly higher in ICD patients than healthy women. There was no association between the degree of spontaneous DNA damage and risk for BC. Ser326Cys hOGG1 allele does not interfere on production of spontaneous DNA lesions. Normal levels of vitamin B12 and folate may cause genomic instability in AE patients.

Les carcinomes rénaux représentent environ 3% des cancers chez l’adulte. Les plus fréquents parmi ces tumeurs sont les carcinomes rénaux à cellules claires (CRCC) (70% des cas). Dans une première partie, nous avons analysé le caryotype de 89 patients ayant subi une néphrectomie pour CRCC et avons corrélé les déséquilibres chromosomiques avec les principaux facteurs histo-pronostiques et cliniques de ces tumeurs. Cette étude nous a permis de confirmer l’impact diagnostique et/ou pronostique d’anomalies chromosomiques. Certaines étaient déja connues dans la littérature comme la perte du bras court d’un chromosome 3 à impact diagnostique ou la perte d’un chromosome 9 ou de son bras court associée à un pronostic défavorable. Nous avons ensuite, dans une seconde partie, sélectionné selon des critères cliniques et histologiques, deux lignées cellulaires R-180 et R-305 établies à partir de prélèvements chirurgicaux de CRCC de patients dont l’évolution clinique était défavorable pour le patient R-180 (survie de 1 an) et favorable pour le patient R-305 (survie de 7 ans). Nous avons analysé les profils cytogénétiques des deux lignées cellulaires et recherché des marqueurs d’intérêt. Nous avons mis en évidence une amplification du gène pard3 dans la lignée R-180 correspondant au patient qui est décédé 1 an après le diagnostic. Cette amplification a été associée à la surexpression de la protéine correspondante PAR-3 et à des modifications de l’organisation du cytosquelette. La diminution de l’expression de PAR-3 par transfection de siRNA dans les cellules R-180 a permis la restauration de l’organisation du cytosquelette et la réduction des capacités de migration cellulaire par rapport aux cellules non transfectées. Ce résultat suggère un rôle de PAR-3 dans la migration cellulaire des cellules R-180. Afin de valider la pertinence de ce nouveau biomarqueur dans le CRCC, nous avons étudié 101 tumeurs par immunohistochimie. Nous avons montré une corrélation significative entre la surexpression de PAR-3 dans la tumeur primitive des patients et une diminution de la survie globale et de la survie sans progression indépendamment d’autres facteurs pronostiques importants comme les métastases. De plus la surexpression de PAR-3 a été significativement associée aux facteurs histopathologiques et cliniques de mauvais pronostic : grades nucléaires de Fuhrman III ou IV, nécrose tumorale, composante sarcomatoide, atteinte surrénale, invasion de la graisse rénale ou hilaire, composante éosinophile, statut non-inactivé du gène VHL, grade tumoral plus élevé, envahissement ganglionnaire ou métastatique, et score ECOG (Eastern Cooperative Oncology Group) péjoratif. L’ensemble de nos résultats suggèrent que la surexpression de PAR-3 est associée à un risque significatif de progression et de mortalité dans le CRCC. Sa mise en évidence par immunohistochimie en routine hospitalière pourrait être utile pour identifier les patients à haut risque de progression, même en l’absence des paramètres pronostiques habituels. Des études complémentaires sont en cours pour intégrer ce biomarqueur dans les nomogrammes ainsi que pour évaluer l’impact de cette dérégulation dans la résistance des CRCC aux thérapies ciblées. / Kidney cancers represent about 3% of all adults’ malignancies. The most common form of kidney cancer is renal carcinoma of which 70 % of cases are defined as clear cell Renal Cell Carcinoma (ccRCC). We undertook a systematic review of all ccRCCs with a total of 89 patients who underwent nephrectomy surgery. We assessed the karyotype profile of all patients that we correlate with an immunohistochemical features and tumor symptoms. This study demonstrates a high impact of chromosomal abnormalities on patients’ diagnosis and prognosis. Some of these abnormalities have been submitted in other publications as the loss of the chromosome 3 p-arm which has a diagnosis impact, and the loss of the chromosome 9 or it s p-arm that have a poor prognosis impact. We selected two cell lines (R-180 and R-305) derived from ccRCC surgical specimens of a patient with unfavorable clinical course (R-180 cells) and a patient with favorable prognosis (R-305 cells) to identify genetic and molecular features that may explain the survival difference of the two patients. The cytogenetic analysis of these cell lines revealed that the pard3 gene was amplified only in the R-180 cell line that was derived from an aggressive ccRCC. The pard3 gene amplification was associated with overexpression of the encoded protein and altered cytoskeleton organization. PAR-3 knockdown in R-180 cell restored the cytoskeleton organisation and reduced cell migration in comparison to non-transfected cells. These results suggest PAR-3 role in R-180 migration cells line. With a view to corroborate the relevance of this new biomarker PAR-3 in ccRCC, we have studied 101 tumors using immunohistochemical methods. We proved a significant correlation between PAR-3 overexpression in the primitive tumor and, the decreasing of overall and free progression survival independently of other risk factors as metastasis. We also fund that the overexpression of PAR-3 is associated with an unfavorable clinical and immunohistochemical prognosis factors such as: stage III -IV in fuhrman system grading ,tumor necrosis, sarcomatoide component, supra renal metastasis, cancer spreading (surrounding fat and hilar), eosinophil component , none inactivate VHL gene, high tumor stage, lymph nodes spread, metastasis and ECOG scale. Our results reveal that the PAR-3 overexpression is associated with significant risk of ccRCCs mortality and spreading tumor. Immunohistochemical screening may be usefulness to identify patient’s high spreading risk whether the lack of the habitual prognosis parameters. Other studies are in progress to integrate this biomarker in nomograms and also to evaluate the impact on ccRCC’s resistance to targeted therapy.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The economic potential of the medicinal species native to Brazil is huge, rendering it important to maintain the available plant genetic diversity via studies characterizing germplasm. Between these studies, highlights the characterization meiotic and pollen viability, genotoxic and antiproliferative activitie, beyond determination of phenolic compounds. Sambucus australis Cham. & Schltdl. ( sabugueiro ) numbers among the native species with medicinal potential, being widely used in the treatment of symptoms of skin eruptions, influenzas and common colds, for its diaphoretic, anti-inflammatory and analgesic actions. The objective of the present study was to investigate the antiproliferative, genotoxic and antigenotoxic activity of aqueous extracts of two accesses of S. australis, using the Allium cepa test, and to determine the phenolic compounds present in these extracts, as well as to analyze the meiotic behavior and estimate the pollen viability of different accesses of S. australis collected in Rio Grande do Sul state. Antiproliferative, genotoxic and antigenotoxic activities were determined by assessing the effect of aqueous extracts from the inflorescences and leaves of two accesses of S. australis, at concentrations of 3 g.L-1 and 12 g.L-1, on the cellular cycle of A. cepa. High performance liquid chromatography (HPLC) was employed to determine the phenolic compounds present in the extracts. For the meiosis analysis, slides were prepared by squashing technique of the anthers removed from the flower buds. The phases of association and distribution of the chromosomes were observed and meiotic indexes determined. To estimate pollen viability, the slides were prepared by squashing the anthers, comparing three stains: 2% acetic-orcein, 2% acetic-carmine, and Alexander´s reaction. All statistical analyses of the data were performed using the Scott-Knott test (p<0.05). Results showed that the aqueous extracts of S. australis exerted antiproliferative activity on the cellular cycle of A. cepa. The extracts prepared from S. australis (12g.L-1) leaves, for both accesses, also exhibited antigenotoxic activity. Chromatographic analysis disclosed the presence of the following compounds: gallic acid, chlorogenic acid, caffeic acid, ellagic acid, rutin, quercitrin, isoquercitrin, quercetin and kaempferol. Ultimately, the accesses of S. australis studied exhibited regular meiotic behavior, gametic number n=19, meiotic index > 90% and high pollen viability, being stain Alexander´s reaction the most efficient to estimate pollen viability in the specie. / O potencial econômico de espécies medicinais nativas no Brasil é imenso, sendo necessário conservar a diversidade genética vegetal disponível através de estudos de caracterização de germoplasma. Entre esses estudos, destacam-se a caracterização meiótica, a viabilidade polínica, a análise da atividade genotóxica e antiproliferativa, além da determinação dos compostos fenólicos. Dentre as espécies nativas com potencial medicinal, Sambucus australis Cham. & Schltdl. (sabugueiro) tem grande popularidade no tratamento sintomático de moléstias eruptivas, gripes e resfriados, por suas ações diaforética, anti-inflamatória e analgésica. O presente trabalho teve por objetivo verificar a atividade antiproliferativa, genotóxica e antigenotóxica de extratos aquosos de dois acessos de S. australis, através do teste de Allium cepa, bem como determinar os compostos fenólicos presentes nesses extratos, além de analisar o comportamento meiótico e estimar a viabilidade polínica de diferentes acessos de S. australis coletados no Rio Grande do Sul. Para determinação das atividades antiproliferativa, genotóxica e antigenotóxica, foi avaliado o efeito dos extratos aquosos das inflorescências e folhas de dois acessos de S. australis, nas concentrações de 3 g.L-1 e 12 g.L-1, sobre o ciclo celular de A. cepa. Os compostos fenólicos presentes nesses extratos foram determinados por meio da técnica de cromatografia líquida de alta eficiência (CLAE). Para a análise da meiose, foram preparadas lâminas pela técnica de esmagamento das anteras retiradas dos botões florais, observando-se as fases de associação e distribuição dos cromossomos, sendo também determinados os índices meióticos. Para estimativa da viabilidade polínica, as lâminas foram preparadas por esmagamento das anteras, comparando-se três corantes: orceína acética 2%, carmim acético 2% e reativo de Alexander. Todos os dados foram analisados estatisticamente pelo teste de Scott-Knott (p<0,05). A partir dos resultados, pode-se observar que os extratos aquosos de S. australis apresentaram atividade antiproliferativa sobre o ciclo celular de A. cepa. Os extratos preparados a partir das folhas de S. australis (12g.L-1), em ambos acessos, também apresentaram atividade antigenotóxica. A partir da análise cromatográfica foi possível determinar a presença dos seguintes compostos: ácido gálico, ácido clorogênico, ácido cafeico, ácido elágico, rutina, quercitrina, isoquercitrina, quercetina e canferol. Por fim, os acessos de S. australis estudados possuem comportamento meiótico regular, número gamético n=19, índice meiótico superior a 90% e alta viabilidade polínica, sendo o corante reativo de Alexander o mais eficiente para estimar a viabilidade polínica na espécie.

1. Clinical studies. Clinical diagnosis of dementia has been made largely on the basis of clinical findings supported by appropriate radiological and laboratory investigations. A minority of patients have treatable or reversible underlying causes for their dementing syndrome. It is important to distinguish between the two main forms of dementia Alzheimer's disease, senile dementia of Alzheimer type (AD/SDAT) and MID so that advantage can be taken of any future progress in treatments. In the clinical study significant differences between several diagnostic procedures were found between patients with AD/SDAT and MID. Blood pressure was significantly lower in the AD/SDAT group and focal neurological signs were seen in 70% of the MID patients but only in 6% of patients with AD/SDAT. Electrocardiogram was normal in all patients with AD/SDAT but pathological in 75% of the MID patients. Electroencephalogram showed generalized slow frequencies in 79% of the AD/SDAT patients and localized changes in 65% of the MID patients. Computerized tomography showed a significantly greater dilation of the ventricular system in MID patients compared to AD/SDAT patients and controls. Monoamine metabolites in the cerebrospinal fluid were lower in AD/SDAT patients and normal in MID patients. Psychopathological signs were found to be more variable and more pronounced in the AD/SDAT group compared with MID patients. 2. Etiological studies. Immunoglobulin and albumin were found changed in serum and CSF of both AD/SDAT and MID, indicating a more active immune response in MID and a less dense cerebrospinal fluid barrier in both MID and AD/SDAT. There appears to be a consumption of IgG in the central nervous system in patients with AD/SDAT. Abnormal chromosomes appearing as acentric fragments, i.e. without visible centromeres, were found in 90% of patients with AD/SDAT, 30% of patients with MID, and not at all in the control group. Increased aneuploidy was also seen both in patients with MID and AD/SDAT. Diabetes mellitus in old age and AD/SDAT do not seem to coexist. Furthermore, patients with AD/SDAT have changed carbohydrate metabolism with decreased fasting blood sugar concentrations, increased glucose tolerance and higher concentration of insulin during an oral glucose tolerance test. / <p>S. 1-47: sammanfattning, s. 49-144: 5 uppsatser</p> / digitalisering@umu.se

This master’s thesis is focused on digital image analysis of mitotic chromosomes. It deals with the design of the processing of digital images - from image preprocessing to clasification of each chromosomes, including testing on a set of images. This work introduces used cytogenetic methods, that are used to visualize chromosomes. In its practical part describes morphology operations and clasification procedure. Classification of the chomosomes was divided into 5 groups (A-G). All algorithms were created in the MATLAB program.

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%.

Cytogenetic analysis of cells of chronic lymphocytic leukemia (CLL) is difficult because of their low proliferative activity. To obtain sufficient number of mitoses for performing chromosomal analysis a suitable stimulation of cell division is needed. Using DSP30/IL2 stimulated cultivation 391 CLL samples were investigated in 5 years' period. The cultivation was showed to have high success rate (96%; 375/391) with also high rate of detection of pathological clones by both karyotype and metaphase FISH analyses (in 84% of samples; 329/391). Almost in half of samples (44%; 171/391) other aberrations than recurrent FISH (i.e. 13q14 deletion, trisomy 12, TP53, ATM genes deletions) were found. Also high frequency of translocations (37%; 144/391), complex karyotypes (28%; 111/391) and clonal evolution, which was detected in one third of all samples (34% of samples with presence of more than two clones; 133/391) and like a new event in disease duration even more frequently (in 39% of samples repeatedly investigated after stimulated cultivation; 21/54), was revealed. The presence of translocations, complex karyotypes and clonal evolution was associated with progressive form of disease (P 0,000003, resp. P 0,0002 and P 0,05/P 0,04). In cases of the recurrent deletions the detailed analysis of metaphase...

In this study, the efficacy of MarrowMAX, a new IVD-certified culture medium for bone marrow samples, was evaluated and compared to the in-house modified culture medium RPMI-1640, which is routinely used at the Academic Hospital in Uppsala for chromosomal analysis. The hypothesis was that the new culture medium MarrowMAX would lead to an increased number of metaphases, resulting in a higher mitotic index, better spreading, and improved metaphase morphology. Twenty patient samples were cultured in parallel using both media and subjected to identical methods for sample preparation and staining for chromosomal analysis. The results demonstrated significant differences between MarrowMAX and RPMI-1640 for one-day, two-day, and three-day cultures. However, no significant differences were observed for directly harvested cultures and overnight colcemid cultures. These findings suggest that MarrowMAX is more effective than RPMI-1640, resulting in a higher frequency of metaphases, which is crucial for detailed chromosomal analysis. This was further confirmed by flow cytometry analysis, showing a higher number of proliferated cells in the MarrowMAX culture compared to the RPMI-1640 culture, in one representative sample. MarrowMAX's enhanced capacity to promote cell growth and structure in a laboratory setting indicates its potential as a promising alternative to RPMI-1640 for future diagnostic applications, potentially leading to faster diagnoses and improved treatment efficacy for patients with hematological conditions.

L’analyse des anomalies génomiques récurrentes est importante pour établir le diagnostic, le pronostic et pour orienter la thérapie des leucémies aiguës pédiatriques. L’objectif de notre étude est d’élaborer une stratégie optimale pour détecter les anomalies chromosomiques dans les leucémies aiguës lymphoblastiques (LAL) et myéloïdes (LAM) des enfants. Pour ce faire, nous avons caractérisé au caryotype, avec des panels d’hybridation in situ en fluorescence (FISH), par RT-PCR et par l’index d’ADN 253 leucémies de novo reçues au CHU Sainte-Justine entre 2005 et 2011 (186 LAL-B, 27 LAL-T et 40 LAM). Nous avons réussi à optimiser la détection des anomalies chromosomiques dans les trois types de leucémies, avec des fréquences de 93,5% dans les LAL-B (174/186), 66,7% dans les LAL-T (18/27) et 90% dans les LAM (36/40). Nos résultats suggèrent d’utiliser plusieurs tests génétiques concomitants afin d’optimiser la détection des anomalies génomiques dans les LAL et les LAM de novo pédiatriques. / Analysis of recurrent genomic abnormalities is important for the diagnosis, prognosis and therapeutic choices in paediatric acute leukemia. The aim of our study is to establish a strategy optimizing the detection of cytogenetic abnormalities in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). To do so, we have characterized childhood AML and ALL cases received in cytogenetic laboratory of CHU Sainte-Justine (Montreal, Canada) between 2005 and 2011. Overall, 253 de novo cases have been analyzed (186 B-ALL, 27 T-ALL and 40 AML) by karyotyping, fluorescence in situ hybridization (FISH) panels, RT-PCR and DNA index. Chromosomal abnormalities detection rates achieved 93,5% in B-ALL (174/186), 66,7% in T-ALL (18/27) and 90% in AML (36/40). Our results suggest the analysis of both molecular and cytogenetic tests to optimize the detection of genomic abnormalities in new cases of childhood AML and ALL.