The application of cDNA and tissue microarray methods in the study of human carcinomas

Currently, numerous high-throughput technologies are available for the study of human carcinomas. In literature, many variations of these techniques have been described. The common denominator for these methodologies is the high amount of data obtained in a single experiment, in a short time period, and at a fairly low cost. However, these methods have also been described with several problems and limitations. The purpose of this study was to test the applicability of two selected high-throughput methods, cDNA and tissue microarrays (TMA), in cancer research. Two common human malignancies, breast and colorectal cancer, were used as examples. This thesis aims to present some practical considerations that need to be addressed when applying these techniques. cDNA microarrays were applied to screen aberrant gene expression in breast and colon cancers. Immunohistochemistry was used to validate the results and to evaluate the association of selected novel tumour markers with the outcome of the patients. The type of histological material used in immunohistochemistry was evaluated especially considering the applicability of whole tissue sections and different types of TMAs. Special attention was put on the methodological details in the cDNA microarray and TMA experiments. In conclusion, many potential tumour markers were identified in the cDNA microarray analyses. Immunohistochemistry could be applied to validate the observed gene expression changes of selected markers and to associate their expression change with patient outcome. In the current experiments, both TMAs and whole tissue sections could be used for this purpose. This study showed for the first time that securin and p120 catenin protein expression predict breast cancer outcome and the immunopositivity of carbonic anhydrase IX associates with the outcome of rectal cancer. The predictive value of these proteins was statistically evident also in multivariate analyses with up to a 13.1- fold risk for cancer specific death in a specific subgroup of patients.

Ultrastructural study of the neovagina following the utilization of human amniotic membrane for treatment of congenital absence of the vagina

We present an ultrastructural study of the utilization of human amniotic membrane in the treatment of congenital absence of the vagina in 10 patients. All patients were surgically treated with application of an amniotic membrane graft using the modified McIndoe and Bannister technique. Sixty days after surgery, samples of the vaginal neo-epithelium were collected for transmission electron microscopy analysis. The ultrastructural findings consisted of a lining of mature squamous epithelium indicating the occurrence of metaplasia of the amniotic epithelium into the vaginal epithelium. The cells were arranged in layers as in the normal vaginal epithelium, i.e., superficial, intermediate and deep layers. There were desmosomes and cytoplasmic intermediate cytokeratin filaments, as well as some remnant features of the previous amniotic epithelium. These findings suggest that human amniotic membrane is able to complete metaplasia into squamous cells but the mechanism of this cellular transformation is unknown

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Manual and semi-automatic quantification of in vivo ¹H-MRS data for the classification of human primary brain tumors

In vivo proton magnetic resonance spectroscopy (¹H-MRS) is a technique capable of assessing biochemical content and pathways in normal and pathological tissue. In the brain, ¹H-MRS complements the information given by magnetic resonance images. The main goal of the present study was to assess the accuracy of ¹H-MRS for the classification of brain tumors in a pilot study comparing results obtained by manual and semi-automatic quantification of metabolites. In vivo single-voxel ¹H-MRS was performed in 24 control subjects and 26 patients with brain neoplasms that included meningiomas, high-grade neuroglial tumors and pilocytic astrocytomas. Seven metabolite groups (lactate, lipids, N-acetyl-aspartate, glutamate and glutamine group, total creatine, total choline, myo-inositol) were evaluated in all spectra by two methods: a manual one consisting of integration of manually defined peak areas, and the advanced method for accurate, robust and efficient spectral fitting (AMARES), a semi-automatic quantification method implemented in the jMRUI software. Statistical methods included discriminant analysis and the leave-one-out cross-validation method. Both manual and semi-automatic analyses detected differences in metabolite content between tumor groups and controls (P < 0.005). The classification accuracy obtained with the manual method was 75% for high-grade neuroglial tumors, 55% for meningiomas and 56% for pilocytic astrocytomas, while for the semi-automatic method it was 78, 70, and 98%, respectively. Both methods classified all control subjects correctly. The study demonstrated that ¹H-MRS accurately differentiated normal from tumoral brain tissue and confirmed the superiority of the semi-automatic quantification method.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

Securin, Cdc20 and Cdc27 in predicting the outcome of human breast cancer

Deregulated proliferation has been recognized among the most important factors promoting breast cancer development and progression. The aim of the project is to gain understanding of the role of specific cell cycle regulators of metaphase-anaphase transition and evaluate their potential in breast cancer prognostication and treatment decisions. Metaphase-anaphase transition is triggered by activation of anaphase promoting complex (APC) which is activated by a cascade of regulatory proteins, among them securin, Cdc20 and Cdc27. These proteins promote the metaphase–anaphase transition and participate in the timely separation of the chromatids. This study is based on a patient material of approximately 600 breast cancer patients and up to 22 years of follow-up. As the main observation, based on DNA cytometric and immunohistochemical methods, securin, Cdc20 and Cdc27 protein expressions were associated with abnormal DNA content and outcome of breast cancer. In the studied patient material, high securin expression alone and in combination with Cdc20 and Cdc27 predicted up to 9.8-fold odds for aneuploid DNA content in human breast cancer. In Kaplan–Meier analyses, high expression of securin systematically indicated decrease in breast cancer survival as compared to low expression cases. The adverse effect of high securin expression was further strengthened by combining it with Cdc20 or Cdc27 expressions, resulting in up to 6.8-fold risk of breast cancer death. High securin and Cdc20 expression was also associated with triple-negative breast cancer type with high statistical significance. Securin, Cdc20 or Cdc27 have not previously been investigated in a clinically relevant large breast cancer patient material or in association with DNA ploidy. The present findings suggest that the studied proteins may serve as potential biomarkers for identification of aggressive course of disease and unfavourable outcome of human breast cancer, and that they may provide a future research aim for understanding abnormal proliferation in malignant disease.

The Midnight express phenomenon : a historical materialist approach to the reception of the film Midnight express /

This thesis examines the Midnight Express phenomenon focusing on the film's reception by audiences in Europe, North America, and Turkey between 1978-2003. Using and enhancing the "historical materialist approach" to film reception developed by Janet Staiger, the thesis considers the historical determinants of the film's nationally and culturally differential readings in different periods and of the transformations in those readings. The thesis argues that while Midnight Express was most likely read in the late 1970s as an attempt to reaffirm American social identity by projecting Turks as an instance of the negative Other, there has been an important shift in the reception of the film in the West during the 1990s due to the changes in the discursive contexts in which the film has been circulating. One does not observe any specific reference to Turkish prisons as a part of the issue of human rights violations in Turkey in the initial reception of the film by European and American critics, whereas these issues appear to be important constituents of a particular reception of the film in the West in the present. The thesis explains this shift by pointing to the constitution of a particular discourse on human rights violations in Turkey after 1980, and especially throughout the 1990s, which has become a part of the discursive repertoires of the Western audience. Therefore, the thesis argues that today, Midnight Express functions as a more legitimate political statement about Turkey in the eyes of some Western audiences than it had been in the 1970s. On the other hand, parallel to the increasing desire of Turkey to connect itself to the West, particularly to become a member of the European Union, one observes an immense increase in the belief in and defense against the negative effects of Midnight Express on Turkey's international representation since the 1990s. The historical and current discourses that audiences, both in Turkey and abroad, bring into play suggest that these audiences engage with Midnight Express by assuming or denying not only the subject positions constructed by the film text but also certain history-specific extra-filmic subject positions produced by other social and discursive formations.

"Re-huminization": the role of human-animal similarity in predicting prejudice towards immigrants and non-human animals

Research implies that there ~ay be an association between attitudes toward margil1alized human outgroups and non-human animals. Very few studies, however, have specifically tested this relation empirically. The general purpose of the present research was to determine if such a relation exists and if perceptions of human-animal similarity avail as a common predictor of both types of attitudes. Ideological orientations associated with prejudiced attitudes (Social Dominance Orientation, Right-Wing Authoritarianism, and Universal Orientation) were also examined as individual differences in predicting perceptions of human-animal similarity. As predicted, people who endorsed prejudiced attitudes toward human outgroups (Study 1) and immigrants in particular (Studies 2 and 3), were more likely to endorse prejudiced attitudes toward non-human animals. In Study 2, perceptions that humans are superior (versus similar) to other animals directly predicted higher levels of prejudice toward non-human animals, whereas the effect of human superiority beliefs on immigrant prejudice was mediated by dehumanization. In other words, greater perceptions of humans as superior (versus similar) to other animals "allowed for" greater dehumanization of immigrants, which in turn resulted in heightened immigrant prejudice. Furthermore, people higher in Social Dominance Orientation or Right-Wing Authoritarianism were particularly likely to perceive humans as superior (versus similar) to other animals, whereas people characterized by a greater Universal Orientation were more likely to perceive humans and non-human animals as similar. Study 3 examined whether inducing perceptions of human-animal similarity through experimental manipulation would lead to more favourable attitudes toward non-human animals and immigrants. Participants were randomly assigned to read one of four 11 editorials designed to highlight either the similarities or differences between humans and other animals (i.e., animals are similar to humans; humans are similar to animals;~~nimals are inferior to humans; humans are superior to animals) or to a neutral control condition. Encouragingly, when animals were described as similar to humans, prejudice towards non-human animals and immigrants was significantly lower, and to some extent this finding was also true for people naturally high in prejudice (i.e., high in Social Dominance Orientation or Right-Wing Authoritarianism). Inducing perceptions that nonhuman animals are similar to humans was particularly effective at reducing the tendency to dehumanize immigrants ("re-humanization"), lowering feelings of personal threat regarding one's animal-nature, and at increasing inclusive intergroup representations and empathy, all of which uniquely accounted for the significant decreases in prejudiced attitudes. Implications for research, theory and prejudice interventions are considered.

Structural bioinformatics analysis of the family of human ubiquitin-specific proteases

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le devoir syndical de représentation sous l'angle de la Charte des droits et libertés de la personne: le cas des clauses" orphelin"

En raison des présentes transformations du marché du travail, le mouvement syndical est à la recherche d’un nouvel équilibre. Dans ce contexte plutôt changeant, réorganisations et flexibilité se négocient en échange d’une protection accrue pour ceux qui demeurent en emploi. Si d’une part, ceci mène à une prolifération de nouvelles formes d’emploi atypiques et de disparités de traitement, d’autre part la constitutionnalisation du droit du travail change le rapport quant à l’universalisme syndical. Nous nous sommes alors interrogée sur les impacts que cela peut avoir sur le devoir syndical de représentation. Prenant cela en considération, nous avons voulu au cours de notre recherche faire la lumière tant sur certains impacts de la négociation de clauses de disparité de traitement dans les milieux de travail syndiqués que sur les défis contemporains du droit du travail et de l’emploi. Cette thèse propre au champ d’études que sont les relations industrielles se distingue des autres recherches dans le même domaine en se concentrant sur l’effet empirique de la mise en œuvre d’une norme, par l’étude de deux cas suggestifs. Plus précisément, notre thèse avait comme principal objectif de répondre à la question suivante : Quels sont les effets sur l’acteur (action) syndical du recours à la norme d’égalité, à la suite la négociation de clauses « orphelin »? Pour y parvenir, nous avons dû reconstituer comment les acteurs syndicaux s’étaient approprié l’espace ouvert par la constitutionnalisation du droit du travail. Nous avons utilisé une méthode qualitative, impliquant une revue de la littérature sociohistorique, théorique et juridique, une analyse de la jurisprudence existant à ce sujet et l’étude de deux cas portant sur la négociation d’une clause « orphelin » le tout représentant soixante entrevues semi-dirigées dans plusieurs casernes et palais de justice. Les connaissances acquises permettent au plan empirique une meilleure compréhension des interactions possibles entre acteurs, institutions et stratégies lors de la mise en œuvre de la norme d’égalité ainsi que des déterminants de l’action (ou de l’inaction) syndicale. Sur le plan pratique, nous espérons que notre thèse puisse guider toute personne ou tout groupe de personnes désirant mettre en œuvre des mesures égalitaires, tant pour éviter certains écueils reliés à ces actions ou réactions suscitées par la mise en œuvre du droit à l’égalité, que pour mesurer l’efficacité des actions entreprises. De surcroît, sur le plan théorique, nous croyons que de mettre à l’épreuve un modèle théorique recoupant stratégies et institutions permettra d’aborder les problèmes à la fois au niveau de l’acteur et au niveau structurel. Les résultats obtenus aident à comprendre en quoi et comment peut se construire l’action syndicale dans certains (2) cas de mise en œuvre de la norme d’égalité. Le choix de différentes stratégies ne peut pas être dissocié des différents contextes économiques, sociaux et juridiques, ni des capacités stratégiques des différents acteurs en cause. Les principales contraintes identifiées ont pu être imputées tant aux structures de l’association syndicale qu’aux différents univers normatifs mobilisés. Les sources de tensions vécues dans les milieux à l’étude ont été rattachées notamment aux perceptions d’injustice et à la méconnaissance des différentes normes juridiques. En conséquence, il faut conclure que pour les associations syndicales (sans se restreindre à celles à l’étude), la question d’iniquité pourrait avoir de graves conséquences et non seulement sur le devoir de représentation. Nous avons spécialement souligné la fragilisation de leurs assises, dans leurs deux pôles d’actions : d’acteur économique (faille dans la capacité d’action traditionnelle) et d’acteur politique (climat hostile au syndicalisme). Nous souhaitons que l’acteur syndical puisse mobiliser, à la lumière des résultats de notre recherche, des arguments concourant à repenser l’action syndicale, que nous croyons être plus pertinente que jamais. Nous croyons également que les différents acteurs du monde du travail pourraient s’approprier certaines de nos recommandations afin de favoriser une citoyenneté au travail plus inclusive.

Human Rights Violations in Police Custody

Human rights are the basic rights of every individual against the state or any other public authority as a member of the human family irrespective of any other consideration. Thus every individual of the society has the inherent right to be treated with dignity in all situations including arrest and keeping in custody by the police. Rights of an individual in police custody are protected basically by the Indian Constitution and by various other laws like Code of Criminal Procedure, Evidence Act, Indian Penal Code and Protection of Human Rights Act. The term `custody' is defined neither in procedural nor in substantive laws. The word custody means protective care. The expression `police custody' as used in sec. 27 of Evidence Act does not necessarily mean formal arrest. In India with special reference to Kerala and evolution and development of the concept of human rights and various kinds of human rights violations in police custody in different stages of history. Human rights activists and various voluntary organisations reveals that there are so many factors contributing towards the causes of violations of human rights by police. Sociological causes like ambivalent outlook of the society with respect to the use of third degree methods by the police, economic causes like meager salary and inadequate living conditions, rampant corruption in police service, unnecessary political interference in the crime investigation, work load of police personnel without any time limit and periodic holidays, unnecessary pressure from superior police officers and the general public for speedy detection causing great mental strain to the investigating officers, defective system of recruitment and training, imperfect system of investigation and lack of public co-operation are some of the factors identified in the field survey towards the causes of violations of human rights in police custody.