Von der Gemischbildung zu den Schadstoffemissionen im Dieselmotor auf direktem Weg

Magdeburg, Univ., Fak. für Maschinenbau, Diss., 2010

Technique for the detection of Toxoplasma gondii antigens in mouse urine

A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis.

A new host of Trypanosoma cruzi from Jujuy, Argentina: octodontomys gliroides (Gervais & D'Orbigny, 1844) (Rodentia, Octodontidae)

To identify wild hosts of Trypanosoma cruzi, surveys were conducted in the subandean valleys of Jujuy Province, Argentina, between June 1986 and March 1987. Seventy two mammals from 13 different species were examined by xenodiagnosis. Fifty two of them were mostly roedents trapped at the localities of Maimará, León and Tilcara, and the remainder had been kept in captivity at the Estación Biológica Experimental, in Jujuy. Trypanosoma cruzi infection was detected only in 2 Octodontomys gliroides (2 pos./8 exam. 25%) from all 72 examined mammals. Isolates were called Octodontomys Argentina 1 and 2 (OA1 and OA2). Both infected animals were caught at the archaelogical ruin of Pucará, at Tilcara. Repeated searches for triatomines in the ruin itself and in neighbour houses rendered negative results. Groups of mice inoculated with either OA1 or OA2 isolates became infected between 7 (OA1) to 12 days (OA2) postinoculation PI. Parasitemia peaks were observed between day 12th - 14th PI. Scarce amastigote nests were found in myocardium and skeletal muscle. Mortality was observed only for mice inoculated with OA1. Isoenzyme patterns of OA1 and OA2 were identical to one found in dogs and slightly different from that of human parasites in Argentina. Bones from Octodontomys sp., were recently found in a cave, dated 10200-8600 BC, in Pumamarca, near Tilcara, Jujuy. There are evidences that O. gliroides cohabited with man in ancient times and was associated to the domestic cycle of T. cruzi transmission, playing a role like that of domestic cavies. in Bolivia.

Dispersive flight and house invasion by Triatoma guasayana and Triatoma sordida in Argentina

Flight activity and invasion of houses by Triatoma sordida and T. guasayana were studied in the Province of Santiago del Estero, Argentina. Spontaneous findings of both species in houses were recorded from 1982 to 1989. Light trap collections were performed in 1982, 1983 and 1984, at the woods surrounding the settlements of Amamá (43 houses) and Trinidad (19 houses). Most of the 101 triatomines collected, were unfed and negative for Trypanosoma cruzi. T. guasayana predominated over T. sordida, and both appeared on the lighted screens between 19-31 min (mean 24) after dusk and the catch time was 30-45 min. Although entomological evaluation of 41 houses at Amamá performed in September 1985, just before insecticidal spraying, showed that Triatoma infestans predominated, adults of T. guasayana were collected in sleeping places, in 7 houses (17%). Most triatomines invading houses from then up to 1990 were flying T. guasayana (20/27) and females outnumbered males. Three non infected T. guasayana females were fed on man and two T. guasayana males positive for "T. cruzi like" trypanosomes were unfed. Therefore, visiting hungry adults could transmit T. cruzi to people and introduce wild parasites to the domestic cycle. T. guasayana stands as the main potential substitute of T. infestans in the studied area, and it might play there the same role as T. sordida in Brazil.

Qualité des soins ambulatoires : opinion des patients infectés par le VIH

Ce travail décrit une étude, qui a visé à évaluer la performance d'un questionnaire de satisfaction en développement, destiné à mesurer l'opinion de patients infectés par le VIH suivis ambulatoirement. 1. Problématique. 1.1. La qualité des soins, une définition. 1.2. L'opinion des patients : comment la mesurer ? Le questionnaire, les questionnaires existants, le focus groups, description de la technique. 2. Patients et méthodes. 2.1. Critères d'inclusion et d'exclusion. 2.2. Les envois et les rappels. 2.3. Stabilité test-retest. 2.4. Le recrutement. 3. Résultats. 3.1. Acceptabilité du questionnaire : participation. 3.2. Représentativité de l'échantillon. 3.3. Scores par dimension. 3. 4. Evaluation de la validité du questionnaire, convergence des items par dimension et cohérence interne. 3.5. Les questions issues du questionnaire SF-36. 4. Discussion. 4.1. Coping et méthodes alternatives. 4.2. Impact sur les service d'une enquête sur la qualité des soins. 5.1. Le questionnaire de satisfaction élaboré. 5.2. Les différents questionnaires des Hospices : patients hospitalisés et ambulatoires. 5.3. Formulaire d'information de l'enquête par questionnaire. 5.4. Formulaire de consentement à l'enquête. 5.5. Lettre d'accompagnement du questionnaire. 5.6. Profil socio-démographique et résultats complets par item.

BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells.

Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.

Expression and role of BORIS/CTCFL in human cancer stem cells

Le cancer est défini comme la croissance incontrôlée des cellules dans le corps. Il est responsable de 20 % des décès en Europe. Plusieurs expériences montrent que les tumeurs sont issues et se développent grâce à un petit nombre de cellules, que l'on appelle cellules souches cancéreuses (CSC). Ces CSC sont également responsables de l'apparition de métastases et de la résistance aux médicaments anticancéreux. De ce fait, l'identification des gènes qui contribuent aux propriétés de ces CSC (comme la survie des tumeurs, les métastases et la résistance aux médicaments) est nécessaire pour mieux comprendre la biologie des cancers et d'améliorer la qualité des soins des patients avec un cancer. A ce jour, de nombreux marqueurs ont été proposés ainsi que de nouvelles thérapies ciblées contre les CSC. Toutefois, et malgré les énormes efforts de la recherche dans ce domaine, la quasi-totalité des marqueurs de CSC connus à ce jour sont aussi exprimés dans les cellules saines. Ce projet de recherche visait à trouver un nouveau candidat spécifique des CSC. Le gène BORIS (pour Brother of Regulator of Imprinted Sites), nommé aussi CTCFL (CTCF-like), semble avoir certaines caractéristiques de CSC et pourrait donc devenir une cible prometteuse pour le traitement du cancer. BORIS/CTCFL est une protéine nucléaire qui se lie à l'ADN, qui est exprimée dans les tissus normaux uniquement dans les cellules germinales et qui est réactivée dans un grand nombre de tumeurs. BORIS est impliqué dans la reprogrammation épigénétique au cours du développement et dans la tumorigenèse. En outre, des études récentes ont montré une association entre l'expression de BORIS et un mauvais pronostic chez des patients atteints de différents types de cancers. Nous avons développé une nouvelle technologie basée sur les Molecular Beacon pour cibler l'ARNm de BORIS et cela dans les cellules vivantes. Grâce à ce système expérimental, nous avons montré que seule une toute petite sous-population (0,02 à 5%) de cellules tumorales exprimait fortement BORIS. Les cellules exprimant BORIS ont pu être isolées et elles présentaient les caractéristiques de CSC, telles qu'une forte expression de hTERT et des gènes spécifiques des cellules souches (NANOG, SOX2 et OCT4). En outre, une expression élevée de BORIS a été mise en évidence dans des populations enrichies en CSC ('side population' et sphères). Ces résultats suggèrent que BORIS pourrait devenir un nouveau et important marqueur de CSC. Dans des études fonctionnelles sur des cellules de cancer du côlon et du sein, nous avons montré que le blocage de l'expression de BORIS altère largement la capacité de ces cellules à former des sphères, démontrant ainsi un rôle essentiel de BORIS dans l'auto- renouvellement des tumeurs. Nos expériences montrent aussi que BORIS est un facteur important qui régule l'expression de gènes jouant un rôle clé dans le développement et la progression tumorale, tels le gène hTERT et ceux impliqués dans les cellules souches, les CSC et la transition épithélio-mésenchymateuse (EMT). BORIS pourrait affecter la régulation de la transcription de ces gènes par des modifications épigénétiques et de manière différente en fonction du type cellulaire. En résumé, nos résultats fournissent la preuve que BORIS peut être classé comme un gène marqueur de cellules souches cancéreuse et révèlent un nouveau mécanisme dans lequel BORIS jouerait un rôle important dans la carcinogénèse. Cette étude ouvre de nouvelles voies pour mieux comprendre la biologie de la progression tumorale et offre la possibilité de développement de nouvelles thérapies anti-tumorales et anti-CSC avec BORIS comme molécule cible. - Cancer is defined as the uncontrolled growth of cells in the body. It causes 20% of deaths in the European region. Current evidences suggest that tumors originate and are maintained thanks to a small subset of cells, named cancer stems cells (CSCs). These CSCs are also responsible for the appearance of metastasis and therapeutic resistance. Consequently, the identification of genes that contribute to the CSC properties (tumor survival, metastasis and therapeutic resistance) is necessary to better understand the biology of malignant diseases and to improve care management. To date, numerous markers have been proposed to use as new CSC- targeted therapies. Despite the enormous efforts in research, almost all of the known CSCs markers are also expressed in normal cells. This project aimed to find a new CSC-specific candidate. BORIS (Brother of Regulator of Imprinted Sites) or CTCFL (CTCF-like) is a DNA binding protein involves in epigenetic reprogramming in normal development and in tumorigenesis. Recent studies have shown an association of BORIS expression with a poor prognosis in different types of cancer patients. Therefore, BORIS seems to have the same characteristics of CSCs markers and it could be a promising target for cancer therapy. BORIS is normally expressed only in germinal cells and it is re-expressed in a wide variety of tumors. We developed a new molecular beacon-based technology to target BORIS mRNA expressing cells. Using this system, we showed that the BORIS expressing cells are only a small subpopulation (0.02-5%) of tumor cells. The isolated BORIS expressing cells exhibited the characteristics of CSCs, with high expression of hTERT and stem cell genes (NANOG, SOX2 and OCT4). Furthermore, high BORIS expression was observed in the CSC-enriched populations (side population and spheres). These results suggest that BORIS might be a novel and powerful CSCs marker. In functional studies, we observed that BORIS knockdown significantly impairs the capacity to form spheres in colon and breast cancer cells, thus demonstrating a critical role of BORIS in the self-renewal of tumors. The results showed in the functional analysis indicate that BORIS is an important factor that regulates the expression of key-target genes for tumor development and progression, such as hTERT, stem cells, CSCs markers and EMT (epithelial mesenchymal transition)-related marker genes. BORIS could affect the transcriptional regulation of these genes by epigenetic modification and in a cell type dependent manner. In summary, our results support the evidence that BORIS can be classified as a cancer stem cell marker gene and reveal a novel mechanism in which BORIS would play a critical role in tumorigenesis. This study opens new prospective to understand the biology of tumor development and provides opportunities for potential anti-tumor drugs.

High Expression of hTERT and Stemness Genes in BORIS/CTCFL Positive Cells Isolated from Embryonic Cancer Cells.

BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.