De novo T cell generation and cell cycle analysis of CD4 and CD8 T cells during HIV-disease

RESUME POUR UN LARGE PUBLIC Parmi les globules blancs, les lymphocytes T 004 jouent un rôle primordial dans la coordination de la réponse immunitaire contre les pathogènes et les lymphocytes T CD8 dans leur élimination. Lors d'une infection par le virus de l'immunodéficience humaine (VIH-1), non seulement les cellules T CD4 sont les principales cibles d'infections, mais aussi elles disparaissent progressivement tout au long de la maladie. Ce phénomène, appelé aussi épuisement des lymphocytes T CD4, est la principale cause provoquant le Syndrome d'Immunodéficience Acquise (SIDA). Malgré de grands efforts de recherche, nous ne sommes toujours pas en mesure de dire si ce phénomène est dû à un défaut dans la production de nouvelles cellules ou à une destruction massive de cellules en circulation. Dans cette étude, nous nous proposions, dans un premier temps, de comparer la production de nouvelles cellules T CD4 et CD8 chez des individus VIH-négatifs et positifs. Les cellules nouvellement produites portent un marqueur commun que l'on appelle TREC et qui est facilement mesurable. En considérant des paramètres cliniques, nous étions en mesure de déterminer le niveau de TRECs de cellules T CD4 et CD8 dans différentes phases de la maladie. De là, nous avons pu déterminer que le niveau de TREC est toujours plus bas dans les cellules T CD8 de patients VIH-positifs comparativement à notre groupe contrôle. Nous avons pu déterminer par une analyse ultérieure que cette différence est due à une forte prolifération de ces cellules chez les patients VIH-positifs, ce qui a pour effet de diluer ce marqueur. En revanche, la production de nouvelles cellules T CD4 chez des patients VIH-positifs est accentuée lors de la phase précoce de la maladie et largement réprimée lors de la phase tardive. Dans un second temps, nous avons effectué une analyse à grande échelle de l'expression de gènes associés à la division cellulaire sur des lymphocytes T CD4 et CD8 d'individus VIH-¬positifs et négatifs, avec comme contrôle des cellules proliférant in vitro. De cette étude, nous avons pu conclure que les cellules T CD8 de patients VIH-positifs étaient en état de prolifération, alors que les lymphocytes T CD4 présentaient des défauts majeurs conduisant à un arrêt de la division cellulaire. Nos résultats montrent que la capacité à produire de nouvelles cellules chez des patients VIH¬positifs reste active longtemps pendant la maladie, mais que l'incapacité des cellules T CD4 à proliférer peut enrayer la reconstitution immunitaire chez ces individus. ABSTRACT The hallmark of HIV-1 infection is the depletion of CD4 T cells. Despite extensive investigation, the mechanisms responsible for the loss of CD4 T cells have been elucidated only partially. In particular, it remains controversial whether CD4 T cell depletion results from a defect in T cell production or from a massive peripheral destruction. In this study, de novo T cell generation has been investigated by measuring T cell receptor rearrangement excision circles (TRECs) on large cohorts of HIV-negative (N=120) and HIV-1 infected (N=298) individuals. Analysis of TREC levels was performed in HIV-infected subjects stratified by the stage of HIV disease based on CD4 T cell counts (early: >500 CD4 T cells/µl; intermediate: <500>200; late: <200) and by age (20 to 60 years, n = 259). Our data show that TREC levels in CD8 T cells were significantly lower in HIV-infected subjects at any stage of disease compared to the control group. In contrast, TREC levels in CD4 T cells were significantly higher in HIV-infected subjects at early stages disease while no significant differences were observed at intermediate stages of the disease and were severely reduced only at late stages of disease. To investigate further the status of cell cycle in peripheral CD4 and CD8 T cells in HIV-1 infections, we determined the pattern of gene expression with the microarray technology. In particular, CD4 and CD8 T cells of HIV-1 infected and HIV-negative subjects were analysed by Cell Cycle cDNA expression array. The patterns of gene expression were compared to in vitro stimulated CD4 and CD8 T cells and this analysis showed that CD8 T cells of HIV-1 infected subjects had a pattern of gene expression very similar to that of in vitro stimulated CD8 T cells thus indicating ongoing cell cycling. In contrast, CD4 T cells of HIV-1 infected subjects displayed a complex pattern of gene expression. In fact, CD4 T cells expressed high levels of genes typically associated with cell activation, but low levels of cell cycle genes. Therefore, these results indicated that activated CD4 T cells of HIV-1 infected subjects were in cell cycle arrest. Taking together these results indicate that thymus function is preserved for long time during HIV- 1 infection and the increase observed in early stage disease may represent a compensatory mechanism to the depletion of CD4 T cells. However, we provide evidence for a cell cycle arrest of peripheral CD4 T cells that may prevent potentially the replenishment of CD4 T cells. RESUME Les mécanismes responsables de la perte des lymphocytes T CD4 lors de l'infection pas VIH n'ont été élucidés que partiellement. Nous ne savons toujours pas si l'épuisement des lymphocytes T CD4 résulte d'un défaut dans la production de cellules ou d'une destruction périphérique massive. Dans cette étude, la production de cellules T a été étudiée en mesurant les cercles d'excision générés lors du réarrangement du récepteur au cellules T (TRECs) chez des individus VIH-négatifs (N=120) et VIH-1 positifs (N=298). L'analyse des niveaux de TREC a été faite chez sujets HIV-infectés en considérant les phases de la maladie sur la base des comptes CD4 (phase précoce: > 500 cellules CD4/µl; intermédiaire: < 500>200; tardive: < 200) et par âge. Nos données démontrent que les niveaux de TRECs des cellules T CD8 étaient significativement plus bas chez les sujets VIH-1 infectés, à tous les stades de la maladie comparativement au groupe contrôle. En revanche, les niveaux de TRECs des cellules T CD4 étaient significativement plus élevés chez les sujets VIH-1 infectés durant la phase précoce de la maladie, tandis qu'aucune différence significative n'était observée durant la phase intermédiaire et étaient très réduits dans la phase tardive. Dans une deuxième partie, nous avons utilisé la technique des biopuces à d'ADN complémentaire pour analyser la régulation du cycle cellulaire chez les lymphocytes T CD4 et CD8 périphériques lors d'une infection au VIH-1. Des profils d'expression ont été déterminés et comparés à ceux de cellules T CD4 et CD8 stimulées in vitro, démontrant que les cellules T CD8 des sujets VIH-positifs avaient un profil d'expression très semblable à celui des cellules stimulées in vitro en prolifération. En revanche, les lymphocytes T CD4 des sujets VIH-1 positifs avaient un profil d'expression de gène plus complexe. En fait, leur profil montrait une sur- expression de gènes associés à une activation cellulaire, mais une sous-expression de ceux induisant une division. Ainsi, ces résultats indiquent que les lymphocytes T CD4 d'individus VIH-positifs présentent des dérégulations qui conduisent à un arrêt du cycle cellulaire. Ces résultats montrent que la fonction thymique est préservée longtemps pendant l'infection au VIH-1 et que l'augmentation de la quantité de TRECs dans la phase précoce de la maladie peut représenter un mécanisme compensatoire à l'épuisement des cellules T CD4. Cependant, nous démontrons aussi un clair dysfonctionnement du cycle cellulaire chez les cellules T CD4 d'individus infectés par VIH-1 ce qui peut enrayer la reconstitution du système immunitaire.

Equilibration and disequilibration between monazite and garnet: indication from phase-composition and quantitative texture analysis

The integration of information which can be gained from accessory [i.e. age (t)] and rock-forming minerals [i.e. temperature (T) and pressure (P)] requires a more profound understanding of the equilibration kinetics during metamorphic processes. This paper presents an approach comparing conventional P-T estimate from equilibrated assemblages of rock-forming minerals with temperature data derived from yttrium-garnet-monazite (YGM) and yttrium-garnet-xenotime (YGX) geothermometry. Such a comparison provides an initial indication on differences between equilibration of major and trace elements. Regarding this purpose, two migmatites, two polycyclic and one monocyclic gneiss from the Central Alps (Switzerland, northern Italy) were investigated. While the polycyclic samples exhibit trace-element equilibration between monazite and garnet grains assigned to the same metamorphic event, there are relics of monazite and garnet obviously surviving independent of their textural position. These observations suggest that surface processes dominate transport processes during equilibration of those samples. The monocyclic gneiss, on the contrary, displays rare isolated monazite with equilibration of all elements, despite comparably large transport distances. With a nearly linear crystal-size distribution of the garnet grain population, growth kinetics, related to the major elements, were likely surface-controlled in this sample. In contrast to these completely equilibrated examples, the migmatites indicate disequilibrium between garnet and monazite with a change in REE patterns on garnet transects. The cause for this disequilibrium may be related to a potential disequilibrium initiated by a changing bulk chemistry during melt segregation. While migmatite environments are expected to support high transport rates (i.e. high temperatures and melt presence), the evolution of equilibration in migmatites is additionaly related to change in chemistry. As a key finding, surface-controlled equilibration kinetics seem to dominate transport-controlled processes in the investigated samples. This may be decisive information towards the understanding of age data derived from monazite.

Logging residues from regeneration fellings for biofuel production - a GIS-based availability and supply cost analysis

Finland has large forest fuel resources. However, the use of forest fuels for energy production has been low, except for small-scale use in heating. According to national action plans and programs related to wood energy promotion, the utilization of such resources will be multiplied over the next few years. The most significant part of this growth will be based on the utilization of forest fuels, produced from logging residues of regeneration fellings, in industrial and municipal power and heating plants. Availability of logging residues was analyzed by means of resource and demand approaches in order to identify the most suitable regions with focus on increasing the forest fuel usage. The analysis included availability and supply cost comparisons between power plant sites and resource allocation in a least cost manner, and between a predefined power plant structure under demand and supply constraints. Spatial analysis of worksite factors and regional geographies were carried out using the GIS-model environment via geoprocessing and cartographic modeling tools. According to the results of analyses, the cost competitiveness of forest fuel supply should be improved in order to achieve the designed objectives in the near future. Availability and supply costs of forest fuels varied spatially and were very sensitive to worksite factors and transport distances. According to the site-specific analysis the supply potential between differentlocations can be multifold. However, due to technical and economical reasons ofthe fuel supply and dense power plant infrastructure, the supply potential is limited at plant level. Therefore, the potential and supply cost calculations aredepending on site-specific matters, where regional characteristics of resourcesand infrastructure should be taken into consideration, for example by using a GIS-modeling approach constructed in this study.

White eye phenotypes and their genetic analysis

An interesting case for undergraduate students of general Genetics is to consider that different genes can produce the same or similar phenotypes. We present here an experiment to discover that the same phenotype could be produced by different genes, and then, to carry out the genetic analysis of these genes. For this laboratory study we have used the following Drosophila melanogaster strains: white (white eyes) and scarlet-brown (white eyes).

White eye phenotypes and their genetic analysis

An interesting case for undergraduate students of general Genetics is to consider that different genes can produce the same or similar phenotypes. We present here an experiment to discover that the same phenotype could be produced by different genes, and then, to carry out the genetic analysis of these genes. For this laboratory study we have used the following Drosophila melanogaster strains: white (white eyes) and scarlet-brown (white eyes).

Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

ABSTRACT Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies.

Customer Research on a Group of European Book Publishers: Trends, Modes of Operation and Decision-Making Processes

Työn päätarkoitus oli tuottaa Stora Enson käyttöön tietoa kirjakustantajista, yhdestä yrityksen asiakassegmentistä. Yritys oli kiinnostunut useista asioista, jotka koskivat asiakkaita ja heidän mielipiteitään. Tarkoitus on, että Stora Enso voi käyttää tutkimuksella koottua tietoa oman toimintansa suunnittelun tukena. Kerätty sekundääritieto esittelee eurooppalaisen kirjakustantamisen nykytilaa ja tulevaisuutta sekä teorioita, jotka tukevat tutkittuja aihealueita. Primääritieto kerättiin henkilökohtaisilla haastatteluilla. Otanta koostui kymmenestä kirjakustantajasta, jotka toimivat Suomessa sekä Stora Enson päämarkkina-alueilla. Tutkimus tarjoaa päivitetyn kuvauksen kirjakustantamisesta. Haastateltavien mielipiteet alan trendeistä olivat yhteneviä yleisen mielipiteen kanssa, eikä suuria mielipide-eroavaisuuksia havaittu. Kustantajien toimintatapoja ja päätöksentekoprosesseja voidaan kuvata monimutkaisiksi, koska useat asiat vaikuttavat kirjan syntyyn ja paperin ostoprosessiin. Lisäksi tutkimus esittelee haastateltavien mielipiteitä paperin merkityksestä heidän liiketoiminnassaan.

Online drawings for dementia diagnose: in-air and pressure information analysis

In this paper we present experimental results comparing on-line drawings for control population (left and right hand) as well as Alzheimer disease patients. The drawings have been acquired by means of a digitizing tablet, which acquires time information angles and pressures. Experimental measures based on pressure and in-air movements appear to be significantly different for both groups, even when control population performs the tasks with the non-dominant hand.

Particle shape and orientation in laser diffraction and static image analysis size distribution analysis of micrometer sized rectangular particles

Laser diffraction (LD) and static image analysis (SIA) of rectangular particles [United States Pharmacopeia, USP30-NF25, General Chapter <776>, Optical Miroscopy.] have been systematically studied. To rule out sample dispersion and particle orientation as the root cause of differences in size distribution profiles, we immobilize powder samples on a glass plate by means of a dry disperser. For a defined region of the glass plate, we measure the diffraction pattern as induced by the dispersed particles, and the 2D dimensions of the individual particles using LD and optical microscopy, respectively. We demonstrate a correlation between LD and SIA, with the scattering intensity of the individual particles as the dominant factor. In theory, the scattering intensity is related to the square of the projected area of both spherical and rectangular particles. In traditional LD the size distribution profile is dominated by the maximum projected area of the particles (A). The diffraction diameters of a rectangular particle with length L and breadth B as measured by the LD instrument approximately correspond to spheres of diameter ØL and ØB respectively. Differences in the scattering intensity between spherical and rectangular particles suggest that the contribution made to the overall LD volume probability distribution by each rectangular particle is proportional to A2/L and A2/B. Accordingly, for rectangular particles the scattering intensity weighted diffraction diameter (SIWDD) explains an overestimation of their shortest dimension and an underestimation of their longest dimension. This study analyzes various samples of particles whose length ranges from approximately 10 to 1000 μm. The correlation we demonstrate between LD and SIA can be used to improve validation of LD methods based on SIA data for a variety of pharmaceutical powders all with a different rectangular particle size and shape.

Investigation and data adaptive analysis of CARS spectra

In this diploma work advantages of coherent anti-Stokes Raman scattering spectrometry (CARS) and various methods of the quantitative analysis of substance structure with its help are considered. The basic methods and concepts of the adaptive analysis are adduced. On the basis of these methods the algorithm of automatic measurement of a scattering strip size of a target component in CARS spectrum is developed. The algorithm uses known full spectrum of target substance and compares it with a CARS spectrum. The form of a differential spectrum is used as a feedback to control the accuracy of matching. To exclude the influence of a background in CARS spectra the differential spectrum is analysed by means of its second derivative. The algorithm is checked up on the simulated simple spectra and on the spectra of organic compounds received experimentally.

The difference of disease perception by juvenile idiopathic arthritis patients and their parents : analysis of the JAMAR questionnaire.

BACKGROUND: The JAMAR (Juvenile Arthritis Multidimensional Assessment Report) has been developed to evaluate the perception of the patient and his parents on different items: well-being, pain, functional status, quality of life, disease activity, disease course, side effects of medication, therapeutic compliance and satisfaction with illness outcome. Our aim was to compare disease's perception by JIA patients and their parents. METHODS: We included into the study 100 consecutive patients over 7 years of age. We asked both parent and child to complete the JAMAR questionnaire. For each patient we recorded demographic and disease related data. We examined the level of disagreement between children and parents for the quantitative items of the JAMAR: VAS Pain, VAS Disease Activity, VAS Well Being, Juvenile Arthritis Functional Score, HRQoL. Then we looked for a relation between discordance-rate and demographic and clinical variables. RESULTS: Children and parents' median scores for all five items were similar. Individual dyads agreement was low, with a large amount of pairs (80) discordant for at least one item. We found higher MD VAS and JADAS in more discordant dyads, suggesting that when the disease is more active discordance between child and parent increase. CONCLUSION: The JAMAR questionnaire is an important tool that helps clinicians to detect divergent child and parent's disease perceptions. It is essential that both patients and parents fill the JAMAR questionnaire for a complete clinical and psychosocial evaluation.