Prevalent role of TCR alpha-chain in the selection of the preimmune repertoire specific for a human tumor-associated self-antigen.

The specificity of recognition of pMHC complexes by T lymphocytes is determined by the V regions of the TCR alpha- and beta-chains. Recent experimental evidence has suggested that Ag-specific TCR repertoires may exhibit a more V alpha- than V beta-restricted usage. Whether V alpha usage is narrowed during immune responses to Ag or if, on the contrary, restricted V alpha usage is already defined at the early stages of TCR repertoire selection, however, has remained unexplored. Here, we analyzed V and CDR3 TCR regions of single circulating naive T cells specifically detected ex vivo and isolated with HLA-A2/melan-A peptide multimers. Similarly to what was previously observed for melan-A-specific Ag-experienced T cells, we found a relatively wide V beta usage, but a preferential V alpha 2.1 usage. Restricted V alpha 2.1 usage was also found among single CD8(+) A2/melan-A multimer(+) thymocytes, indicating that V alpha-restricted selection takes place in the thymus. V alpha 2.1 usage, however, was independent from functional avidity of Ag recognition. Thus, interaction of the pMHC complex with selected V alpha-chains contributes to set the broad Ag specificity, as underlined by preferential binding of A2/melan-A multimers to V alpha 2.1-bearing TCRs, whereas functional outcomes result from the sum of these with other interactions between pMHC complex and TCR.

Autoreactive T cells that bypass negative selection are not tolerant and respond to self-antigen stimulation during infection

Le répertoire cellulaire Τ a pour but d'être tolérant aux antigènes du soi afin d'éviter l'induction de maladies autoimmunes. C'est pourquoi les lymphocytes Τ autoréactifs sont éliminés dans le thymus lors de leur développement par le processus de sélection négative. La plupart des recherches étudient les lymphocytes Τ de haute avidité. Ces lymphocytes Τ de haute avidité sont très sensibles et réagissent fortement à un antigène du soi. En conséquence, ces cellules induisent le développement de maladies autoimmunes lorsqu'elles ciblent des organes exprimant l'antigène du soi. Plusieurs études ont montré que les lymphocytes Τ qui réagissent faiblement aux antigènes spécifiques à un tissu, nommé lymphocytes Τ de faible avidité, peuvent contourner les mécanismes de tolérance centrale et périphérique. J'ai utilisé des souris Rip-mOva qui expriment l'Ovalbumine comme antigène du soi spécifique à un tissu. Dans ces souris transgéniques Rip-mOva, les lymphocytes Τ de faible avidité survivent à la sélection négative. Une fois stimulés à la périphérie, ces lymphocytes Τ CD8+ de faible avidité ont la capacité d'infiltrer les organes qui expriment l'antigène du soi chez les souris Rip-mOva et peuvent induire une destruction tissulaire. L'objectif principal de mon projet de thèse était de comprendre les caractéristiques phénotypiques et fonctionnelles de ces lymphocytes Τ dans un état d'équilibre et dans un contexte infectieux. Pour étudier ces cellules dans un modèle murin bien défini, nous avons généré des souris exprimant un récepteur de cellule Τ transgénique appelé OT-3. Ces souris transgéniques OT-3 ont des lymphocytes Τ CD8+ de faible avidité spécifiques à l'épitope SIINFEKL de l'antigène Ovalbumine. Nous avons démontré qu'un grand nombre de lymphocytes Τ CD8+ OT-3 ne sont pas éliminés lors de la sélection négative dans le thymus après avoir rencontré l'antigène du soi. Par conséquent, les lymphocytes Τ OT-3 de faible avidité sont présents dans une fenêtre de sélection comprise entre la sélection positive et négative. Cette limite se définie comme le seuil d'affinité et est impliquée dans l'échappement de certains lymphocytes Τ OT- 3 autoréactifs. A la périphérie, ces cellules sont capables d'induire une autoimmunité après stimulation au cours d'une infection, ce qui nous permet de les définir comme étant non tolérante et non dans un état anergique à la périphérie. Nous avons également étudié le seuil d'activation des lymphocytes Τ OT-3 à faible avidité à la périphérie et avons constaté que des ligands peptidiques plus faibles que l'épitope natif SIINFEKL sont capables de les activer au cours d'une infection ainsi que de les différencier en lymphocytes Τ effecteurs et mémoires. Les données illustrent une déficience lors de la sélection négative dans le thymus de lymphocytes Τ CD8+ autoréactifs de faible avidité contre un antigène du soi spécifique à tissu et montrent que ces cellules sont entièrement compétentes lors d'une infection. - The diverse Τ cell repertoire needs to be tolerant to self-antigen to avoid the induction of autoimmunity. This is why autoreactive developing Τ cells are deleted in the thymus. The deletion of self-reactive Τ cells occurs through the process of negative selection. Most studies investigated high avidity Τ cells. These high avidity Τ cells are very sensitive and strongly react to a self-antigen. As a consequence, these cells induce the development of autoimmunity when they target organs which express the self-antigen. High avidity autoreactive CD8+ Τ cells are deleted in the thymus. However, several studies have shown Τ cells that weakly respond to tissue-restricted antigen, referred to as low avidity Τ cells, can bypass central and peripheral tolerance mechanisms. I used Rip-mOva mice that expressed Ovalbumin as a neo self-antigen in a tissue-restricted fashion. In these transgenic Rip-mOva mice low avidity CD8+ Τ cells survive negative selection. Upon stimulation in the periphery, these low avidity CD8+ Τ cells have the ability to infiltrate organs that express the self-antigen in the Rip-mOva mice and can also induce the destruction of the tissue. The major aim of my PhD project was to understand the phenotypic and functionality characteristics of these Τ cells in a steady-state condition and in a context of an infection. To study these cells in a well-defined mouse model, we generated OT-3 Τ cell receptor transgenic mice that express low avidity CD8+ Τ cells that are specific for the SIINFEKL epitope of the Ovalbumin antigen. We have been able to demonstrate that a large number of OT-3 CD8+ Τ cells survive negative selection in the thymus after encountering the self-antigen. Thus, low avidity OT-3 Τ cells are present in a window of selection comprised between positive and negative selection. This boundary defined as the affinity threshold is involved in the escape of some autoreactive low avidity OT-3 Τ cells. Once they circulate in the periphery, they are able to induce autoimmunity after stimulation during an infection, allowing us to allocate these cells as being non-tolerant and not in an anergic state in the periphery. We have also looked at the threshold of activation of low avidity OT-3 CD8+ Τ cells in the periphery and found that peptide ligands that are weaker than the native SIINFEKL epitope are able to activate OT-3 Τ cells during an infection and to differentiate them into effector and memory Τ cells. The data illustrate the impairment of negatively selecting low avidity autoreactive CD8+ Τ cells against a tissue-restricted antigen in the thymus and shows that these cells are fully competent upon an infection.

Autoreactive T cells bypass negative selection and respond to self-antigen stimulation during infection.

Central and peripheral tolerance prevent autoimmunity by deleting the most aggressive CD8(+) T cells but they spare cells that react weakly to tissue-restricted antigen (TRA). To reveal the functional characteristics of these spared cells, we generated a transgenic mouse expressing the TCR of a TRA-specific T cell that had escaped negative selection. Interestingly, the isolated TCR matches the affinity/avidity threshold for negatively selecting T cells, and when developing transgenic cells are exposed to their TRA in the thymus, only a fraction of them are eliminated but significant numbers enter the periphery. In contrast to high avidity cells, low avidity T cells persist in the antigen-positive periphery with no signs of anergy, unresponsiveness, or prior activation. Upon activation during an infection they cause autoimmunity and form memory cells. Unexpectedly, peptide ligands that are weaker in stimulating the transgenic T cells than the thymic threshold ligand also induce profound activation in the periphery. Thus, the peripheral T cell activation threshold during an infection is below that of negative selection for TRA. These results demonstrate the existence of a level of self-reactivity to TRA to which the thymus confers no protection and illustrate that organ damage can occur without genetic predisposition to autoimmunity.

Treatment of rats with a self-selected hyperlipidic diet, increases the lipid content of the main adipose tissue sites in a proportion similar to that of the lipids in the rest of organs and tissues

Adipose tissue (AT) is distributed as large differentiated masses, and smaller depots covering vessels, and organs, as well as interspersed within them. The differences between types and size of cells makes AT one of the most disperse and complex organs. Lipid storage is partly shared by other tissues such as muscle and liver. We intended to obtain an approximate estimation of the size of lipid reserves stored outside the main fat depots. Both male and female rats were made overweight by 4-weeks feeding of a cafeteria diet. Total lipid content was analyzed in brain, liver, gastrocnemius muscle, four white AT sites: subcutaneous, perigonadal, retroperitoneal and mesenteric, two brown AT sites (interscapular and perirenal) and in a pool of the rest of organs and tissues (after discarding gut contents). Organ lipid content was estimated and tabulated for each individual rat. Food intake was measured daily. There was a surprisingly high proportion of lipid not accounted for by the main macroscopic AT sites, even when brain, liver and BAT main sites were discounted. Muscle contained about 8% of body lipids, liver 1-1.4%, four white AT sites lipid 28-63% of body lipid, and the rest of the body (including muscle) 38-44%. There was a good correlation between AT lipid and body lipid, but lipid in"other organs" was highly correlated too with body lipid. Brain lipid was not. Irrespective of dietary intake, accumulation of body fat was uniform both for the main lipid storage and handling organs: large masses of AT (but also liver, muscle), as well as in the"rest" of tissues. These storage sites, in specialized (adipose) or not-specialized (liver, muscle) tissues reacted in parallel against a hyperlipidic diet challenge. We postulate that body lipid stores are handled and regulated coordinately, with a more centralized and overall mechanisms than usually assumed.

Combined selection in early generation testing of self-pollinated plants

Seven selection indexes based on the phenotypic value of the individual and the mean performance of its family were assessed for their application in breeding of self-pollinated plants. There is no clear superiority from one index to another although some show one or more negative aspects, such as favoring the selection of a top performing plant from an inferior family in detriment of an excellent plant from a superior family

Self-assembly of two-component gels: stoichiometric control and component selection

Two-component systems capable of self-assembling into soft gel-phase materials are of considerable interest due to their tunability and versatility. This paper investigates two-component gels based on a combination of a L-lysine-based dendron and a rigid diamine spacer (1,4-diaminobenzene or 1,4-diaminocyclohexane). The networked gelator was investigated using thermal measurements, circular dichroism, NMR spectroscopy and small angle neutron scattering (SANS) giving insight into the macroscopic properties, nanostructure and molecular-scale organisation. Surprisingly, all of these techniques confirmed that irrespective of the molar ratio of the components employed, the "solid-like" gel network always consisted of a 1:1 mixture of dendron/diamine. Additionally, the gel network was able to tolerate a significant excess of diamine in the "liquid-like" phase before being disrupted. In the light of this observation, we investigated the ability of the gel network structure to evolve from mixtures of different aromatic diamines present in excess. We found that these two-component gels assembled in a component-selective manner, with the dendron preferentially recognising 1,4-diaminobenzene (>70%). when similar competitor diamines (1,2- and 1,3-diaminobenzene) are present. Furthermore, NMR relaxation measurements demonstrated that the gel based oil 1,4-diaminobenzene was better able to form a selective ternary complex with pyrene than the gel based oil 1,4-diaminocyclohexane, indicative of controlled and selective pi-pi interactions within a three-component assembly. As such, the results ill this paper demonstrate how component selection processes in two-component gel systems call control hierarchical self-assembly.

Diet and food selection by small mammals in an old-growth Atlantic forest of south-eastern Brazil

Here we describe the stomach contents of nine small mammal species (seven rodents and two didelphid marsupials) co-occurring in an old-growth Atlantic forest area. For four terrestrial rodents, we also compared the importance of arthropods in the diet and the selection of arthropod groups by comparing consumption with availability. Small mammals and arthropods were sampled in a 36-ha grid containing 25 sampling stations spaced every 150 m, and 47 stomach contents were analysed. While plant matter was the predominant item in the stomach contents of two rodents (Oligoryzomys nigripes and Rhipidomys mastacalis), four species presented arthropods as the main food item (the rodents Brucepattersonius soricinus and Oxymycterus dasytrichus, and the marsupials Monodelphis n. sp. and Marmosops incanus) and three consumed more plant matter than arthropods, but had significant amounts of both items (the rodents Delomys sublineatus, Euryoryzomys russatus and Thaptomys nigrita). Our results suggest that differences in diet, coupled with differences in habit and microhabitat preferences, are important factors allowing resource partition among species of the diverse group of co-occurring terrestrial small mammals in Atlantic forest areas. Moreover, arthropods were not preyed opportunistically by any of the four terrestrial rodents, since consumption was not proportional to availability. Rather, selection or rejection of arthropod groups seems to be determined by aspects other than availability, such as nutritional value, easiness of capture and handling or palatability.

Free diet selection by broilers as influenced by dietary macronutrient ratio and corticosterone supplementation. 1. Diet selection, organ weights, and plasma metabolites

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Self-esteem, narcissism, and stressful life events: Testing for selection and socialization

We examined whether self-esteem and narcissism predict the occurrence of stressful life events (i.e., selection) and whether stressful life events predict change in self-esteem and narcissism (i.e., socialization). The analyses were based on longitudinal data from 2 studies, including samples of 328 young adults (Study 1) and 371 adults (Study 2). The effects of self-esteem and narcissism were mutually controlled for each other and, moreover, controlled for effects of depression. After conducting the study-level analyses, we meta-analytically aggregated the findings. Self-esteem had a selection effect, suggesting that low self-esteem led to the occurrence of stressful life events; however, this effect became nonsignificant when depression was controlled for. Regardless of whether depression was controlled for or not, narcissism had a selection effect, suggesting that high narcissism led to the occurrence of stressful life events. Moreover, stressful life events had a socialization effect on self-esteem, but not on narcissism, suggesting that the occurrence of stressful life events decreased self-esteem. Analyses of trait–state models indicated that narcissism consisted almost exclusively of perfectly stable trait variance, providing a possible explanation for the absence of socialization effects on narcissism. The findings have significant implications because they suggest that a person’s level of narcissism influences whether stressful life events occur, and that self-esteem is shaped by the occurrence of stressful life events. Moreover, we discuss the possibility that depression mediates the selection effect of low self-esteem on stressful life events.

The Choosing Healthy Options of Starches (CHOOS) diet and dietary intake self-monitoring for weight loss among adolescent females

Previous research has shown dietary intake self-monitoring, and culturally tailored weight loss interventions to be effective tools for weight loss. Technology can be used to tailor weight loss interventions to better suit adolescents. There is a lack of research to date on the use of personal digital assistants (PDAs) to self-monitor dietary intake among adolescents. The objective of this study was to determine the difference in dietary intake self-monitoring frequency between using a Personal Digital Assistant (PDA) or paper logs as a diet diary in obese adolescent females; and to describe differences in diet adherence, as well as changes in body size and self-efficacy to resist eating. We hypothesized dietary intake self-monitoring frequency would be greater during PDA use than during paper log use. This study was a randomized crossover trial. Participants recorded their diet for 4 weeks: 2 weeks on a PDA and 2 weeks on paper logs. Thirty-four obese females ages 12-20 were recruited for participation. Thirty were included in analyses. Participants recorded more entries/day while using the paper logs (4.10 entries/day ± 0.63) than while using the PDA (3.01 entries/day ±0.75) (p<0.001). Significantly more meals and snacks were skipped during paper log use (0.81/day ± 0.65) than during PDA use (0.23/day ± 0.22) (p=0.011). Changes in body size (BMI, weight, and waist circumference) and self-efficacy to resist eating did not differ significantly between PDA and paper log use. When compared to paper logs, participants felt the PDA was more convenient (p=0.020), looked forward to using the PDA more (p=0.008), and would rather continue using the PDA than the paper logs (p=0.020). The findings of this study indicate use of a PDA as a dietary intake self-monitoring tool among adolescents would not result in increased dietary intake self-monitoring to aid in weight loss. Use of paper logs would result in greater data returned to clinicians, though use of PDAs would likely get adolescents more excited about adhering to recommendations to record their diet. Future research should look at updated communication devices, such as cell phones and other PDAs with additional features, and the role they can play in increasing dietary intake self-monitoring among adolescents.^

Diet selection by the lesser rhea (Rhea pennata pennata) in Payunia, Northern Patagonia (Mendoza, Argentina)

The lesser rhea (family Rheidae) is a flightless large bird of South America, threatened due to habitat loss, hunting and egg collecting, with special concern in Northern Patagonia. Diet and food availability were estimated throughout the year by micro-histological analysis and point-quadrat transects in a landscape inside and another outside the Payunia Reserve, the northernmost part of the Rhea pennata pennata distribution. Significant differences were detected by Kruskall-Wallis ANOVA, food selection by Chi-square test and Bailey’s confidence interval. A strong food selection characterized the diet of lesser rheas, dominated by leaves of shrubs and forbs, complemented by dicot seeds and a few insects. This agrees with the documented low dietary overlap with other herbivores in Payunia. Dietary changes agree with the expected from the selective quality hypothesis. Food availability was better inside than outside the protected area, with probable conservation effects for lesser rheas. Seeds, forbs and soft grasses could be for lesser rheas some key food resources to survive during unfavorable seasons in arid environments without "mallines", as Payunia. Shrubby patches, with high availability of preferred food items (tall shrubs and forbs), stood out as key habitats. Therefore, avoiding fire and woody plant removal is crucial for the conservation of lesser rheas in the northern of its range.

In vitro selection of self-cleaving RNAs with a low pH optimum

RNAs that undergo a rapid site-specific cleavage at low pH have been selected by in vitro selection (the SELEX process). The cleavage does not require the addition of any divalent metal ions, and is in fact inhibited by divalent metal ions, spermine, or high concentrations of monovalent metal ions. This low pH catalyzed cleavage results in a 2′,3′-cyclic phosphate at the 3′ end and a free hydroxyl at the 5′ end. The reaction proceeds with a calculated rate of 1.1 min−1 at room temperature in cacodylate buffer at pH 5.0. The rate of cleavage is dependent on the pH and shows an optimum around pH 4.0. The rate constant is independent of RNA concentration, indicating to an intramolecular reaction. Autocatalytic cleavage at low pH, in the absence of a metal ion requirement, adds to the reaction possibilities that may have existed on the prebiotic earth.

Feature selection by multi-objective optimisation: Application to network anomaly detection by hierarchical self-organising maps

Feature selection is an important and active issue in clustering and classification problems. By choosing an adequate feature subset, a dataset dimensionality reduction is allowed, thus contributing to decreasing the classification computational complexity, and to improving the classifier performance by avoiding redundant or irrelevant features. Although feature selection can be formally defined as an optimisation problem with only one objective, that is, the classification accuracy obtained by using the selected feature subset, in recent years, some multi-objective approaches to this problem have been proposed. These either select features that not only improve the classification accuracy, but also the generalisation capability in case of supervised classifiers, or counterbalance the bias toward lower or higher numbers of features that present some methods used to validate the clustering/classification in case of unsupervised classifiers. The main contribution of this paper is a multi-objective approach for feature selection and its application to an unsupervised clustering procedure based on Growing Hierarchical Self-Organising Maps (GHSOMs) that includes a new method for unit labelling and efficient determination of the winning unit. In the network anomaly detection problem here considered, this multi-objective approach makes it possible not only to differentiate between normal and anomalous traffic but also among different anomalies. The efficiency of our proposals has been evaluated by using the well-known DARPA/NSL-KDD datasets that contain extracted features and labelled attacks from around 2 million connections. The selected feature sets computed in our experiments provide detection rates up to 99.8% with normal traffic and up to 99.6% with anomalous traffic, as well as accuracy values up to 99.12%.