Potential and limitations of regulatory T-cell therapy in solid organ transplantation.

Over the past few years, the therapeutic potential of Treg has been highlighted in the field of autoimmune diseases and after allogeneic transplantation. The first hurdle for the therapeutic use of Treg is their insufficient numbers in non-manipulated individuals, in particular when facing strong immune activation and expanding effector cells, such as in response to an allograft. Here we review current approaches being explored for Treg expansion in the perspective of clinical therapeutic protocols. We describe different Treg subsets that could be suitable for clinical application, as well as discuss factors such as the required dose of Treg, their antigen-specificity and in vivo stability, that have to be considered for optimal Treg-based immunotherapy in transplantation. Since Treg may not be sufficient as stand-alone therapy for solid organ transplantation in humans, we draw attention to possible hurdles and combination therapy with immunomodulatory drugs that could possibly improve the in vivo efficacy of Treg.

Implementing statistical learning methods through Bayesian networks. Part 1: a guide to Bayesian parameter estimation using forensic science data

As a thorough aggregation of probability and graph theory, Bayesian networks currently enjoy widespread interest as a means for studying factors that affect the coherent evaluation of scientific evidence in forensic science. Paper I of this series of papers intends to contribute to the discussion of Bayesian networks as a framework that is helpful for both illustrating and implementing statistical procedures that are commonly employed for the study of uncertainties (e.g. the estimation of unknown quantities). While the respective statistical procedures are widely described in literature, the primary aim of this paper is to offer an essentially non-technical introduction on how interested readers may use these analytical approaches - with the help of Bayesian networks - for processing their own forensic science data. Attention is mainly drawn to the structure and underlying rationale of a series of basic and context-independent network fragments that users may incorporate as building blocs while constructing larger inference models. As an example of how this may be done, the proposed concepts will be used in a second paper (Part II) for specifying graphical probability networks whose purpose is to assist forensic scientists in the evaluation of scientific evidence encountered in the context of forensic document examination (i.e. results of the analysis of black toners present on printed or copied documents).

High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

THE INFLUENCE OF THE REGULATORY ENVIRONMENT ON THE DEFINITION OF PERFORMANCE: THE EXAMPLE OF THE SPORT BETTING AND LOTTERY SECTORS

This thesis examines the interplay between state regulation and the way organisations define performance. Performance is generally understood to be a multidimensional concept, but the extent to which its different facets are shaped by regulation remains an understudied question. This thesis aims to address this question and provide at least a partial answer to it. To do so, it examines whether the level of regulation amplifies or abates the multidimensionality of regulated entities' performance definition, i.e. the way they define the concept of performance. The leading question is whether an organisation's performance definition can be associated with the regulatory intensity its environment confronts it with. Moreover, the study explores whether the type of ownership-public or private-plays a role in regard to how a regulated entity defines performance. In order to undertake this investigation, the thesis focuses on the performance definitions of organisations in six different sport betting and lottery regulations. Qualitative data is gathered from primary and secondary documents as well as through semi-structured interviews with chief executive officers (CEO), members of executive management and gambling experts in each of these countries. The thesis concludes that the performance definitions of the organisations under study are indeed multidimensional, as well as clearly influenced by their respective regulatory environments. However, not all performance dimensions identified in the literature are present, nor can they all be estimated to be part of the performance definition. In addition, the public-private difference in defining performance-as conceptualised in the literature- seems to be abated in a regulated environment. The central role played by regulation in regard to the multidimensionality of the performance definition partially outweighs the effect of the nature of ownership.

Light intensity modulates the regulatory network of the shade avoidance response in Arabidopsis.

Plants such as Arabidopsis thaliana respond to foliar shade and neighbors who may become competitors for light resources by elongation growth to secure access to unfiltered sunlight. Challenges faced during this shade avoidance response (SAR) are different under a light-absorbing canopy and during neighbor detection where light remains abundant. In both situations, elongation growth depends on auxin and transcription factors of the phytochrome interacting factor (PIF) class. Using a computational modeling approach to study the SAR regulatory network, we identify and experimentally validate a previously unidentified role for long hypocotyl in far red 1, a negative regulator of the PIFs. Moreover, we find that during neighbor detection, growth is promoted primarily by the production of auxin. In contrast, in true shade, the system operates with less auxin but with an increased sensitivity to the hormonal signal. Our data suggest that this latter signal is less robust, which may reflect a cost-to-robustness tradeoff, a system trait long recognized by engineers and forming the basis of information theory.

Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Effects of maintaining ditch networks on the development of Scots pine stands

Tiivistelmä: Kunnostusojituksen vaikutus rämemänniköiden kehitykseen

Regional deterministic characterization of fracture networks and its application to GIS-based rock fall risk assessment

The study investigates the possibility to incorporate fracture intensity and block geometry as spatially continuous parameters in GIS-based systems. For this purpose, a deterministic method has been implemented to estimate block size (Bloc3D) and joint frequency (COLTOP). In addition to measuring the block size, the Bloc3D Method provides a 3D representation of the shape of individual blocks. These two methods were applied using field measurements (joint set orientation and spacing) performed over a large field area, in the Swiss Alps. This area is characterized by a complex geology, a number of different rock masses and varying degrees of metamorphism. The spatial variability of the parameters was evaluated with regard to lithology and major faults. A model incorporating these measurements and observations into a GIS system to assess the risk associated with rock falls is proposed. The analysis concludes with a discussion on the feasibility of such an application in regularly and irregularly jointed rock masses, with persistent and impersistent discontinuities.

Discharge water quality from old ditch networks in Finnish peatland forests

Tiivistelmä: Vanhoilta metsäojitusalueilta valuvan veden ominaisuudet

Adjuvants that improve the ratio of antigen-specific effector to regulatory T cells enhance tumor immunity.

Antitumor immunity is strongly influenced by the balance of tumor antigen-specific effector and regulatory T cells. However, the impact that vaccine adjuvants have in regulating the balance of antigen-specific T cell populations is not well understood. We found that antigen-specific T regulatory cells (Treg) were induced following subcutaneous vaccination with either OVA or melanoma-derived peptides, with a restricted expansion of effector T cells. Addition of the adjuvants CpG-ODN or Poly(I:C) preferentially amplified effector T cells over Tregs, dramatically increasing the antigen-specific T effector:Treg ratios and inducing polyfunctional effector cells. In contrast, two other adjuvants, imiquimod and Quil A saponin, favored an expansion of antigen-specific Tregs and failed to increase effector T cell:Treg ratios. Following therapeutic vaccination of tumor-bearing mice, high ratios of tumor-specific effector T cells:Tregs in draining lymph nodes were associated with enhanced CD8+ T cell infiltration at the tumor site and a durable rejection of tumors. Vaccine formulations of peptide+CpG-ODN or Poly(I:C) induced selective production of pro-inflammatory Type I cytokines early after vaccination. This environment promoted CD8+ and CD4+ effector T cell expansion over that of antigen-specific Tregs, tipping the effector T cell to Treg balance to favor effector cells. Our findings advance understanding of the influence of different adjuvants on T cell populations, facilitating the rational design of more effective cancer vaccines.

ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

Generation of biological association networks: A novel strategy to detect new targets in cancer therapy

The aim of this work was to design a novel strategy to detect new targets for anticancer treatments. The rationale was to build Biological Association Networks from differentially expressed genes in drug-resistant cells to identify important nodes within the Networks. These nodes may represent putative targets to attack in cancer therapy, as a way to destabilize the gene network developed by the resistant cells to escape from the drug pressure. As a model we used cells resistant to methotrexate (MTX), an inhibitor of DHFR. Selected node-genes were analyzed at the transcriptional level and from a genotypic point of view. In colon cancer cells, DHFR, the AKR1 family, PKC¿, S100A4, DKK1, and CAV1 were overexpressed while E-cadherin was lost. In breast cancer cells, the UGT1A family was overexpressed, whereas EEF1A1 was overexpressed in pancreatic cells. Interference RNAs directed against these targets sensitized cells towards MTX.