From Violation to Reconstruction: The Process of Self-Renewal Associated with Chronic Fatigue Syndrome

Chronic Fatigue Syndrome (CFS) is a contested condition that generates scepticism and occupies a marginalised position within medical and social contexts. The thesis examines the illness experiences, and specifically the experiences of self, for people affected with CFS. Using qualitative inquiry, a substantive theory related to the process of self-renewal and adaptation associated with CFS is explicated. The theory encompasses the trajectory of CFS from onset to chronicity, and in exceptional instances, recovery. Illness narratives were derived from in-depth, semi-structured interviews of 19 adults, including 16 people affected with, and 3 people recovered from, CFS. Data was coded and analysed using a grounded theory approach. Analysis generated two parallel narratives that defined the illness experience of CFS: the narrative of the illness biographies and the narrative of self, specifically the struggling and diminished self seeking renewal. The illness biographies encompassed the stories of symptoms and their explanations, the encounters that ensued and their contentious milieu. The narrative of self was the primary narrative. It articulated the negative consequences to self and personhood associated with CFS, named the Violation of Self, and the consequent efforts of participants to decrease the struggle and violation by use of the Guardian Response and the Reconstructing Response. The Guardian Response provided protection and self-reclamation. The Reconstructing Response fostered self-renewal and meaning. The two narratives were bridged by the threats of CFS. That is, the illness biographies were accompanied by threats of disruption related to chronic illness, and by threats of invalidation that arose from CFS as a contested condition. In turn, these threats provided the catalyst to the violation and responses as described in the narrative of self. Under different conditions the relative strengths of violation, guardianship or reconstruction fluctuated, and it was these fluctuations that presented the participants with the ongoing struggle of CFS

Peptide Ligands for Pro-survival Protein Bfl-1 from Computationally Guided Library Screening

Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-x[subscript L], Bcl-2, Bcl-w, Mcl-1, and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides, and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-x[subscript L] and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma, and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with K[subscript d] < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ and may guide the development of targeted Bfl-1 inhibitors.National Institute of General Medical Sciences (U.S.) (Award GM084181)National Institute of General Medical Sciences (U.S.) (Award P50-GM68762

The role of kinetic context in apparent biased agonism at GPCRs

Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that ‘kinetic context’, as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism

Registered replication report on Fischer, Castel, Dodd, and Pratt (2003)

The attentional spatial-numerical association of response codes (Att-SNARC) effect (Fischer, Castel, Dodd, & Pratt, 2003)—the finding that participants are quicker to detect left-side targets when the targets are preceded by small numbers and quicker to detect right-side targets when they are preceded by large numbers—has been used as evidence for embodied number representations and to support strong claims about the link between number and space (e.g., a mental number line). We attempted to replicate Experiment 2 of Fischer et al. by collecting data from 1,105 participants at 17 labs. Across all 1,105 participants and four interstimulus-interval conditions, the proportion of times the effect we observed was positive (i.e., directionally consistent with the original effect) was .50. Further, the effects we observed both within and across labs were minuscule and incompatible with those observed by Fischer et al. Given this, we conclude that we failed to replicate the effect reported by Fischer et al. In addition, our analysis of several participant-level moderators (finger-counting habits, reading and writing direction, handedness, and mathematics fluency and mathematics anxiety) revealed no substantial moderating effects. Our results indicate that the Att-SNARC effect cannot be used as evidence to support strong claims about the link between number and space

TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance

Abstract Ligands addressed to the mitochondrial Translocator Protein (TSPO) have been suggested as cell death/life and steroidogenesis modulators. Thus, TSPO ligands have been proposed as drug candidates in several diseases; nevertheless, a correlation between their binding affinity and in vitro efficacy has not been demonstrated yet, questioning the specificity of the observed effects. Since drug-target residence time is an emerging parameter able to influence drug pharmacological features, herein, the interaction between TSPO and irDE-MPIGA, a covalent TSPO ligand, was investigated in order to explore TSPO control on death/life processes in a standardized glioblastoma cell setting. After 90 min irDE-MPIGA cell treatment, 25 nM ligand concentration saturated irreversibly all TSPO binding sites; after 24 h, TSPO de-novo synthesis occurred and about 40 % TSPO binding sites resulted covalently bound to irDE-MPIGA. During cell culture treatments, several dynamic events were observed: (a) early apoptotic markers appeared, such as mitochondrial membrane potential collapse (at 3 h) and externalization of phosphatidylserine (at 6 h); (b) cell viability was reduced (at 6 h), without cell cycle arrest. After digitonin-permeabilized cell suspension treatment, a modulation of mitochondrial permeability transition pore was evidenced. Similar effects were elicited by the reversible TSPO ligand PIGA only when applied at micromolar dose. Interestingly, after 6 h, irDE-MPIGA cell exposure restored cell survival parameters. These results highlighted the ligand-target residence time and the cellular setting are crucial parameters that should be taken into account to understand the drug binding affinity and efficacy correlation and, above all, to translate efficiently cellular drug responses from bench to bedside

Computational Methods Used in Hit-to-Lead and Lead Optimization Stages of Structure-Based Drug Discovery

GPCR modeling approaches are widely used in the hit-to-lead (H2L) and lead optimization (LO) stages of drug discovery. The aims of these modeling approaches are to predict the 3D structures of the receptor-ligand complexes, to explore the key interactions between the receptor and the ligand and to utilize these insights in the design of new molecules with improved binding, selectivity or other pharmacological properties. In this book chapter, we present a brief survey of key computational approaches integrated with hierarchical GPCR modeling protocol (HGMP) used in hit-to-lead (H2L) and in lead optimization (LO) stages of structure-based drug discovery (SBDD). We outline the differences in modeling strategies used in H2L and LO of SBDD and illustrate how these tools have been applied in three drug discovery projects