During skeletal muscle stimulation, there is a summation of local events of Ca2+ release from the sarcoplasmic reticulum, known as Ca2+ sparks. Ca2+ sparks originate from groups of skeletal ryanodine receptors (RyR1) that activate and close in synchrony. This synchrony allows for the rapid and massive release of Ca2+ from the sarcoplasmic reticulum to initiate contraction and, more important, would provide a mechanism to terminate Ca2+ release under conditions where independent RyR1 are normally active. RyR1 mutations can result in abnormal intracellular Ca2+ signaling that is associated with numerous skeletal muscle disorders including malignant hyperthermia and central core disease. Therefore, investigating the mechanisms that control RyR1 function can help identify how these mutations cause deleterious Ca2+ handling. Currently, most published research on RyR1s gating utilizes single RyR1 reconstituted into planar lipid bilayers to test isolated RyR1. However, in vivo, arrays of RyR1 function in synchrony. Attempts to reconstitute RyR1s into planar lipid bilayers result in experiments that contain multiple channels, which under specific conditions may gate in synchrony, also known as coupled gating. Coupled RyR1 gating was first reported by A. Marks' laboratory and attributed to FK-506 binding protein 12 (FKBP12) associating with neighboring RyR1s the stabilization of RyR1-RyR1 interactions that promote coupled gating. Previous studies suggested that ATP is required for coupled RyR1 gating; however, the mechanism by which ATP promotes the coordinated activity of RyR1s has not been elucidated and is the focus of this thesis. Therefore, my hypothesis is that the agonist action of ATP and FKBP12 bound to RyR1 are required for coupled RyR1 gating. In addition, new pharmacological tools are required to better understand coupled RyR gating. Thus, an additional goal is to identify pharmacological agents that modulate RyR1s in an innovative manner, i.e., help to uncover novel aspects of RyR1 gating and conduction. This investigation suggests that the adenosine based nucleotides, ATP, ADP and AMP, are agonists of RyR1s and promote coupled RyR1 gating in planar lipid bilayers. However, ADP and AMP were unable to maintain coupled RyR1 gating with physiological levels of Mg2+. This suggests that coupled gating would be impaired when the levels ATP decrease, as in muscle fatigue. When ATP was compared to other nucleotides (GTP, ITP, and TTP), the results suggest that the nucleotide agonist action on RyR1s is dependent on the phosphate groups and amino group on the nucleobase. As ATP is the most efficient nucleotide for coupled gating, I also investigated the indirect action of ATP to act as a kinase substrate or alter the cytoskeletal network. The addition of kinases, phosphatases and cytoskeletal modulators did not produce a significant disruption of coupled RyR1 gating. I also tested the role of addition of exogenous FKBP12 to RyR1s that gated independently or had partial coupling, but coupled gating was never improved. Also, the addition of high doses of rapamycin to remove FKBP12 from coupled RyR1 failed to functionally uncouple the channels. Finally, I attempted to find pharmacological agents that could aid in the understanding of coupled RyR1. Some agents were found to modulate RyR1s; however, I did not find a probe that would affect kinetics/conductance of RyR1s and was suitable for comparing coupled gating in bilayers with Ca2+ sparks in cells. Overall, coupled RyR gating is dependent on the physiological modulators ATP and Mg2+. This thesis represents a step forward in identifying the requirements for coupled RyR1 gating and understanding how RyR1s function in cells. Until an understanding of how these receptors communicate in cells is obtained, how different mutations alter the Ca2+ leak will continue to be quite difficult to study.

The overall objective of the work described in this thesis was to characterise the three genes Hv14.3.3c, HvMAPKK1 and HvFKBP41, in terms of a role in defence and stress response signalling. These genes had previously been found to be differentially expressed in compatible versus incompatible interactions of barley with the fungus Rhynchosporium secalis, suggesting a possible role in the plant defence response, while current literature suggests these genes may also play a role in signal transduction, possibly under a broad range of stresses, including abiotic as well as biotic. Two main approaches were undertaken to characterise gene function: expression analysis and the identification of protein-protein interactions. To facilitate expression analysis, full length cDNA fragments of each gene were first obtained using bioinformatics, RACE and genomic walking techniques. Expression was then investigated using quantitative real-time RT-PCR. The results of the expression analysis confirmed that the candidate genes were in fact differentially expressed during infection, suggesting a role in the defence response of barley against R. secalis. Analysing their expression in the context of other stresses and treatments, namely frost, drought and ABA, indicated their role may not be limited only to biotic stress, but include abiotic stress as well. To investigate the possibility that these genes are involved in signalling during the defence response, protein-protein interaction techniques such as yeast two-hybrid and affinity pulldowns were used to identify interacting proteins in an attempt to place the genes within a known signalling network and build and extend on these networks. Y2H screening was used successfully to identify two putative interactors of Hv14.3.3c; an EPSP (5-enolpyruvylshikimate-3-phosphate) synthase and a putative wound-induced protein, and two interactors of HvFKBP41; a Rab-type GTPase and the same wound-induced protein. From what is known about the function of these genes in the literature, they fit well with a role in stress response signalling and the potential to be involved in signalling networks with the candidate gene products and also with each other. Through the trial of many different affinity pulldown techniques, a method for identifying interacting proteins from plant extracts was successfully established, however, issues with protein identification meant that interacting proteins were not identified using this technique. Steps were then made towards confirming the interactions identified using the Y2H system. Full length cDNA sequences of the identified interactors were obtained and expression analysis performed, in the aim of investigating co-expression patterns between the genes encoding the interacting proteins and the three candidate genes, to support a potential interaction. To confirm the Hv14.3.3c-HvEPSP interaction, co-immunoprecipitation and BRET were then used, however confirmation was unsuccessful due to issues with non-specific binding in co-immunoprecipitation and technical issues trying to establish the BRET analysis system in barley. In summary, the results of this study place the candidate genes Hv14.3.3c, HvMAPKK1 and HvFKBP41 as players in signal transduction during the plant defence/stress response. With the identification of previously uncharacterised protein interactions, some progress has also been made towards placing these genes within known signalling networks and identifying potential downstream genes that could possibly play a more specific role in defence response signalling and therefore be potential targets for the generation of resistant or stress tolerant plants. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2009

Most Adoptive Cell Therapies (ACT), including CAR T cell therapies, suffer failure because of the severe side effects due to loss-of-control of the therapeutic cells once they are inside the patient’s body, suggesting that novel strategies must be developed for a better in vivo control of these engineered cells. In the meantime, CAR T cell therapies targeting solid tumors have not experienced the remarkable success achieved with hematopoietic cancers, mainly due to continuous tumor antigen exposure and a suppressive tumor microenvironment. Here we designed a private passageway fusion receptor, which is composed of a ligand binding domain and a glycosylphosphatidylinositol (GPI) anchoring domain, to be expressed and localized to the surface of CAR T cells independently to the classical CAR T construct. These ligand binding domains preserve high binding affinity towards their cognate ligands and are only expressed on the CAR T cells that have been transduced. Therefore, cytotoxic drugs or immunosuppressants linked to the corresponding targeting ligands are shown to be specifically delivered to these fusion receptor positive CAR T cells for lowering the activity of the over-activated CAR T cells. On the other hand, we discovered that a potent TLR7 agonist is able to enhance the lysis effect of the exhausted CAR T cells in a co-culture model. Serial releasable and non-releasable targeted TLR7 agonists were prepared and tested. Based on these data, we suggest that our secret passageway fusion receptor platform provides a better control of the activity of CAR T cells using the corresponding targeting ligand-payload conjugates in a dose dependent manner and function as a doorway for the delivery of instructions to CAR T cells for versatile purposes.

L'unité structurale de la chromatine, nommée nucléosome, est composée d'un double brin d'ADN enroulé autour d'un octamère d'histone, et subit une pléthore de modifications post-traductionnelles. Les conséquences biologiques de l’acétylation des lysines et de l’isomérisation des liaisons peptidyl-prolyl ont été étudiées à travers une analyse à l’échelle atomique par RMN de systèmes d'intérêt reconstitués in vitro. Les liaisons peptidyl-prolyl du domaine N-terminal de l'histone H3 sont substrats in vitro d’une isomérase chez S. cerevisiae nommée Fpr4p, laquelle exerce un contrôle catalyse-dépendant de la transcription. La résolution de la structure du domaine catalytique de Fpr4p, à partir de contraintes géométriques mesurées par RMN, révéla un domaine canonique de la famille FKBP (FK506-binding protein). Grâce à l'analyse de la séquence primaire et aux expériences RMN, nous proposons un modèle structural préliminaire de Fpr4p entière. L'analyse fonctionnelle est réalisée grâce à trois décapeptides construits à partir de la séquence primaire de H3 chez S. cerevisiae. Ils sont tous substrats de Fpr4p et la catalyse est équivalente pour Pro16 et Pro30. La proportion à l'équilibre du conformère cis fut déterminée pour les trois peptides et celle-ci n'est pas affectée par l'activité catalytique de Fpr4p. Les structures en solution des substrats en conformation trans ont été résolues par spectroscopie RMN, et seront utilisées pour des appariements moléculaires in silico sur le domaine catalytique de Fpr4p. Pour étudier le rôle biologique de l'acétylation des histones, une méthodologie de production de protéines acétylées a été développée. Le protocole repose sur la mutation d'une lysine en cystéine d'une protéine recombinante, suivie d'une alkylation contrôlée exploitant la nucléophilie du groupe thiol préalablement introduit. La production de l'agent alkylant adéquat est simple, rapide, réalisable dans un laboratoire de biologie et permet différents marquages isotopiques du groupe acétyle. L'alkylation d'une protéine repliée fut réalisée avec succès en conditions natives. Le dimère d'histone H2A-H2B, un intermédiaire de l'assemblage du nucléosome et siège d'acétylation in vivo, fut reconstruit in vitro. Les déplacements chimiques des domaines N et C-terminaux de H2A sont cohérents avec un état intrinsèquement déstructuré bien que leurs dynamiques moléculaires ne soient pas équivalentes. / The structural unit of chromatin, the nucleosome, is composed of double-stranded DNA wrapped around a histone octamer and is subject to a plethora of post-translational modifications. The biological consequences of peptidyl-prolyl isomerization and lysine acetylation were investigated at atomic scale through analysis of in vitro reconstituted systems by NMR. Peptidyl-prolyl bonds of histone H3 N-terminal domain are substrates in vitro of an isomerase from S. cerevisiae named Fpr4p, which underlies transcriptional control dependent on its catalytic activity. The solution structure of the catalytic domain of Fpr4p was calculated based on restraints from NMR spectroscopy, and reveals a canonical catalytic domain belonging to the FK506-binding protein (FKBP) family. Based on primary sequence analysis and NMR experiments, a preliminary structural model of full length Fpr4p is also presented. Functional analyses were performed with three decapeptides designed from the primary sequence from the N-terminal tail of S. cerevisiae histone H3. All three constitute substrates of Fpr4p, with equivalent catalysis observed for Pro16 and Pro30. The equilibrium proportion of the cis-proline conformer has been determined for all three decapeptides, and these populations are unaffected by Fpr4p catalytic activity. Structural ensembles of the substrates with proline in the trans conformation were determined by using NMR spectroscopy, and will be subsequently used for in silico molecular docking onto Fpr4p. To study a second form of histone regulation, a semi-synthetic method to produce acetylated protein was developed. The protocol relies on the site-specific mutation of lysine to cysteine in recombinant proteins followed by controlled alkylation thanks to nucleophilicity of the introduced thiol. The production of the required alkylation reagent is easy, quick, and suitable for biology laboratory and allows diverse isotopic labeling within the acetyl group. Alkylation of folded proteins has also been achieved in native conditions. As one target of acetylation in vivo, the histone H2A-H2B dimer is an intermediate of nucleosome assembly and was reconstituted in vitro. Chemical shift values of the N- and C-terminal domains of H2A are in agreement with an intrinsically disordered state although they display differences in dynamic mobility.

The Sun is the ultimate energy source on Earth. Photosynthetic organisms are able to catalyze the conversion of solar energy to chemical energy by a reaction called photosynthesis. In plants, this process occurs inside a green organelle called the chloroplast. The protein complexes involved in the photosynthetic light reactions are situated in the thylakoid membrane, which encloses a tiny space called lumen. The Peptidyl-Prolyl cis-trans Isomerase (PPIase) family is the most abundant protein family in the thylakoid lumen. The three PPIase subfamilies, cyclophilins, FKBPs (FK506 binding proteins) and parvulins form a group by their enzymatic activity despite lack of sequence similarity between the subfamilies. Cyclophilins and FKBPs, collectively called immunophilins, were originally discovered as the targets of the immunosuppressive drugs cyclosporine A and FK506, respectively. By suppressing the immune response in humans, these immunophilin-drug complexes revolutionized the field of organ transplantation by preventing graft rejection. Cis-trans isomerization of peptide bonds preceding the amino acid proline is the rate-limiting step of protein folding and several immunophilins have been shown to be important for catalysis of protein folding in vivo. PPIases have been found to be part of large protein complexes as well as in functions such as signalling, protein secretion, RNA processing and cell cycle control. A picture is therefore emerging in which the actual interaction between the PPIase and its target is perhaps more important than the PPIase activity. In the present work, PPIases have been characterized in the chloroplast thylakoid lumen of Spinacia oleracea (spinach) and Arabidopsis thaliana (Arabidopsis). The most active PPIase in the spinach lumen was identified as the cyclophilin TLP20. AtCYP20-2, the Arabidopsis homologue of TLP20, was found to be upregulated at high light and attached to the thylakoid membrane, more precisely to the outer regions of photosystem II supercomplexes. In Arabidopsis, up to 5 cyclophilins and 11 FKBPs were predicted to reside in the lumen. Of these 16 immunophilins, only 2 were identified as active PPIases and significant differences were observed between the two plant species. AtCYP20-2, like TLP20, is an active isomerase although AtFKBP13 is the most active PPIase in the lumen of Arabidopsis. Mutant Arabidopsis plants deficient in AtCYP20-2 displayed no phenothypical changes or decrease in total lumenal PPIase activity. Being the only active PPIase in the mutants, the redox sensitive AtFKBP13 is proposed to compensate for the lack of AtCYP20-2 by oxidative activation. In agreement with the experimental data, the sequence analyses of catalytic domains of lumenal immunophilins demonstrate that only AtCYP20-2 and AtFKBP13 possess the amino acids found essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. It is concluded that with the exception of AtCYP20-2 and AtFKBP13 most immunophilins in the lumen of Arabidopsis lost their PPIase activity on peptide substrates and developed other specialized functions.

No description available.