Tracking and analysis of movement at different scales: from endosomes to humans

Movement is apparent across all spatio-temporal scales in biology and can have a significant effect on the survival of the individual. For this reason, it has been the object of study in a wide range of research fields, i.e. in molecular biology, pharmaceutics, medical research but also in behavioural biology and ecology. The aim of the thesis was to provide methodologies and insight on the movement patterns seen at different spatio-temporal scales in biology; the intra-cellular, the cellular and the organism level. At the intra-cellular level, current thesis studied the compartmental inheritance in Human Osteosarcoma (U2-OS) cells. The inheritance pattern of the endosomal quantum dot fluorescence across two consecutive generations was for first time empirically revealed. In addition, a in silico model was developed to predict the inheritance across multiple generations. At the cellular level, a semi-automated routine was developed that can realize long-term nuclei tracking in U2-OS cell populations labeled with a cell cycle marker in their cytoplasm. A method to extract cell cycle information without the need to explicitly segment the cells was proposed. The movement behaviour of the cellular population and their possible inter-individual differences was also studied. Lastly, at the organism level, the focus of the thesis was to study the emergence of coordination in unfamiliar free-swimming stickleback fish shoals. It was demonstrated that there exist two different phases, the uncoordinated and the coordinated. In addition, the significance of uncoordinated phase to the establishment of the group’s social network was for first time evinced. The adaptation of the stickleback collectives was also studied over time, i.e. the effect of group’s repeated interactions on the emergence of coordination. Findings at the intra-cellular and cellular level can have significant implications on medical and pharmaceutical research. Findings at the organism level can also contribute to the understanding of how social interactions are formed and maintained in animal collectives

Isothermal Titration Calorimetry Studies Of Protein-Mediated Interactions And Preliminary Structural Studies Of Tandem Pdz1-2 Domain Of Psd-95 Protein

Protein-mediated interactions that involve multiple ligands in their binding mechanisms are critical for many cellular functions. The primary focus of this dissertation research was to investigate such interactions for two proteins, the PDZ domain and frataxin, involving peptide and metal binding ligands, respectively. The three component projects of this work comprised (1) thermodynamic analysis of PDZ domain binding using calorimetry; (2) X-ray crystallographic structural studies of a PDZ dual domain; and (3) thermodynamic analysis of frataxin binding to iron. The specific goal of the research conducted with the PDZ domains was to understand the mechanism of action of multiple/tandem protein domains. The protein chosen for study was the dual domain PDZ1-2 of postsynaptic density-95 kDa (PSD-95) protein, a mammalian neuronal protein. The individual constructs, PDZ1 and PDZ2, were also prepared for comparative studies. Challenging the individual PDZ1 and PDZ2 and dual domains with short peptides derived form the C-terminal region of the domains\u27 natural binding partners (8-mer to 6-mer) sequences, it was found that only one of the domains remains functional when expressed separately. Binding of PDZ1 and tandem PDZ1-2 could be characterized, whereas PDZ2 could not be stabilized under solution conditions to yield a valid binding curve. The use of bivalent peptides that would bind with 1:1 stoichiometry to the tandem PDZ1-2 domain was problematic due to ligand insolubility. In an ongoing effort to develop new cellular probes for in vivo investigation of multiprotein assembly, we studied modified ligand CN2180 which targets the tandem PDZ1-2 of PSD-95. Our in vitro testing by ITC of the same peptide after integrating the binding isoterm, yielded a stoichiometry of n=2, indicating that both binding sites are fully occupied, and dissociation constants for each of the two interaction sites were in the single digit micromolar range. In terms of progress on that structural front, the dual PDZ domain protein construct PDZ1-2 of PSD-95 was successfully crystallized. Identifying the conditions required to grow crystals is a major step toward the goal of solving the structure of this tandem domain by X-ray methods. Optimization of the purification protocol resulted in high purity protein that allowed for increased volume of protein crystallization attempts. The optimized conditions yielded well-shaped hexagonal crystals for PDZ1-2, which crystallizes in cubic P23 group. Data reduction resulted in a Rmerge value of 11.2 %. A final structure could not be solved by molecular replacement methods, and additional work will be needed to accomplish this. In the last project, calorimetry was employed to investigate the binding of frataxin to iron ions. This protein is essential for the effective regulation of cellular iron homeostasis. Frataxin, is essential for cellular iron control and is believed to serve as an iron chaperone that delivers mitochondrial Fe(II) to the enzymes ferrochelatase and the ISU apparatus for completion of heme and Fe-S cluster biosynthesis. Monomeric frataxin protein has a high affinity for ferrous ion, completely saturating at 2:1 iron to protein ratio. The micromolar dissociation constants measured for yeast frataxin, with respect to ferrous iron (Kd\u27s of 2.0 and 3.0 uM) were obtained from calorimetric titrations. The weak interaction is consistent with the hypothesis of frataxin acting as an iron chaperone. Frataxin must be able to form a favorable interface with its protein partners and also easily release the metal. Although this thermodynamic binding study increases understanding of the underlying metal-binding behavior, additional structural characterization will be critical to help elucidate how frataxin binds iron and docks with its protein partners to promote metal delivery

Isothermal Titration Calorimetry Studies Of Protein-Mediated Interactions And Preliminary Structural Studies Of Tandem Pdz1-2 Domain Of Psd-95 Protein

Protein-mediated interactions that involve multiple ligands in their binding mechanisms are critical for many cellular functions. The primary focus of this dissertation research was to investigate such interactions for two proteins, the PDZ domain and frataxin, involving peptide and metal binding ligands, respectively. The three component projects of this work comprised (1) thermodynamic analysis of PDZ domain binding using calorimetry; (2) X-ray crystallographic structural studies of a PDZ dual domain; and (3) thermodynamic analysis of frataxin binding to iron. The specific goal of the research conducted with the PDZ domains was to understand the mechanism of action of multiple/tandem protein domains. The protein chosen for study was the dual domain PDZ1-2 of postsynaptic density-95 kDa (PSD-95) protein, a mammalian neuronal protein. The individual constructs, PDZ1 and PDZ2, were also prepared for comparative studies. Challenging the individual PDZ1 and PDZ2 and dual domains with short peptides derived form the C-terminal region of the domains\u27 natural binding partners (8-mer to 6-mer) sequences, it was found that only one of the domains remains functional when expressed separately. Binding of PDZ1 and tandem PDZ1-2 could be characterized, whereas PDZ2 could not be stabilized under solution conditions to yield a valid binding curve. The use of bivalent peptides that would bind with 1:1 stoichiometry to the tandem PDZ1-2 domain was problematic due to ligand insolubility. In an ongoing effort to develop new cellular probes for in vivo investigation of multiprotein assembly, we studied modified ligand CN2180 which targets the tandem PDZ1-2 of PSD-95. Our in vitro testing by ITC of the same peptide after integrating the binding isoterm, yielded a stoichiometry of n=2, indicating that both binding sites are fully occupied, and dissociation constants for each of the two interaction sites were in the single digit micromolar range. In terms of progress on that structural front, the dual PDZ domain protein construct PDZ1-2 of PSD-95 was successfully crystallized. Identifying the conditions required to grow crystals is a major step toward the goal of solving the structure of this tandem domain by X-ray methods. Optimization of the purification protocol resulted in high purity protein that allowed for increased volume of protein crystallization attempts. The optimized conditions yielded well-shaped hexagonal crystals for PDZ1-2, which crystallizes in cubic P23 group. Data reduction resulted in a Rmerge value of 11.2 %. A final structure could not be solved by molecular replacement methods, and additional work will be needed to accomplish this. In the last project, calorimetry was employed to investigate the binding of frataxin to iron ions. This protein is essential for the effective regulation of cellular iron homeostasis. Frataxin, is essential for cellular iron control and is believed to serve as an iron chaperone that delivers mitochondrial Fe(II) to the enzymes ferrochelatase and the ISU apparatus for completion of heme and Fe-S cluster biosynthesis. Monomeric frataxin protein has a high affinity for ferrous ion, completely saturating at 2:1 iron to protein ratio. The micromolar dissociation constants measured for yeast frataxin, with respect to ferrous iron (Kd\u27s of 2.0 and 3.0 uM) were obtained from calorimetric titrations. The weak interaction is consistent with the hypothesis of frataxin acting as an iron chaperone. Frataxin must be able to form a favorable interface with its protein partners and also easily release the metal. Although this thermodynamic binding study increases understanding of the underlying metal-binding behavior, additional structural characterization will be critical to help elucidate how frataxin binds iron and docks with its protein partners to promote metal delivery

Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 174

This bibliography lists 181 reports, articles, and other documents introduced into the NASA scientific and technical information system in November 1977

Investigation of the deformation of in-vessel components of a nuclear fusion experiment using optical strain sensors

A fibre-optic, EM-insensitive measurement for the deformation of in-vessel components has successfully been installed and operated at the nuclear fusion experiment ASDEX Upgrade. The sensors were tested for their neutron tolerance and vacuum compatibility. Installation was done by copper-steel laser beam welding. Measurements of in-service oscillations due to all three existing types of load cases show good agreement with theory and simulations. A fatigue lifetime assessment was performed

Dendritic spines and memory formation in the chick

Passive avoidance training results in a number of biochemical, morphological, and electrophysiological changes in the forebrain of the one day-old chick. One particular region in which these alterations occur is the intermediate part of the medial hyperstriatum ventrale (IMHV). This thesis reports several morphological experiments on the effects of passive avoidance training on dendritic spines and dendritic branching patterns of large, multipolar, projection neurons (abbreviated LMPNs) in the IMHV, in order to determine structural correlates of memory formation for this task, at the light microscope level. The chicks were trained on a passive avoidance training task by presentation of a shiny chrome bead coated with a bitter tasting substance (methylanthranilate). The chicks will spontaneously peck at the bead, but show a characteristic disgust response on the first peck and avoid a similar (dry) bead subsequently (trained or M-chicks). Control chicks were presented with a water coated bead, which they do not find aversive (W-chicks). 24-26h later, the chicks were perfused with an aldehyde fixative, and the left and right IMHV regions were dissected out and Golgi-impregnated by the rapid Golgi method. The blocks were sectioned with a tissue chopper at 90-120p.m, and permanently mounted onto slides, in DPX, which were coded so that subsequent procedures were performed "blind". LMPNs in the left and right IMHV regions from trained and control chicks were identified and examined for changes in spine density, spine shape and in dendritic branching patterns (which were analysed by the Sholl concentric ring method, by the number of dendrites at each br^ch order and by vertex analysis). Training resulted in (1) a significant increase in spine density in the left and right hemispheres and (2) an increase in the mean diameter of the spine heads with concomitant shortening of the spine stems, but only significantly in. the left hemisphere. These changes occured without a significant change in the mean overall spine length and also without significant alterations in the lengths or the diameters of the dendrite branches. A significant hemispheric asymmetry was also observed between the left (L) and right (R) hemispheres of control chicks: R>L, but no asymmetry was found in trained chicks. No significant differences in branching patterns were found after passive avoidance training. Because the changes in spine density and shape may have been caused by nonspecific factors associated with the training experience, such as stress, arousal, or the taste of the methyl anthranilate, per se, a further experiment was conducted in which trained chicks were given a brief, subconvulsive, trahscranial electroshock, 5 min after showing the disgust response. This rendered approximately half of the chicks amnesic, the rest showed recall when tested 24h after training. Control chicks receiving the same treatment showed no change in pecking behaviour. A significant increase in spine density was found in the recall group compared with either the amnesic or the shocked water control groups, but only in the left hemisphere. However, no alterations in spine shape and no significant differences in dendrite lengths or dendrite diameters were noted. In conclusion: (1) passive avoidance training is associated with an increase in spine density that is specifically related to long-term memory formation for the task; (2) spine density and shape changes can occur within 24h of a single-trial learning experience; (3) these alterations occur without significant differences in branching patterns of the LMPNs, suggesting either that it may take longer than 24h for observable changes in branching patterns to occur, or that they may not be involved in memory storage for the training task, and (4) because the shape changes were only found on LMPNs in the left hemisphere of trained chicks, this hemisphere may be predominantly involved in the memory storage processes for this task. This is also supported by qualitative results from the dendritic branching investigation