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The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.

Oligomeric forms of Aβ peptides are implicated in Alzheimer's disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca2+) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca2+-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca2+influx in Xenopus laevis oocytes. Aβ1-42 oligomers evoked single-channel Ca2+fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (Po) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high Po, large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca2+-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longerlived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo. JEM © The Rockefeller University Press.

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+T cells and in vivo-labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11-12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events.

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Bibliography: leaves 100-103., The Discrete Element Method (DEM) is a collection of numerical techniques based on a discontinuum idealisation of a physical system. Discrete Element Methods have been successfully applied to the simulation of the dynamic behaviour of granular media. Using the Finite Element Method to model the behaviour of each discrete element has resulted in the Combined Finite Discrete Element Method. This combination allows researchers to investigate the behaviour of assemblies of particles in which each particle need not behave in an entirely elastic mamrer. More importantly, discrete crack constitutive models may now be applied to each discrete element. The purpose of this thesis is to simulate the macroscopic behaviour of an assembly of brittle particles, using the Combined Finite Discrete Element Method. Brittle behaviour is incorporated via the use of a Mode I and Mode II brittle failure constitutive models. The Mode I and Mode II failure models used are the Rankine and Tresca failure models, respectively. The development of a pre-processor to generate the initial configuration of the particle assembly also formed a major component of the thesis. Algorithms to fill a plane with randomly shaped polygons were developed and implemented for the pre-processor. The cold compaction process is idealised as the compaction of a granular medium, in which each particle is a deformable polygon. Two algorithms are proposed and implemented to generate randomly shaped polygons. A further algorithm was developed to fill a simple bounding polygon with smaller polygons. A_commercial CFDEM code, ELFENExplicit, was used to investigate the effect of particle constitutive model and particle interaction on the macroscopic behaviour of the granular assembly. The built-in linear elastic and Mode I Rankine failure models were used in the initial simulations while a Tresca failure model was implemented to investigate the effect of a Mode II failure model.

We present device model calculations for the current–voltage (I–V) characteristics of organic diodes and compare them with measurements of structures fabricated using MEH‐PPV. The measured I–V characteristics have a Fowler–Nordheim (FN) functional form, but are more than three orders of magnitude smaller than the calculated FN tunneling current. We find that the low mobility of the organic materials leads to a large backflow of injected carriers into the injecting contact. These results account for the experimental observations and also demonstrate how transport layers in multilayer organic light‐emitting diodes can be used to improve carrier injection.