Cellular and network mechanisms generating spontaneous population events in the immature rat hippocampus

Distinct endogenous network events, generated independently of sensory input, are a general feature of various structures of the immature central nervous system. In the immature hippocampus, these type of events are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings in vitro. GABA, the major inhibitory neurotransmitter of the adult brain, has a depolarizing action in immature neurons, and GDPs have been proposed to be driven by GABAergic transmission. Moreover, GDPs have been thought to reflect an early pattern that disappears during development in parallel with the maturation of hyperpolarizing GABAergic inhibition. However, the adult hippocampus in vivo also generates endogenous network events known as sharp (positive) waves (SPWs), which reflect synchronous discharges of CA3 pyramidal neurons and are thought to be involved in cognitive functions. In this thesis, mechanisms of GDP generation were studied with intra- and extracellular recordings in the neonatal rat hippocampus in vitro and in vivo. Immature CA3 pyramidal neurons were found to generate intrinsic bursts of spikes and to act as cellular pacemakers for GDP activity whereas depolarizing GABAergic signalling was found to have a temporally non-patterned facilitatory role in the generation of the network events. Furthermore, the data indicate that the intrinsic bursts of neonatal CA3 pyramidal neurons and, consequently, GDPs are driven by a persistent Na+ current and terminated by a slow Ca2+-dependent K+ current. Gramicidin-perforated patch recordings showed that the depolarizing driving force for GABAA receptor-mediated actions is provided by Cl- uptake via the Na-K-C1 cotransporter, NKCC1, in the immature CA3 pyramids. A specific blocker of NKCC1, bumetanide, inhibited SPWs and GDPs in the neonatal rat hippocampus in vivo and in vitro, respectively. Finally, pharmacological blockade of the GABA transporter-1 prolonged the decay of the large GDP-associated GABA transients but not of single postsynaptic GABAA receptor-mediated currents. As a whole the data in this thesis indicate that the mechanism of GDP generation, based on the interconnected network of bursting CA3 pyramidal neurons, is similar to that involved in adult SPW activity. Hence, GDPs do not reflect a network pattern that disappears during development but they are the in vitro counterpart of neonatal SPWs.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Dynamic Bayesian network inferencing for non-homogeneous complex systems

Dynamic Bayesian Networks (DBNs) provide a versatile platform for predicting and analysing the behaviour of complex systems. As such, they are well suited to the prediction of complex ecosystem population trajectories under anthropogenic disturbances such as the dredging of marine seagrass ecosystems. However, DBNs assume a homogeneous Markov chain whereas a key characteristics of complex ecosystems is the presence of feedback loops, path dependencies and regime changes whereby the behaviour of the system can vary based on past states. This paper develops a method based on the small world structure of complex systems networks to modularise a non-homogeneous DBN and enable the computation of posterior marginal probabilities given evidence in forwards inference. It also provides an approach for an approximate solution for backwards inference as convergence is not guaranteed for a path dependent system. When applied to the seagrass dredging problem, the incorporation of path dependency can implement conditional absorption and allows release from the zero state in line with environmental and ecological observations. As dredging has a marked global impact on seagrass and other marine ecosystems of high environmental and economic value, using such a complex systems model to develop practical ways to meet the needs of conservation and industry through enhancing resistance and/or recovery is of paramount importance.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.

A composition dependent thermal behavior of GexSe35-xTe65 glasses

Alternating differential scanning calorimetric (ADSC) studies have been performed to understand the thermal behavior of bulk GexSe35-xTe65 glasses (17 <= x <= 25); it is found that the glasses with x <= 20 exhibit two crystallization exotherms (T-c1 & T-c2). On the other hand, those with x >= 20.5, show a single crystallization reaction upon heating. The exothermic reaction at T-c1 has been found to correspond to the partial crystallization of the glass into hexagonal Te and the reaction at T-c2 is associated with the additional crystallization of rhombohedral Ge-Te phase. The glass transition temperature of GexSe35-xTe65 glasses is found to show a linear but not-steep increase, indicating a progressive, but a gradual increase in network connectivity with Ge addition. It is also found that T-c1 of GexSe35-xTe65 glasses with x <= 20, increases progressively with Ge content and eventually merges with T-c2 at x approximate to 20.5 (< r > = 2.41); this behavior has been understood on the basis of the reduction in Te-Te bonds of lower energy and increase in Ge-Te bonds of higher energy, with increasing Ge content. Apart from the interesting composition dependent crystallization, an anomalous melting behavior is also exhibited by the GexSe35-xTe65 glasses.

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.

Investigation of the effects of extracellular osmotic pressure on morphology and mechanical 1 properties of individual chondrocyte

It has been demonstrated that most cells of the body respond to osmotic pressure in a systematic manner. The disruption of the collagen network in the early stages of osteoarthritis causes an increase in water content of cartilage which leads to a reduction of pericellular osmolality in chondrocytes distributed within the extracellular environment. It is therefore arguable that an insight into the mechanical properties of chondrocytes under varying osmotic pressure would provide a better understanding of chondrocyte mechanotransduction and potentially contribute to knowledge on cartilage degeneration. In this present study, the chondrocyte cells were exposed to solutions with different osmolality. Changes in their dimensions and mechanical properties were measured over time. Atomic Force Microscopy (AFM) was used to apply load at various strain-rates and the force-time curves were logged. The thin-layer elastic model was used to extract the elastic stiffness of chondrocytes at different strain-rates and at different solution osmolality. In addition, the porohyperelastic (PHE) model was used to investigate the strain-rate dependent responses under the loading and osmotic pressure conditions. The results revealed that the hypo-osmotic external environment increased chondrocyte dimensions and reduced Young’s modulus of the cells at all strain-rates tested. In contrast, the hyper-osmotic external environment reduced dimensions and increased Young’s modulus. Moreover, by using the PHE model coupled with inverse FEA simulation, we established that the hydraulic permeability of chondrocytes increased with decreasing extracellular osmolality which is consistent with previous work in the literature. This could be due to a higher intracellular fluid volume fraction with lower osmolality.

Individuals on the Move : Body Condition Dependent Dispersal and Quasi-Local Competition in Metapopulations

Individual movement is very versatile and inevitable in ecology. In this thesis, I investigate two kinds of movement body condition dependent dispersal and small-range foraging movements resulting in quasi-local competition and their causes and consequences on the individual, population and metapopulation level. Body condition dependent dispersal is a widely evident but barely understood phenomenon. In nature, diverse relationships between body condition and dispersal are observed. I develop the first models that study the evolution of dispersal strategies that depend on individual body condition. In a patchy environment where patches differ in environmental conditions, individuals born in rich (e.g. nutritious) patches are on average stronger than their conspecifics that are born in poorer patches. Body condition (strength) determines competitive ability such that stronger individuals win competition with higher probability than weak individuals. Individuals compete for patches such that kin competition selects for dispersal. I determine the evolutionarily stable strategy (ESS) for different ecological scenarios. My models offer explanations for both dispersal of strong individuals and dispersal of weak individuals. Moreover, I find that within-family dispersal behaviour is not always reflected on the population level. This supports the fact that no consistent pattern is detected in data on body condition dependent dispersal. It also encourages the refining of empirical investigations. Quasi-local competition defines interactions between adjacent populations where one population negatively affects the growth of the other population. I model a metapopulation in a homogeneous environment where adults of different subpopulations compete for resources by spending part of their foraging time in the neighbouring patches, while their juveniles only feed on the resource in their natal patch. I show that spatial patterns (different population densities in the patches) are stable only if one age class depletes the resource very much but mainly the other age group depends on it.

Collaboration on social network sites: Amateurs, professionals and celebrities

Amateurs are found in arts, sports, or entertainment, where they are linked with professional counterparts and inspired by celebrities. Despite the growing number of CSCW studies in amateur and professional domains, little is known about how technologies facilitate collaboration between these groups. Drawing from a 1.5-year field study in the domain of bodybuilding, this paper describes the collaboration between and within amateurs, professionals, and celebrities on social network sites. Social network sites help individuals to improve their performance in competitions, extend their support network, and gain recognition for their achievements. The findings show that amateurs benefit the most from online collaboration, whereas collaboration shifts from social network sites to offline settings as individuals develop further in their professional careers. This shift from online to offline settings constitutes a novel finding, which extends previous work on social network sites that has looked at groups of amateurs and professionals in isolation. As a contribution to practice, we highlight design factors that address this shift to offline settings and foster collaboration between and within groups.

"Hey world, take a look at me!": Appreciating the human body on social network sites

Social network sites (SNSs) such as Facebook have the potential to persuade people to adopt a lifestyle based on exercise and healthy nutrition. We report the findings of a qualitative study of an SNS for bodybuilders, looking at how bodybuilders present themselves online and how they orchestrate the SNS with their offline activities. Discussing the persuasive element of appreciation, we aim to extend previous work on persuasion in web 2.0 technologies.

Biofilm formation by multidrug resistant Escherichia coli ST131 is dependent on type 1 fimbriae and assay conditions.

Escherichia coli sequence type 131 (ST131) have emerged as a pandemic lineage of important multidrug resistant pathogens worldwide. Despite many studies examining the epidemiology of ST131, only a few studies to date have investigated the capacity of ST131 strains to form biofilms. Some of these studies have reported contrasting findings, with no specific ST131 biofilm-promoting factors identified. Here we examined a diverse collection of ST131 isolates for in vitro biofilm formation in different media and assay conditions, including urine from healthy adult women. We found significant differences among strains and assay conditions, which offers an explanation for the contrasting findings reported by previous studies using a single condition. Importantly, we showed that expression of type 1 fimbriae is a critical determinant for biofilm formation by ST131 strains and that inhibition of the FimH adhesin significantly reduces biofilm formation. We also offer direct genetic evidence for the contribution of type 1 fimbriae in biofilm formation by the reference ST131 strain EC958, a representative of the clinically dominant H30-Rx ST131 subgroup. This is the first study of ST131 biofilm formation in biologically relevant conditions and paves the way for the application of FimH inhibitors in treating drug resistant ST131 biofilm infections.

Field-Effect and Frequency Dependent Transport in Semiconductor-Enriched Single-Wall Carbon Nanotube Network Device

The electrical and optical response of a field-effect device comprising a network of semiconductor-enriched single-wall carbon nanotubes, gated with sodium chloride solution is investigated. Field-effect is demonstrated in a device that uses facile fabrication techniques along with a small-ion as the gate electrolyte-and this is accomplished as a result of the semiconductor enhancement of the tubes. The optical transparency and electrical resistance of the device are modulated with gate voltage. A time-response study of the modulation of optical transparency and electrical resistance upon application of gate voltage suggests the percolative charge transport in the network. Also the ac response in the network is investigated as a function of frequency and temperature down to 5 K. An empirical relation between onset frequency and temperature is determined.

Ligand dependent intra and inter subunit communication in human tryptophanyl tRNA synthetase as deduced from the dynamics of structure networks

Homodimeric protein tryptophanyl tRNA synthetase (TrpRS) has a Rossmann fold domain and belongs to the 1c subclass of aminoacyl tRNA synthetases. This enzyme performs the function of acylating the cognate tRNA. This process involves a number of molecules (2 protein subunits, 2 tRNAs and 2 activated Trps) and thus it is difficult to follow the complex steps in this process. Structures of human TrpRS complexed with certain ligands are available. Based on structural and biochemical data, mechanism of activation of Trp has been speculated. However, no structure has yet been solved in the presence of both the tRNA(Trp) and the activated Trp (TrpAMP). In this study, we have modeled the structure of human TrpRS bound to the activated ligand and the cognate tRNA. In addition, we have performed molecular dynamics (MD) simulations on these models as well as other complexes to capture the dynamical process of ligand induced conformational changes. We have analyzed both the local and global changes in the protein conformation from the protein structure network (PSN) of MD snapshots, by a method which was recently developed in our laboratory in the context of the functionally monomeric protein, methionyl tRNA synthetase. From these investigations, we obtain important information such as the ligand induced correlation between different residues of this protein, asymmetric binding of the ligands to the two subunits of the protein as seen in the crystal structure analysis, and the path of communication between the anticodon region and the aminoacylation site. Here we are able to elucidate the role of dimer interface at a level of detail, which has not been captured so far.

A Combinatorial Construct for Keyless Message Dispersal (Work in Progress)

We propose a keyless and lightweight message transformation scheme based on the combinatorial design theory for the confidentiality of a message transmitted in multiple parts through a network with multiple independent paths, or for data stored in multiple parts by a set of independent storage services such as the cloud providers. Our combinatorial scheme disperses a message into v output parts so that (k-1) or less parts do not reveal any information about any message part, and the message can only be recovered by the party who possesses all v output parts. Combinatorial scheme generates an xor transformation structure to disperse the message into v output parts. Inversion is done by applying the same xor transformation structure on output parts. The structure is generated using generalized quadrangles from design theory which represents symmetric point and line incidence relations in a projective plane. We randomize our solution by adding a random salt value and dispersing it together with the message. We show that a passive adversary with capability of accessing (k-1) communication links or storage services has no advantage so that the scheme is indistinguishable under adaptive chosen ciphertext attack (IND-CCA2).

Influence of tool rake angle on the quality of pure magnesium chip-consolidated product

Chips were produced by orthogonal Cutting of cast pure magnesium billet with three different tool rake angles viz., -15 degrees, -5 degrees and +15 degrees on a lathe. Chip consolidation by solid state recycling technique involved cold compaction followed by hot extrusion. The extruded products were characterized for microstructure and mechanical properties. Chip-consolidated products from -15 degrees rake angle tools showed 19% increase in tensile strength, 60% reduction ingrain size and 12% increase in hardness compared to +15 degrees rake chip-consolidated product indicating better chip bonding and grain refinement. Microstructure of the fracture specimen Supports the abovefinding. On the overall, the present work high lights the importance of tool take angle in determining the quality of the chip-consolidated products. (C) 2009 Elsevier B.V. All rights reserved.