82 research outputs found
Charge reversal of colloidal particles
A theory is presented for the effective charge of colloidal particles in
suspensions containing multivalent counterions. It is shown that if colloids
are sufficiently strongly charged, the number of condensed multivalent
counterion can exceed the bare colloidal charge leading to charge reversal.
Charge renormalization in suspensions with multivalent counterions depends on a
subtle interplay between the solvation energies of the multivalent counterions
in the bulk and near the colloidal surface. We find that the effective charge
is {\it not} a monotonically decreasing function of the multivalent salt
concentration. Furthermore, contrary to the previous theories, it is found that
except at very low concentrations, monovalent salt hinders the charge reversal.
This conclusion is in agreement with the recent experiments and simulations
Numerical electrokinetics
A new lattice method is presented in order to efficiently solve the
electrokinetic equations, which describe the structure and dynamics of the
charge cloud and the flow field surrounding a single charged colloidal sphere,
or a fixed array of such objects. We focus on calculating the electrophoretic
mobility in the limit of small driving field, and systematically linearise the
equations with respect to the latter. This gives rise to several subproblems,
each of which is solved by a specialised numerical algorithm. For the total
problem we combine these solvers in an iterative procedure. Applying this
method, we study the effect of the screening mechanism (salt screening vs.
counterion screening) on the electrophoretic mobility, and find a weak
non-trivial dependence, as expected from scaling theory. Furthermore, we find
that the orientation of the charge cloud (i. e. its dipole moment) depends on
the value of the colloid charge, as a result of a competition between
electrostatic and hydrodynamic effects.Comment: accepted for publication in Journal of Physics Condensed Matter
(proceedings of the 2012 CODEF conference
Valproic acid enhances the efficacy of radiation therapy by protecting normal hippocampal neurons and sensitizing malignant glioblastoma cells
Neurocognitive deficits are serious sequelae that follow cranial irradiation used to treat patients with medulloblastoma and other brain neoplasms. Cranial irradiation causes apoptosis in the subgranular zone of the hippocampus leading to cognitive deficits. Valproic acid (VPA) treatment protected hippocampal neurons from radiation-induced damage in both cell culture and animal models. Radioprotection was observed in VPA-treated neuronal cells compared to cells treated with radiation alone. This protection is specific to normal neuronal cells and did not extend to cancer cells. In fact, VPA acted as a radiosensitizer in brain cancer cells. VPA treatment induced cell cycle arrest in cancer cells but not in normal neuronal cells. The level of anti-apoptotic protein Bcl-2 was increased and the pro-apoptotic protein Bax was reduced in VPA treated normal cells. VPA inhibited the activities of histone deacetylase (HDAC) and glycogen synthase kinase-3β (GSK3β), the latter of which is only inhibited in normal cells. The combination of VPA and radiation was most effective in inhibiting tumor growth in heterotopic brain tumor models. An intracranial orthotopic glioma tumor model was used to evaluate tumor growth by using dynamic contrast-enhanced magnetic resonance (DCE MRI) and mouse survival following treatment with VPA and radiation. VPA, in combination with radiation, significantly delayed tumor growth and improved mouse survival. Overall, VPA protects normal hippocampal neurons and not cancer cells from radiation-induced cytotoxicity both in vitro and in vivo. VPA treatment has the potential for attenuating neurocognitive deficits associated with cranial irradiation while enhancing the efficiency of glioma radiotherapy
Time- and region-dependent blood-brain barrier impairment in a rat model of organophosphate-induced status epilepticus
Acute organophosphate (OP) intoxication can trigger seizures that progress to status epilepticus (SE), and survivors often develop chronic morbidities, including spontaneous recurrent seizures (SRS). The pathogenic mechanisms underlying OP-induced SRS are unknown, but increased BBB permeability is hypothesized to be involved. Previous studies reported BBB leakage following OP-induced SE, but key information regarding time and regional distribution of BBB impairment during the epileptogenic period is missing. To address this data gap, we characterized the spatiotemporal progression of BBB impairment during the first week post-exposure in a rat model of diisopropylfluorophosphate-induced SE, using MRI and albumin immunohistochemistry. Increased BBB permeability, which was detected at 6 h and persisted up to 7 d post-exposure, was most severe and persistent in the piriform cortex and amygdala, moderate but persistent in the thalamus, and less severe and transient in the hippocampus and somatosensory cortex. The extent of BBB leakage was positively correlated with behavioral seizure severity, with the strongest association identified in the piriform cortex and amygdala. These findings provide evidence of the duration, magnitude and spatial breakdown of the BBB during the epileptogenic period following OP-induced SE and support BBB regulation as a viable therapeutic target for preventing SRS following acute OP intoxication
NMR and in silico studies of fucosylated chondroitin sulfate (fCS) and its interactions with selectins
This thesis describes structural studies on the interactions between the fucosylated
chondroitin sulfate (fCS) oligosaccharides and human proteins known as selectins. fCS is a
carbohydrate obtained from sea cucumbers, that can be classified as a branched
glycosaminoglycan (GAG). It has attracted much attention due to its anti-coagulant, anti-inflammatory,
antimetastatic and anti-HIV properties and its structure was previously
determined by NMR. Selectins constitute a family of proteins involved in cell adhesion
processes, such as inflammation, attachment of viral particles and migration of tumour cells.
fCS oligosaccharides have been shown to bind to selectins, which is likely a reason behind
their biological activity. However, the mechanism of this interaction is currently unknown.
The initial part of the thesis describes the experimental work on expression and purification
of the recombinant L- and P-selectin constructs in Pichia pastoris, Escherichia coli and HEK
293 cells. The aim of these experiments was to produce two constructs for each selectin, a
single domain construct, consisting of the C-type lectin domain only, and a double domain
construct, consisting of both the C-type lectin and the EGF-like domains. The intention was
that the recombinant proteins would be labelled with 13C and 15N to allow for the in-depth
structural NMR studies on the fCS-selectin interaction. Various experimental approaches
have been explored, including the use of different cell lines, modifications to construct design,
as well as alterations to expression and purification conditions. Although it was not possible
to produce soluble selectin constructs in either bacterial or yeast cells, protein expression
tests in HEK293 cells, performed in collaboration with the Oxford Protein Production facility
(OPPF), led to production of a soluble L-selectin construct, consisting of the L-selectin C-type
lectin domain.
The produced L-selectin construct, as well as two commercially available constructs of the Land
P-selectin extracellular domains, were used in the Saturation Transfer Difference (STD)
NMR experiments to provide new information about the nature of the fCS-selectin binding.
The STD experiments allowed to identify the regions within the fCS oligosaccharides that are
in direct contact with the protein and likely play an important role in this interaction.
Experiments on different protein constructs allowed the comparison of fCS binding to P-selectin
and to two different recombinant constructs of L-selectin. Results of these studies
suggest that the binding occurs via a similar mechanism for both L- and P-selectins and that
the fCS oligosaccharides bind to one-domain L-selectin construct with similar affinity as to a
larger construct, consisting of the entire extracellular region of the protein.
Alongside the experimental work, theoretical in silico studies on the fCS-selectin binding
were undertaken as part of this project. The existing X-ray structures of selectin complexes
were subjected to Molecular Dynamics (MD) simulations, which allowed to explore the
dynamic behaviour of E-selectin upon binding to sialyl Lewis x (sLex). It was found that sLex
forms a more favourable interaction with the extended conformation of E-selectin and that
the protein in this conformation is characterised by a high degree of interdomain flexibility,
with a new type of interdomain movement observed in the MD studies on this complex.
In further in silico studies, the fCS oligosaccharides were docked to the existing P-selectin
structures. The docking tests were performed on the computationally produced fCS
trisaccharides with fucose branches either 2,4 or 3,4-sulfated. Results were evaluated with
MD simulations and analysed in the light of current knowledge of selectin-ligand binding and
the STD NMR experimental results. The in silico studies allowed to identify a subset of P-selectin
residues that are likely involved in the interaction with fCS oligosaccharides in vivo.
The conformational behaviour of P-selectin upon binding to fCS was also explored and it was
found that the interdomain hinge is flexible during this interaction and allows transition from
bent to extended conformational state.
Finally, a new NMR method was developed to facilitate the studies of complex carbohydrates,
incorporating the concepts of G-matrix Fourier Transform (GFT) NMR into 2D HSQC and 2D
HSQC-TOCSY experiments. The method allows to separate peaks in the regions of high
spectral overlap, providing information that can simplify the assignment process. The new
experiments facilitated the structural evaluation of a sample containing a mixture of
oligosaccharides resulting from the depolymerisation of fCS polysaccharide
Intermediate-Range Order of Alkali Disilicate Glasses and Its Relation to the Devitrification Mechanism
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