38 research outputs found
Following Suberization in Potato Wound Periderm by Histochemical and Solid-State 13C Nuclear Magnetic Resonance Methods
Computed Tomography Imaging of Primary Lung Cancer in Mice Using a Liposomal-Iodinated Contrast Agent
To investigate the utility of a liposomal-iodinated nanoparticle contrast agent and computed tomography (CT) imaging for characterization of primary nodules in genetically engineered mouse models of non-small cell lung cancer.Primary lung cancers with mutations in K-ras alone (Kras(LA1)) or in combination with p53 (LSL-Kras(G12D);p53(FL/FL)) were generated. A liposomal-iodine contrast agent containing 120 mg Iodine/mL was administered systemically at a dose of 16 µl/gm body weight. Longitudinal micro-CT imaging with cardio-respiratory gating was performed pre-contrast and at 0 hr, day 3, and day 7 post-contrast administration. CT-derived nodule sizes were used to assess tumor growth. Signal attenuation was measured in individual nodules to study dynamic enhancement of lung nodules.A good correlation was seen between volume and diameter-based assessment of nodules (R(2)>0.8) for both lung cancer models. The LSL-Kras(G12D);p53(FL/FL) model showed rapid growth as demonstrated by systemically higher volume changes compared to the lung nodules in Kras(LA1) mice (p<0.05). Early phase imaging using the nanoparticle contrast agent enabled visualization of nodule blood supply. Delayed-phase imaging demonstrated significant differential signal enhancement in the lung nodules of LSL-Kras(G12D);p53(FL/FL) mice compared to nodules in Kras(LA1) mice (p<0.05) indicating higher uptake and accumulation of the nanoparticle contrast agent in rapidly growing nodules.The nanoparticle iodinated contrast agent enabled visualization of blood supply to the nodules during the early-phase imaging. Delayed-phase imaging enabled characterization of slow growing and rapidly growing nodules based on signal enhancement. The use of this agent could facilitate early detection and diagnosis of pulmonary lesions as well as have implications on treatment response and monitoring
Non-Invasive Quantification of White and Brown Adipose Tissues and Liver Fat Content by Computed Tomography in Mice
OBJECTIVES: Obesity and its distribution pattern are important factors for the prediction of the onset of diabetes in humans. Since several mouse models are suitable to study the pathophysiology of type 2 diabetes the aim was to validate a novel computed tomograph model (Aloka-Hitachi LCT-200) for the quantification of visceral, subcutaneous, brown and intrahepatic fat depots in mice. METHODS: Different lean and obese mouse models (C57BL/6, B6.V-Lep(ob), NZO) were used to determine the most adequate scanning parameters for the detection of the different fat depots. The data were compared with those obtained after preparation and weighing the fat depots. Liver fat content was determined by biochemical analysis. RESULTS: The correlations between weights of fat tissues on scale and weights determined by CT were significant for subcutaneous (r(2) = 0.995), visceral (r(2) = 0.990) and total white adipose tissue (r(2) = 0.992). Moreover, scans in the abdominal region, between lumbar vertebrae L4 to L5 correlated with whole-body fat distribution allowing experimenters to reduce scanning time and animal exposure to radiation and anesthesia. Test-retest reliability and measurements conducted by different experimenters showed a high reproducibility in the obtained results. Intrahepatic fat content estimated by CT was linearly related to biochemical analysis (r(2) = 0.915). Furthermore, brown fat mass correlated well with weighted brown fat depots (r(2) = 0.952). In addition, short-term cold-expose (4 °C, 4 hours) led to alterations in brown adipose tissue attributed to a reduction in triglyceride content that can be visualized as an increase in Hounsfield units by CT imaging. CONCLUSION: The 3D imaging of fat by CT provides reliable results in the quantification of total, visceral, subcutaneous, brown and intrahepatic fat in mice. This non-invasive method allows the conduction of longitudinal studies of obesity in mice and therefore enables experimenters to investigate the onset of complex diseases such as diabetes and obesity
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
Comparison of neutralino and sneutrino dark matter in a model with spontaneous CP violation
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