Assessment of the nanomechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy

Coronary atherosclerosis is a major cause of mortality and morbidity worldwide. Despite its systemic nature, atherosclerotic plaques form and develop at “predilection” sites often associated with disturbed biomechanical forces. Therefore, computational approaches that analyse the biomechanics (blood flow and tissue mechanics) of atherosclerotic plaques have come to the forefront over the last 20 years. Assignment of appropriate material properties is an integral part of the simulation process. Current approaches for derivation of material properties rely on macro-mechanical testing and are agnostic to local variations of plaque stiffness to which collagen microstructure plays an important role. In this work we used Atomic Force Microscopy to measure the stiffness of healthy and atherosclerotic coronary arteries and we hypothesised that are those are contingent on the local microstructure. Given that the optimal method for studying mechanics of arterial tissue with this method has not been comprehensively established, an indentation protocol was firstly developed and optimised for frozen tissue sections as well as a co-registration framework with the local collagen microstructure utilising the same tissue section for mechanical testing and histological staining for collagen. Overall, the mechanical properties (Young’s Modulus) of the healthy vessel wall (median = 11.0 kPa, n=1379 force curves) were found to be significantly stiffer (p=1.3410-10) than plaque tissue (median=4.3 kPa, n=1898 force curves). Within plaques, lipid-rich areas (median=2.2 kPa, n=392 force curves) were found significantly softer (p=1.4710-4) than areas rich in collagen, such as the fibrous cap (median=4.9 kPa, n=1506 force curves). No statistical difference (p=0.89) was found between measurements in the middle of the fibrous cap (median=4.8 kPa, n=868 force curves) and the cap shoulder (median=5.1 kPa, n=638 force curves). Macro-mechanical testing methods dominate the entire landscape of material testing techniques. Plaques are very heterogenous in composition and macro-mechanical methods are agnostic to microscale variations in plaque stiffness. Mechanical testing by indentation may be better suited to quantify local variations in plaque stiffness, that are potent drivers of plaque rupture.Open Acces

Alexandrea in Aegypto. The role of the Egyptian tradition in the Hellenistic and Roman periods : ideology, culture, identity, and public life

Alexandria, the capital of Egypt during the Hellenistic and Roman periods is often hailed as the ancient cosmopolitan center of Mediterranean par excellence. Since the foundation of the city by Alexander the Great in 331 BC, several traditions- along with their representatives, mainly Greek and Egyptian- coexisted and interacted with each other, resulting in a multiculturalism in Alexandrian society. However, in the past scholarship, the Greek cultural aspect of the city has been extensively discussed, while the Egyptian part has never been fully overviewed and interpreted. Such interpretations caused in a large extent, from the one side, a deformed picture of the Alexandria’s Greek-ness, almost equal to this of a Greek “colony” separated from Egypt –Alexandria ad Aegyptum- and from the other side a blur picture about the role of the Egyptian tradition in the Greco-Egyptian interaction and the life of Alexandrian society. However, over the last decade an alternative framework of understanding has been developed in several case studies, while discoveries from the underwater missions indicate that the city had much more Egyptian characteristics than hitherto believed. Therefore, the thesis aims to provide an overview and interpretation of the Egyptian elements and influences in Alexandria, focusing on issues of ideology, culture, identity and public life. In this way, it has been attempted to offer a better understanding of the multicultural life of Alexandria in the Hellenistic and Roman periods.LEI Universiteit LeidenClassical and Mediterranean Archaeolog

CEST MRI for the characterisation of human brain tumours

Chemical Exchange Saturation Transfer (CEST) is a novel MRI technique that amplifies the detection of otherwise undetectable compounds. Under certain physico-chemical and experimental conditions, CEST allows the detection of compounds containing amide, amine and hydroxyl proton groups, that are in chemical exchange with free water molecules. The main focus of this work is to gain an understanding of the underlying principles of CEST, both in-vitro and in-vivo. After introducing essential MRI principles in chapter 1, chapter 2 contains a review of the CEST theory, optimisation techniques and applications of CEST, mainly in characterising brain tumours. Chapter 3 studies the impact of experimental parameters in generating and quantifying the APT signal, in-silico. The parameters investigated were: the B_{1} irradiation power, the APT pool size, the saturation length and finally the SNR. For each of those parameters, the relationship between the parameter and APT signal was derived and its implications in optimising CEST experiments were discussed. For the model under investigation, it was found that the optimal parameters were: B_{1} = 1µT, APT pool size of 0.01 and saturation time of 3 seconds, at most. Furthermore, it is shown that the presence of noise diminishes the ability to quantify the APT effect accurately and that this loss varies linearly with SNR. In conclusion, the model studied here should be imaged with an SNR of ~70 so that the percentage error and relative accuracy in the measurements is ~10% and 0.8, respectively. Chapter 4 contains a feasibility comparison of measuring extracellular pH with CEST, by using three clinically approved iodinated contrast agents, at 7T, in-vitro. The contrast agents tested were: Iodixanol (brand name - Visipaque, GE Healthcare), Iopamidol (brand name - Niopam, Bracco UK Ltd) and Iopromide (brand name - Ultravist, Bayer Healthcare). Phantoms of the agents, in phosphate buffer and at different pH levels were prepared and subsequently scanned using a 2D gradient echo CEST sequence. The CEST effect was calculated by asymmetry analysis and pH maps were derived on a pixel by pixel basis. In addition, calibration curves were determined by fitting the in-vitro CEST signal of all three agents, against pH. Under the experimental conditions used, Niopam and Ultravist showed two, while Visipaque showed one, CEST exchange peaks. Consequently, Visipaque was found unsuitable in determining pH, by ratiometric CEST analysis. On the contrary, Niopam and Ultravist showed great potential in determining extracellular pH, as the ratiometric analysis produced linear calibration curves, for both agents. Moreover, Ultravist showed superior linearity to Niopam (R^{2} = 0.99, R^{2} = 0.88). Next steps could involve, measuring the CEST effects, of both Niopam and Ultravist, in-vivo. First, on a pre-clinical model and second, in humans. The latter will be at 3T, instead of 7T. Patients could be immediately scanned after routine CT procedures, where either of these contrast agents is used. Before assessing the pH, calibration curves, of both agents, will be derived at 3T, in-vitro, with experiments similar to those reported at 7T. Chapter 5 contains the comparison between a modified single slice and volumetric CEST gradient echo sequences, in generating CEST contrast, in a series of phantoms, at 3T. The single slice sequence was successfully modified by myself, whereas the volumetric sequence was prepared by a team from Institute of Psychiatry, Psychology and Neuroscience in King’s College, London. Besides allowing for different spatial excitation, the two sequences also differ in the pre-saturation pulse type. The latter is a Fermi pulse, in the 2D sequence and a Gaussian pulse, in the 3D sequence. A common limitation of both sequences is the maximum allowed pulse duration, which is in the order of milli-seconds. However, CEST effects were successfully measured in phantoms of the iodinated contrast agent, Ultravist, creatine and glutamate, under physiological conditions (pH =7). Asymmetry analysis was used to quantify the CEST effect, in all cases. Despite post-processing corrections for B_{0} inhomogeneities, some datasets were noisy. This is perhaps due to small sample size and inefficient shimming. The Gaussian pulse of the 3D sequence was found to produce a 2-fold increased CEST effect in Ultravist and a 7-fold CEST effect increase in creatine, as compared to the Fermi pulse of the 2D sequence. Furthermore, increasing the duty-cycle of the 2D sequence did not result in greater CEST effect magnitude. Analysis of the main field homogeneity, after linear and high order shimming, showed that the latter results in frequency shifts within 0.5 ppm, whereas the shifts, after linear shimming, extended beyond that range. As the CEST effect is highly sensitive on frequency shifts, a high order shimming is more beneficial, especially when characterising CEST effects in-vivo. Future work could include studying the pH dependence of Ultravist and Niopam, in-vitro. Even if only one CEST peak, instead of the two found at 7T, can be measured at 3T, calibration curves can still be derived by implementing an RF power based ratiometric method. Subsequent translation, of measuring pH, in patients that have received iodinated contrast injection, will require further ethical approval. Besides measuring pH, typical CEST acquisitions could be optimised in more complex phantoms, in-vitro. For example, the CEST effects of three-pool model phantoms (agar gel + creatine or ammonium chloride) could be tested, using the 3D gradient echo CEST sequence. Existing analysis tools could be updated to allow for Lorentzian fitting separation of co-existing CEST effects. New tools, allowing fitting of Bloch equations to CEST data, should also be developed. In addition, besides B_{0} inhomogeneity corrections, the impact of RF inhomogeneity effects, on CEST signals, should be studied. The latter correction might be crucial in detecting weaker CEST effects at 3T. It would also be interesting to measure the relaxation compensated CEST effect at 3T. That would be possible if our sequences allow for a pre-saturation time in the order of seconds (t_{sat}>>T_{1w}). Ultimately, the efficacy of the 3D CEST sequence could be tested in healthy volunteers and finally in brain tumour patients, after acquisition of appropriate ethical approval. Chapter 6 contains the details of a CEST study, of cerebral gliomas at 7T, where the APT and NOE signals, between low and high grade brain tumours and normal white matter, are compared. In addition, the correlation between, APT and NOE signals with apparent diffusion coefficient (ADC), is also investigated. More specifically, a 3D CEST MRI scan, targeting amide and aliphatic protons, was acquired, at 7T, in seven untreated glioma patients, who had previously undergone routine clinical 3T MRI. APT and NOE maps were produced by Lorentzian fitting of the CEST data. Significantly higher tumour APT signal was found in both low grades and glioblastomas, as compared to healthy white matter. In addition, the APT signal, was 3 to 4-fold elevated in glioblastoma versus low grades. On the contrary, significantly lower tumour NOE was found in both low grades and glioblastomas, as compared normal tissue, with the latter being ~7-fold decreased in GBMs. Further, investigation of the correlation of the APT and NOE, tumour and control signals, with ADC, did not show any particular trend, with the exception of the NOE - ADC signals in controls, which showed a weak, negative correlation. Further analysis of our data could include asymmetry analysis, as a comparison to Lorentzian fitting, in cerebral gliomas. Also, quantification of the conventional magnetisation transfer (MT), along with APT and NOE effects, in oedema, necrotic and haemorrhagic regions, that often accompany gliomas. Chapter 7 contains an overall discussion of the work on this thesis, how it relates to the literature and presents some ideas on what should be done next

Architecture and DSP Implementation of a DVB-S2 Baseband Demodulator

This paper presents the design and implementation of a baseband demodulator for DVB-S2 satellite receivers. In order to meet the requirements of different complex and multi-domain signal processing stages of the DVB-S2 baseband signal-flow, the presented architecture is based on efficient fixed-point implementation of the various demodulation algorithms and on the use of a dynamic time-sharing scheduler for the various DSP software tasks. The prototyping of the demodulator and its verification in the design of a complete digital DVB-S2 satellite receiver using a versatile testbed is also presented

Considering the influence of coronary motion on artery-specific biomechanics using fluid-structure interaction simulation

The endothelium in the coronary arteries is subject to wall shear stress and vessel wall strain, which influences the biology of the arterial wall. This study presents vessel-specific fluid-structure interaction (FSI) models of three coronary arteries, using directly measured experimental geometries and boundary conditions. FSI models are used to provide a more physiologically complete representation of vessel biomechanics, and have been extended to include coronary bending to investigate its effect on shear and strain. FSI both without- and with-bending resulted in significant changes in all computed shear stress metrics compared to CFD (p = 0.0001). Inclusion of bending within the FSI model produced highly significant changes in Time Averaged Wall Shear Stress (TAWSS) + 9.8% LAD, + 8.8% LCx, - 2.0% RCA; Oscillatory Shear Index (OSI) + 208% LAD, 0% LCx, + 2600% RCA; and transverse wall Shear Stress (tSS) + 180% LAD, + 150% LCx and + 200% RCA (all p < 0.0001). Vessel wall strain was homogenous in all directions without-bending but became highly anisotropic under bending. Changes in median cyclic strain magnitude were seen for all three vessels in every direction. Changes shown in the magnitude and distribution of shear stress and wall strain suggest that bending should be considered on a vessel-specific basis in analyses of coronary artery biomechanics

Lithium chloride therapy fails to improve motor function in a transgenic mouse model of Machado-Joseph disease

The accumulation of misfolded proteins in neurons, leading to the formation of cytoplasmic and nuclear aggregates, is a common theme in age-related neurodegenerative diseases, possibly due to disturbances of the proteostasis and insufficient activity of cellular protein clearance pathways. Lithium is a well-known autophagy inducer that exerts neuroprotective effects in different conditions and has been proposed as a promising therapeutic agent for several neurodegenerative diseases. We tested the efficacy of chronic lithium 10.4 mg/kg) treatment in a transgenic mouse model of Machado-Joseph disease, an inherited neurodegenerative disease, caused by an expansion of a polyglutamine tract within the protein ataxin-3. A battery of behavioral tests was used to assess disease progression. In spite of activating autophagy, as suggested by the increased levels of Beclin-1, Atg7, and LC3II, and a reduction in the p62 protein levels, lithium administration showed no overall beneficial effects in this model concerning motor performance, showing a positive impact only in the reduction of tremors at 24 weeks of age. Our results do not support lithiumchronic treatment as a promising strategy for the treatment of Machado-Joseph disease (MJD).FCT -Fundação para a Ciência e a Tecnologia(SFRH/BD/51059/2010

A Personalized Framework for Dynamic Modeling of Disease Trajectories in Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is the most common peripheral blood and bone marrow cancer in the developed world. This manuscript proposes mathematical model equations representing the disease dynamics of B-cell CLL. We interconnect delay differential cell cycle models in each of the tumor-involved disease centers using physiologically-relevant cell migration. We further introduce 5 hypothetical case studies representing CLL heterogeneity commonly seen in clinical practice and demonstrate how the proposed CLL model framework may capture disease pathophysiology across patient types. We conclude by exploring the capacity of the proposed temporally- and spatially-distributed model to capture the heterogeneity of CLL disease progression. By using Global Sensitivity Analysis, the critical parameters influencing disease trajectory over space and time are: (i) the initial number of CLL cells in peripheral blood, the number of involved lymph nodes, the presence and degree of splenomegaly; (ii) the migratory fraction of nonproliferating as well as proliferating CLL cells from bone marrow into blood and of proliferating CLL cells from blood into lymph nodes; (iii) the parameters inducing nonproliferative cells to proliferate. The proposed model offers a practical platform which may be explored in future personalized patient protocols once validated

CEST MRI for the characterisation of human brain tumours

Chemical Exchange Saturation Transfer (CEST) is a novel MRI technique that amplifies the detection of otherwise undetectable compounds. Under certain physico-chemical and experimental conditions, CEST allows the detection of compounds containing amide, amine and hydroxyl proton groups, that are in chemical exchange with free water molecules. The main focus of this work is to gain an understanding of the underlying principles of CEST, both in-vitro and in-vivo. After introducing essential MRI principles in chapter 1, chapter 2 contains a review of the CEST theory, optimisation techniques and applications of CEST, mainly in characterising brain tumours. Chapter 3 studies the impact of experimental parameters in generating and quantifying the APT signal, in-silico. The parameters investigated were: the B_{1} irradiation power, the APT pool size, the saturation length and finally the SNR. For each of those parameters, the relationship between the parameter and APT signal was derived and its implications in optimising CEST experiments were discussed. For the model under investigation, it was found that the optimal parameters were: B_{1} = 1µT, APT pool size of 0.01 and saturation time of 3 seconds, at most. Furthermore, it is shown that the presence of noise diminishes the ability to quantify the APT effect accurately and that this loss varies linearly with SNR. In conclusion, the model studied here should be imaged with an SNR of ~70 so that the percentage error and relative accuracy in the measurements is ~10% and 0.8, respectively. Chapter 4 contains a feasibility comparison of measuring extracellular pH with CEST, by using three clinically approved iodinated contrast agents, at 7T, in-vitro. The contrast agents tested were: Iodixanol (brand name - Visipaque, GE Healthcare), Iopamidol (brand name - Niopam, Bracco UK Ltd) and Iopromide (brand name - Ultravist, Bayer Healthcare). Phantoms of the agents, in phosphate buffer and at different pH levels were prepared and subsequently scanned using a 2D gradient echo CEST sequence. The CEST effect was calculated by asymmetry analysis and pH maps were derived on a pixel by pixel basis. In addition, calibration curves were determined by fitting the in-vitro CEST signal of all three agents, against pH. Under the experimental conditions used, Niopam and Ultravist showed two, while Visipaque showed one, CEST exchange peaks. Consequently, Visipaque was found unsuitable in determining pH, by ratiometric CEST analysis. On the contrary, Niopam and Ultravist showed great potential in determining extracellular pH, as the ratiometric analysis produced linear calibration curves, for both agents. Moreover, Ultravist showed superior linearity to Niopam (R^{2} = 0.99, R^{2} = 0.88). Next steps could involve, measuring the CEST effects, of both Niopam and Ultravist, in-vivo. First, on a pre-clinical model and second, in humans. The latter will be at 3T, instead of 7T. Patients could be immediately scanned after routine CT procedures, where either of these contrast agents is used. Before assessing the pH, calibration curves, of both agents, will be derived at 3T, in-vitro, with experiments similar to those reported at 7T. Chapter 5 contains the comparison between a modified single slice and volumetric CEST gradient echo sequences, in generating CEST contrast, in a series of phantoms, at 3T. The single slice sequence was successfully modified by myself, whereas the volumetric sequence was prepared by a team from Institute of Psychiatry, Psychology and Neuroscience in King’s College, London. Besides allowing for different spatial excitation, the two sequences also differ in the pre-saturation pulse type. The latter is a Fermi pulse, in the 2D sequence and a Gaussian pulse, in the 3D sequence. A common limitation of both sequences is the maximum allowed pulse duration, which is in the order of milli-seconds. However, CEST effects were successfully measured in phantoms of the iodinated contrast agent, Ultravist, creatine and glutamate, under physiological conditions (pH =7). Asymmetry analysis was used to quantify the CEST effect, in all cases. Despite post-processing corrections for B_{0} inhomogeneities, some datasets were noisy. This is perhaps due to small sample size and inefficient shimming. The Gaussian pulse of the 3D sequence was found to produce a 2-fold increased CEST effect in Ultravist and a 7-fold CEST effect increase in creatine, as compared to the Fermi pulse of the 2D sequence. Furthermore, increasing the duty-cycle of the 2D sequence did not result in greater CEST effect magnitude. Analysis of the main field homogeneity, after linear and high order shimming, showed that the latter results in frequency shifts within 0.5 ppm, whereas the shifts, after linear shimming, extended beyond that range. As the CEST effect is highly sensitive on frequency shifts, a high order shimming is more beneficial, especially when characterising CEST effects in-vivo. Future work could include studying the pH dependence of Ultravist and Niopam, in-vitro. Even if only one CEST peak, instead of the two found at 7T, can be measured at 3T, calibration curves can still be derived by implementing an RF power based ratiometric method. Subsequent translation, of measuring pH, in patients that have received iodinated contrast injection, will require further ethical approval. Besides measuring pH, typical CEST acquisitions could be optimised in more complex phantoms, in-vitro. For example, the CEST effects of three-pool model phantoms (agar gel + creatine or ammonium chloride) could be tested, using the 3D gradient echo CEST sequence. Existing analysis tools could be updated to allow for Lorentzian fitting separation of co-existing CEST effects. New tools, allowing fitting of Bloch equations to CEST data, should also be developed. In addition, besides B_{0} inhomogeneity corrections, the impact of RF inhomogeneity effects, on CEST signals, should be studied. The latter correction might be crucial in detecting weaker CEST effects at 3T. It would also be interesting to measure the relaxation compensated CEST effect at 3T. That would be possible if our sequences allow for a pre-saturation time in the order of seconds (t_{sat}>>T_{1w}). Ultimately, the efficacy of the 3D CEST sequence could be tested in healthy volunteers and finally in brain tumour patients, after acquisition of appropriate ethical approval. Chapter 6 contains the details of a CEST study, of cerebral gliomas at 7T, where the APT and NOE signals, between low and high grade brain tumours and normal white matter, are compared. In addition, the correlation between, APT and NOE signals with apparent diffusion coefficient (ADC), is also investigated. More specifically, a 3D CEST MRI scan, targeting amide and aliphatic protons, was acquired, at 7T, in seven untreated glioma patients, who had previously undergone routine clinical 3T MRI. APT and NOE maps were produced by Lorentzian fitting of the CEST data. Significantly higher tumour APT signal was found in both low grades and glioblastomas, as compared to healthy white matter. In addition, the APT signal, was 3 to 4-fold elevated in glioblastoma versus low grades. On the contrary, significantly lower tumour NOE was found in both low grades and glioblastomas, as compared normal tissue, with the latter being ~7-fold decreased in GBMs. Further, investigation of the correlation of the APT and NOE, tumour and control signals, with ADC, did not show any particular trend, with the exception of the NOE - ADC signals in controls, which showed a weak, negative correlation. Further analysis of our data could include asymmetry analysis, as a comparison to Lorentzian fitting, in cerebral gliomas. Also, quantification of the conventional magnetisation transfer (MT), along with APT and NOE effects, in oedema, necrotic and haemorrhagic regions, that often accompany gliomas. Chapter 7 contains an overall discussion of the work on this thesis, how it relates to the literature and presents some ideas on what should be done next