521 research outputs found
Three-dimensional tissue models and available probes for multi-parametric live cell microscopy : a brief overview
No full textIn recent years, the advances in tissue engineering and regenerative medicine have resulted in introduction of novel 3D tissue models, materials and methods to the regular practice of cell biologists, material scientists and specialists from related areas. 3D tissue models allow mimicking in vivo cell and tissue organization. However, the efficient work in three dimensions has significant challenges, such as compatibility with conventional cell biology methods, live cell imaging and quantification readouts. Here, we briefly discuss the applicability of 3D tissue models to different live cell microscopy modalities and the available range of fluo-and phosphorescent probes and sensors allowing for multi-parametric imaging
Fabrication and handling of 3D scaffolds based on polymers and decellularized tissues
No full textPolymeric, ceramic and hybrid material-based three-dimensional (3D) scaffold or matrix structures are important for successful tissue engineering. While the number of approaches utilizing the use of cell-based scaffold and matrix structures is constantly growing, it is essential to provide a framework of their typical preparation and evaluation for tissue engineering. This chapter describes the fabrication of 3D scaffolds using -two--photon polymerization, decellularization and cell encapsulation methods and easy-to-use protocols allowing assessing the cell morphology, cytotoxicity and viability in these scaffolds
Role of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture
No full textRole of preconditioning with oxygen and glucose deprivation in promoting differentiation of dental pulp stem cells in 3D culture
Irina A. Okkelman, Winter Vandenberghe, Ruslan I. Dmitriev
Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University, Ghent 9000, Belgium
Dental pulp stem cells (DPSC) are promising for bone tissue engineering and repair. To support angiogenesis and long-term viability within microfabricated and 3D-printed tissue constructs, they are often grown and differentiated together with endothelial cells in multicellular spheroids. However, this is often associated with the decline of endothelial cells and slow rate of DPSC differentiation towards osteoblasts. Interestingly, hypoxia affects cell viability and at the same time promotes angiogenesis with osteoblast differentiation.
Here, we hypothesized that oxygen and glucose deprivation pre-conditioning can improve the process of DPSC differentiation in 3D culture with endothelial HUVEC cells. To do this, we performed oxygen and glucose deprivation experiments with 2D cultures of DPSC and HUVEC and tested effects on cell viability, redox potential and morphology. Interestingly, glucose deprivation did not significantly affect redox state of both HUVEC and DPSC. We therefore preconditioned these cell types with 0% O2, followed by 24 h reperfusion, and produced heterocellular spheroids. To understand the effects of preconditioning in this 3D culture, we performed fluorescence microscopy analysis of cell death (Sytox Green) together with monitoring of spheroid oxygenation (ratiometric red/near-infrared O2-sensing nanoparticles) during 7 days of differentiation. We found that cell death was not significantly different in the spheroids from non-differentiated and differentiated groups of cells. However, the periphery-to-core O2 gradients were significantly lower (p=0.05) in non-differentiated hDPSC 21% / HUVEC 0% and hDPSC 0% / HUVEC 0% groups in comparison to hDPSC 21% / HUVEC 21% spheroids. During differentiation, this difference in spheroid oxygenation was slightly ameliorated, keeping the same trend as in non-differentiated spheroids. Thus under the deeper hypoxia, the heterocellular spheroids formed with 0% O2-preconditioned endothelial cells demonstrated cell viability comparable to spheroids formed from non-preconditioned cells. This data correlates with the results on the alteration of redox potential in adherent (2D) DPSC and HUVEC cultures, where hypoxia-conditioned HUVEC cells demonstrated more profound response. We also found that preconditioned cells displayed stronger accumulation of hydroxyapatite and osteogenic differentiation. Collectively, our preliminary data indicates that hypoxia preconditioning, combined with imaging of oxygenation and cell death, can improve the quality of the spheroid-based ‘tissue building blocks’ in biofabrication.
Funding: supported by the Special Research Fund (BOF) grant of Ghent University (BOF/STA/202009/003)
On the History of the Name Ruslan
No full textВ публикации постулируется тезис о существовании общего пласта российского антропонимикона, формирующегося в результате взаимодействия национальных именников. Основная часть сообщения посвящена истории прецедентного в российском антропонимиконе имени Руслан. Прослеживается генетическая связь антропонима с широко распространенным в тюркских языках именем Арслан, имеющим доономастическое значение ‘лев’, выявляются варианты имени в архивных документах, исторических источниках XV–XVII вв., русском сказочном фольклоре. Оценивается роль поэмы А. С. Пушкина «Руслан и Людмила» в формировании ассоциативного фона исследуемого антропонима. Особое внимание уделено производным от имени патрониму и фамилии, а также образованному от него женскому имени Руслана. Прослеживается динамика именования, определяется период его наибольшей популярности в советское и постсоветское время. Отмечается, что имя приобретает определенную семантическую ауру и популярность особенно на Кавказе; оно становится своеобразным маркером российской идентичности, что способствует объединению российского социума и формированию единого антропонимического пространства. Этот вывод подтверждается данными социологического опроса, в котором участвовало около 40 респондентов — представителей разных народов Кавказа. Участники опроса отмечают положительные коннотации имени, ощущают его связь и с исходным тюркским антропонимом Арслан, и с именем пушкинского героя. Ряд респондентов воспринимает имя как «немусульманское», многие авторы анкет оценивают его как русское и подчеркивают, что именно так оно воспринимается за границами России. Культурная история имени Руслан, его трансонимизация представляются перспективными для научного исследования, особенно в условиях новых возможностей интернет-коммуникации и формирования новых ономастических дискурсов.The authors postulate that there exists a common stock of Russian personal names resulting from a partial blending of national anthroponymicons. The main part of the paper focuses on the history of the personal name Ruslan which has etymological ties with the widespread Turkic name Arslan having the pre-onomastic meaning ‘lion’. The authors study the variation of the name in Russian folklore and in the 15th–17th centuries documents and historical sources. They also pay particular attention to the role of Pushkin’s poem Ruslan and Ludmila in the formation of the associative background of the studied name and to various onomastic derivatives, the latter include patronyms, surnames and the female name Ruslana. The author conclude that the name Ruslan became especially popular in Soviet and post-Soviet periods when it acquired a specific “semantic aura”, namely, in Caucasus where Ruslan became a kind of mark of Russian identity and, thus, contributed to the unification of the anthroponymic space. This conclusion was verified in the course of a survey done among 40 respondents representing different peoples of Caucasus. For most respondents the name has positive connotations and is associated with the Turkic name Arslan and the name of Pushkin’s character. However, some respondents consider it as a “non-Muslim”, Russian name and point out that it is often perceived as such outside Russia. The history of the name Ruslan and the ways of its transonymisation can be an interesting object for further research, especially due to the emergence of new communication technologies and onomastic discourses
Geostrategic Competition of Non-State Actors in Africa: The Roman Catholic Church in the Fight for the «Continent of Hope»)
No full textThe Catholic Church of Africa understand the spiritual and material needs of Africans, which allows Catholicism to compete quite successfully with other religions for their souls and hearts. The Vatican takes into account that modern Africa is a continent with a huge and rapidly growing population, facing such serious challenges as global poverty and disease, corruption, economic instability, political crises. This explains the humanitarian focus of the Catholic Church on the continent, which offers Africans, along with spiritual support, educational and medical services. The Catholic hierarchs of Africa and the Vatican face a number of painful issues, the future of Catholicism on the «continent of hope» depends to a large extent on their solution. Africa consists of more than 50 independent states, and the Catholic Church needs to build correct relations with completely different political regimes on the continent – from the extreme left to the right and nationalist. Since the colonial times, the Catholic hierarchy, to a much greater degree than the Protestant churches organized according to the network principle, was distinguished by its political commitment. However, in modern Africa, the Catholic Church must remain above political fights, to affirm its status as an arbiter in conflicts tearing up African society. Despite a completely optimistic picture of the development of Catholicism in Africa, the alarming signal for Catholic hierarchs is the fact that with an increase in the share of Catholics in the population of the continent, since 1970 there has been a decrease in their share in the Christian population. This trend persists for all the largest Catholic countries in Africa. Thus, Protestantism so far wins in the competition with Catholicism for the African flock. One of the most effective ways to strengthen the authority of the Catholic Church remains regular visits of the pontiff to African countries. It is during these visits that the Pope can establish contacts with the governments of the countries at the highest level, as well as with representatives of religious, public circles, cultural and business figures, communicating to them the opinion of the leadership of the Catholic Church on the broadest issues – not only humanitarian, but also socio-political character. Moreover, the visits of the Pope, organized as pilgrimages, are not only of great political, but also religious and emotional significance for the population of the country he is visiting, attracting large masses of people to participate in this event
Fatty acid binding protein 7 regulates beige fat-like differentiation of breast cancer cells and thermogenesis
No full textNew biosensors for metabolic imaging
No full textTissue engineering is a multi-disciplinary field that involves three-dimensional cell and tissue models, live cell microscopy and related imaging modalities, along with fluorescence and phosphorescence-based biosensors. These technologies can work together in developing biologically relevant 3D tissue models for the modelling of complex physiological and diseased states. One of the main challenges facing such models is the lack of non-invasive strategies for quantitative real time monitoring of cellular and tissue physiology, metabolism and viability, that are compatible with live cell microscopy. This thesis presents the design and development of new biosensor, scaffold and nanoparticle materials, with the aim of facilitating quantitative metabolic imaging in cell and tissue culture, via fluorescence lifetime microscopy and phosphorescence lifetime microscopy. Thus, we have developed protein-based biosensor probes, sensitive to pH and calcium in intensity and fluorescence lifetime modalities for the labelling of cellulose scaffold materials, producing a hybrid scaffold material for tissue engineering applications. This was done by genetically engineering of recombinant proteins expressing the cellulose-binding domain (CBD) CenA protein, derived from the fungus C. fimi, fused to pH-sensitive enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), forming CBD-ECFP and CBD-EYFP biosensors, respectively. A third biosensor was also developed with CBD and the genetically encoded calcium indicator known as circularly permutated EGFP (cpEGFP)/M13/Calmodulin (CaM) fusion protein (GCaMP2) forming CBD-GCaMP2. For all three CBD constructs we observed responses in fluorescence intensity to changes in calcium for GCaMP2 and pH for both CBD-ECFP and CBD-EYFP, achieving efficient and stable labelling of various cellulose scaffolds including nanofibrillar, GrowDex, bacterial cellulose and decellularised plant materials. CBD-ECFP labelled GrowDex produced a biosensor scaffold material capable of supporting the growth of 3D cultured human colon cancer cells HCT116, with the ability to measure real-time changes in extracellular pH. The developed labelling strategy allows for the design of biosensor scaffold materials with potential multi-parametric fluorescence lifetime microscopy modalities, which can be used to achieve the controlled production of 3D tissue models with measurable pH and metabolic gradients. Intracellular metabolic imaging is currently dominated by synthetic nanoparticle constructs that suffer from suboptimal intracellular staining, along with high toxicity and immunogenicity. Here we developed several self-assembling protein nanoparticle constructs based on viral like particle, elastin like polypeptide-cowpea chlorotic mottle virus capsid protein (ELP-CP) and protein nanocage ferritin. Such constructs hold promise due to their biological nature making them more biocompatible and biodegradable, thereby reducing toxic and immunogenic effects. Such self-assembling protein nanoparticles are also amenable to multiple strategies of functionalisation such as metallochelate coupling, genetic engineering, chemical modification, and encapsulation. We evaluated metallochelate coupling to design intracellular O2-sensitive biosensors, where oligohistidine-tagged recombinant proteins are bound to nitrilotriacetate (NTA) or iminodiacetic acid (IDA) groups on dyes and small molecules. The NTA or IDA groups form a complex with transition metal ions such as: Zn2+, Ni2+, Co2+, or Cu2+. This complex then co-ordinates to histidine amino acids on the recombinant protein. We successfully produced ratiometric phosphorescent probes from enhanced green fluorescent protein (EGFP), enhanced monomeric blue fluorescent protein 2 (mTagBFP2) and Discosoma red fluorescent protein (DsRed express) coupled to tetracarboxylic platinum (II)-coproporphyrin I (PtCP) PtCP-NTA. Such complexes can be used for ratiometric-based measurements of O2, where fluorescent proteins (FPs) can be used as O2-insensitive references. Most notably we demonstrated the first example of a phosphorescent O2-senstive viral like particle (VLP) structure, ELPCP-H6-PtCP and in comparing to commercial O2-sensitive probe MitoXpress, we observed higher phosphorescence brightness, similar lifetime responses and increased sensitivity in response to O2. The potential to couple a range of FPs or self-assembling protein nanoparticles to O2 sensitive phosphorescent dyes demonstrates that metallochelate coupling is a highly attractive strategy in the design of new intracellular O2 sensors. Using genetic engineering and encapsulation strategies we successful produced both pH and O2- sensitive ferritin nanoparticles. Genetic engineering enabled the expression of multiple cell targeting and penetrating peptides, such as bactenecin 7 and α-enolase, along with fluorescent proteins EGFP or ECFP, without affecting spectral properties of the fluorescent proteins or ferritin self-assembly. Genetic engineered ECFP-FTN construct demonstrated pH sensitivity in fluorescence intensity and lifetime across a physiological range of pH, potentially allowing for applications in fluorescence lifetime microscopy-based measurements of intracellular pH. Through the strategy of pH dependent disassembly and reassembly encapsulation of phosphorescent O2 sensitive probe Pt-Glc, we successfully produced O2-sensitive horse ferritin-based (hoFTN) nanoparticles. The resulting hoFTNPt-Glc displayed a higher phosphorescence intensity signal than free Pt-Glc, possibly due to the concentrated number of Pt-Glc molecules in close proximity within the ferritin structure, and demonstrated responses to oxygenation, increasing phosphorescence intensity when deoxygenated. However, in characterisation of hoFTN-Pt-Glc with MEF cells we observed poor intracellular staining confined to endosomes, similar to free Pt-Glc. These results showed that encapsulation here does not improve intracellular staining or phosphorescence lifetime responses. Despite poor characterisation of ferritin constructs in HCT116 and MEF cell lines, the strategies evaluated here show promise and demonstrate an interchangeable approach to functionalising self-assembling protein nanoparticles and fluorescent proteins for applications in fluorescence lifetime microscopy and phosphorescence lifetime microscopy-based quantitative and ratiometric live cell imaging
Intracellular label-free detection of mesenchymal stem cell metabolism within a perivascular niche-on-a-chip
No full textThe stem cell niche at the perivascular space in human tissue plays a pivotal role in dictating the overall fate of stem cells within it. Mesenchymal stem cells (MSCs) in particular, experience influential microenvironmental conditions, which induce specific metabolic profiles that affect processes of cell differentiation and dysregulation of the immunomodulatory function. Reports focusing specifically on the metabolic status of MSCs under the effect of pathophysiological stimuli - in terms of flow velocities, shear stresses or oxygen tension - do not model heterogeneous gradients, highlighting the need for more advanced models reproducing the metabolic niche. Organ-on-a-chip technology offers the most advanced tools for stem cell niche modelling thus allowing for controlled dynamic culture conditions while profiling tuneable oxygen tension gradients. However, current systems for live cell detection of metabolic activity inside microfluidic devices require the integration of microsensors. The presence of such microsensors poses the potential to alter microfluidics and their resolution does not enable intracellular measurements but rather a global representation concerning cellular metabolism. Here, we present a metabolic toolbox coupling a miniaturised in vitro system for human-MSCs dynamic culture, which mimics microenvironmental conditions of the perivascular niche, with high-resolution imaging of cell metabolism. Using fluorescence lifetime imaging microscopy (FLIM) we monitor the spatial metabolic machinery and correlate it with experimentally validated intracellular oxygen concentration after designing the oxygen tension decay along the fluidic chamber by in silico models prediction. Our platform allows the metabolic regulation of MSCs, mimicking the physiological niche in space and time, and its real-time monitoring representing a functional tool for modelling perivascular niches, relevant diseases and metabolic-related uptake of pharmaceuticals
Protected quantum bits and Josephson junction arrays
No full textIn this thesis we consider a Josephson junction device whose symmetry is described by the point group Td. It can be visualized as a tetrahedron that contains two Josephson junctions on each edge. We find the conditions under which the ground state of the system is degenerate or almost degenerate. In this case, the low-energy degrees of freedom can be mapped to the Hilbert space of a quantum spin 1/2. We evaluate effects of different physical perturbations on the degenerate ground state and find that they are small for most perturbations. We argue that this system can be considered as a very promising candidate for a protected quantum bit with built-in error correction. We propose and discuss an experimental method that allows to test validity of some of the theoretical results obtained for the tetrahedral Josephson junction array and other similar symmetric circuits. We have chosen a simpler pyramidal array to demonstrate the main ideas of our method. Even though the noise resistance and theoretical decoherence time of the pyramidal array are worse than those of the more complex tetrahedral systems, it is much easier to realize the pyramid experimentally. The proposed design can be used with any symmetric Josephson junction circuit. We explore a natural generalization of the tetrahedral quantum bit and consider devices whose symmetry can be described by one of the higher-order permutation groups Sn. We study the level structure and the associated built-in protection of some conceptually simple circuits and show that these circuits have many interesting properties. In particular, their ground state can be highly degenerate and stable with respect to perturbations violating the symmetry. Unfortunately, these highly symmetric systems consist of a large number of identical Josephson junctions. This makes them too complicated for experimental realization.Ph.D.Includes bibliographical referencesIncludes vitaby Ruslan Usmano
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