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Rinderpest virus is an enveloped Nonsegmented Negative Stranded RNA Virus (NNSV) belonging to the genus Morbillivirus in the Family Paramyxoviridae and the causative organism for “cattle plague”. The virion has a transport component and a replication component. The transport component consists of a lipid membrane with two external membrane-anchored glycoproteins, namely Hemagglutinin (H) and Fusion (F) proteins that are necessary for cell entry and release of newly formed virus particles. The replication component consists of viral genomic RNA encapsidated by the nucleoprotein (N) and a RNA polymerase complex (Large subunit L and phosphoprotein P). These two components are linked together by the matrix protein (M) that is believed to play a crucial role in the assembly and maturation of the virion particle by bringing the two major viral components together at the budding site in the host cell. To perform this function, M protein should be able to interact with the host cellular membrane, especially the plasma membrane in the case of Rinderpest virus, should be able to interact with itself to form multimers as well as with the nucleocapsid core. The function might include the interaction of M protein with the cytoplasmic tail of the other two envelope proteins namely F and H. To understand the role of matrix protein in Rinderpest virus life cycle, the following functions were characterized – 1) Matrix protein association with the host cell membrane. 2) Matrix protein association with nucleocapsid protein. Matrix protein association cellular membranes in rinderpest virus infected cells could be a result of its interaction with the cytoplasmic tails of the viral glycoproteins. Hence, this association was characterized in the absence of other viral proteins. In transiently transfected cells, M protein existed in two isoforms namely the soluble cytosolic form and membrane-bound form. The membrane-bound M protein associated stably with the membranes, most likely by a combination of electrostatic and hydrophobic interactions, which is inhibited at high salt or high pH, but not completely. Confocal microscopy analysis showed the presence of M protein in plasma membrane protrusions. When GFP was tagged with this protein, GFP was absent from nucleus and was present predominantly in the cytosol and the plasma membrane protrusions. However, M protein expression did not result in the release of membrane vesicles (Virus-like particles) into the culture supernatant implicating the requirement of other viral proteins in envelope acquisition. Matrix protein of RPV has been shown to co-sediment with nucleocapsid during mild preparation of RNP from virus-infected cells. This association was further investigated by virus solubilization. The matrix protein could be solubilised completely from virion only in the presence of detergent and high salt. This is in agreement with the previous observation from the laboratory that the purified matrix protein remained soluble in the presence of detergent and 1M NaCl. This suggested that M protein could oligomerise or associate with nucleocapsid. The purified M protein when visualized by Electron microscopy showed the presence of globular structures, which may be due to self association of M protein, which may be due to self-aggregation of M protein. The presence of GFPM in filamentous structures in transfected cells, as visualized by confocal microscopy could also be due to self-assembly of M protein. Interaction of matrix protein RPV nucleocapsid was confirmed using co- sedimentation and floatation gradient analysis. Results obtained from M-N binding assay using C-terminal deletions of nucleocapsid protein suggested that the matrix protein interacted with the conserved N-terminal core of nucleocapsid and non- conserved C-terminus 20% is dispensable. This is in agreement with the report that RPV M protein could be replaced with that of Peste-des-petits-ruminants virus(a closely related morbillivirus). The observation that the nucleocapsid protein interacts with both soluble and membrane-bound form suggests that the matrix protein can possibly interact itself to facilitate the assembly of replication component at the site of budding where the transport component is already assembled. Viral proteins of many RNA viruses interact with detergent-resistant host components that facilitate their transport inside the cell to the sits of assembly or replication. Rinderpest viral proteins acquire detergent resistance in infected cells. This acquisition is mediated by viral N protein. The relevance of this interaction in virus life cycle was studied using small molecule drugs that disrupt host cytoskeleton and lipid raft. The results obtained suggested that the host cytoskeleton, especially actin-filaments facilitate virus release from the plasma membrane. RPV matrix protein acquired detergent resistance in infected cells as well as in transfected cells. The pattern of detergent resistance suggested an association with the cytoskeleton or cytoskeleton associated proteins. However, results obtained from co-localisation studies in the presence of actin inhibitor and cold-ionic detergents are not consistent with the above observation. This property could be due to self-association of matrix protein.

Stem cell differentiation is regulated by the complex interplay of multiple parameters, including adhesive intercellular interactions, cytoskeletal and extracellular matrix remodeling, and gradients of agonists and antagonists that individually and collectively vary as a function of spatial locale and temporal stages of development. Directed differentiation approaches have traditionally focused on the delivery of soluble morphogens and/or the manipulation of culture substrates in two-dimensional, monolayer cultures, with the objective of achieving large yields of homogeneously differentiated cells. However, a more complete understanding of stem cell niche complexity motivates tissue engineering approaches to inform the development of physiologically relevant, biomimetic models of stem cell differentiation. The capacity of pluripotent stem cells to simultaneously differentiate toward multiple tissue-specific cell lineages has prompted the development of new strategies to guide complex, three-dimensional morphogenesis of functional tissue structures. The objective of this project was to characterize the spatiotemporal dynamics of stem cell biophysical characteristics and morphogenesis, to inform the development of ESC culture technologies to present defined and tunable cues within the three-dimensional spheroid microenvironment. The hypothesis was that the biophysical and biochemical cues present within the 3D microenvironment are altered in conjunction with morphogenesis as a function of stem cell differentiation stage. Understanding biochemical and physical tissue morphogenesis, including the relationships between remodeling of cytoskeletal elements and intercellular adhesions, associated developmentally relevant signaling pathways, and the physical properties of the EB structure together elucidate fundamental cellular interactions governing embryonic morphogenesis and cell specification. Together, this project has established a foundation for controlling, characterizing, and systematically perturbing aspects of stem cell microenvironments in order to guide the development of complex, functional tissue structures for regenerative therapies.

Stem cell-based therapies are an important developing technology for treating cardiovascular ischemic disease, including subsequent co-morbidities such as ulcerative wounds. Mesenchymal stem cells (MSCs) have a proven ability to augment wound healing and neovascularization processes and have been more recently investigated for their endothelial-like behavior. This doctoral work aims to understand mechanisms underlying matrix-driven MSC tubulogenesis within PEGylated fibrin gels, specifically (1) why this behavior occurs and (2) if this behavior has clinical utility. Briefly, a three-dimensional morphological quantification pipeline was first developed for analyzing the maturity of vascular networks (Chapter 2). This method was applied in later studies that examined the full spectrum of MSC behavior in PEGylated fibrin gels, linking biomaterial properties with network development (Chapter 3). Mechanisms underlying the cell-matrix relationship were more fully clarified through gain-of-function cell studies. These studies indicated that PEGylated fibrin promotes endothelial-like MSC behavior through a combination of hypoxic stress and bioactive fibrin cues (Chapter 4). Notably, this endothelial-like MSC behavior closely mirrored vasculogenic mimicry, a process whereby tumors establish non-endothelialized vasculature in response to hypoxic stress. The functionality of these tumor vessels suggests that mature endothelial differentiation of MSCs may not be necessary to achieve therapeutically beneficial tissue perfusion. This hypothesis opens up new mechanisms for exploitation in vascular tissue engineering strategies. / text

The Shroom family of proteins is currently comprised of four members, Shroom1, 2, 3 and 4. Since Shroom3 was shown to be a critical protein for neural tube closure, the other three proteins are also expected to play an important role for proper development. However, their functions during development were not clear. To address this, my study started with Shroom3 function in the neural plate. Shroom3 had been previously known to induce apical constriction by controlling actin filaments in neuroepithelial cells. My studies show that Shroom3 induces apico-basal cell heightening by controlling parallel microtubule assembly. Shroom3 is able to change the distribution of γ-tubulin, suggesting that Shroom3 controls apical constriction and apico-basal cell elongation via both actin filaments and microtubules. The ability to control γ-tubulin distribution is possessed not only by Shroom3, but also by all other Shroom proteins, although they can not induce apical constriction. In addition, they are expressed in tissues which contain apico-basally elongated cells. Data from functional assays with Shroom2 show that it induces cell elongation and is required for proper cell shape in deep layer neuroepithelial cells in Xenopus. These data suggest that Shroom family proteins control cell architecture during morphogenetic development. I have discovered another role for Shroom2. By comparative analysis with Xenopus and Physalaemus, which have different pigment patterns in eggs, I show that a high level of maternal Shroom2 mRNA is important for pigment polarity in Xenopus. Furthermore, Shroom2 controls the distribution of spectrin which plays a role in pigment granule movement. Thus, Shroom2 is suggested to be a key molecule to control the pigment polarity in amphibian eggs. Together all these data suggest that Shroom family proteins play a role in cell morphogenesis and polarization via controlling the cytoskeleton during Xenopus development. / text

The bacterial cell wall, composed of peptidoglycan (PG), is an essential component of the cell envelope. This macromolecular structure fortifies the cell membrane, determines cell shape, and helps prevent osmotic lysis. The synthesis and remodeling/recycling of this polymer is mediated by PG synthases and hydrolases, respectively. Proper control of the PG hydrolases is particularly important since misregulation of these enzymes can lead to lethal breaches in the cell wall. Surprisingly, however, the precise molecular mechanisms governing the activities of these enzymes remain poorly understood. To help understand how PG hydrolases are regulated, I examined how their activity is controlled during cytokinesis in Escherichia coli. One important class of PG hydrolases necessary for cell division is the LytC-type amidases (AmiA, AmiB and AmiC). These enzymes require activation by the LytM factors EnvC and NlpD. My work focused on elucidating the mechanism by which the LytM factors activate the amidases. Using a genetic enrichment strategy, I isolated amiB misregulation mutants. Interestingly, the mutations mapped to a region of AmiB found only in cell separation amidases. Structural analysis of an AmiB ortholog indicates that this region corresponds to an alpha-helical domain that appears to occlude the active site. Thus, activation of the amidases by the LytM factors likely occurs via a conformational change that displaces the regulatory helix from the active site. In addition to amidase regulation, I also investigated how the LytM activators are recruited to, and regulated at the site of division. Using genetic and biochemical approaches, I showed that EnvC is directly recruited to the division site by FtsEX, an ATP-binding transporter- like complex. Interestingly, ATPase-defective FtsEX derivatives can still recruit EnvC to the divisome, but fail to promote cytokinesis. These results support a model where conformational changes induced by the ATPase activity of FtsE are directly and specifically transmitted to the amidases via FtsX and EnvC. This model is attractive because it provides a mechanism for converting the potentially dangerous activity of septal PG splitting into a discrete process which can be cycled on and off in coordination with the division process.

Patterning, morphogenesis, and cell divisions are distinct processes during development yet are concurrent and likely highly integrated. However, it has been challenging to investigate them as a whole. Recent advances in imaging and labeling tools make it possible to observe live tissues with high coverage and resolution. In this dissertation work, we developed a novel imaging platform that allowed us to fully capture the early neural tube formation process in live zebrafish embryos at cellular resolution. Importantly, these datasets allow us to reliably track single neural progenitors. These tracks carry information on the history of cell movement, shape change, division, and gene expression all together. By comparing tracks of different progenitor fates, we found they show a spatially noisy response to Sonic hedgehog (Shh) and become specified in a positionally mixed manner, in surprising contrast to the "French Flag" morphogen patterning model. Both cell movement and division contribute to cell mixing. In addition, we decoupled the temporal and genetic regulatory network (GRN) noises in Shh interpretation using tracks that carry both Shh signaling and cell fate reporters. Our tracks suggest that, after specification, progenitors undergo sorting to self-assemble a sharp pattern. Consistent with this hypothesis, we found ectopically induced progenitors move to correct locations. Furthermore, we show that proper adhesion is required for cell sorting to happen (Chapters 2 and 3). In the cleavage stage embryos, the cells on the surface undergo shape changes followed by lineage separation and differentiation. We quantitatively measured this morphogenesis process and tracked cell divisions. By applying a mathematical model we uncover a predictive, and perhaps general link between cell division orientation, mechanical interaction, and the morphogenetic behavior of the whole surface layer (Chapter 4). Finally, we discuss the concepts and tools of cell tracking including a multi-color cell labeling method we developed by modifying the "Brainbow" system (Chapter 5). Together this dissertation showcases the importance and promise of live observation based, quantitative and integrated analysis in our understanding of complex multi-cellular developmental processes.

The fascinating capability of cellular self-organization during tissue development and repair is a central question in developmental biology and regenerative medicine. Understanding the dynamic morphogenic and regenerative processes of biological tissues will have important implications in biology and medicine. Nevertheless, the elucidation of the cellular self-organization processes is hindered by a lack of effective tools for monitoring the spatiotemporal gene expression distribution and a lack of ability to perturb the self-organization processes in living cells and tissues. Multimodal modularities that allow both single cell perturbation and gene detection are required to enable a new paradigm in the investigation of complex tissue morphogenic processes. To address this critical challenge in the field of developmental and regenerative medicine, we are developing a multimodal gold nanorod-locked nucleic acid (GNR-LNA) composite for single cell gene expression analysis in living cells and tissues at the transcriptional level. Using antisense RNA sequences, we design LNA probes for detecting specific molecular targets in living cells. The LNA probes bind to the GNR spontaneously due to the intrinsic affinity between the GNR and LNA. In close proximity, the fluorescent probes are effectively quenched by the GNR. Therefore, a fluorescent signal is only observed when the specific target thermodynamically displaces the LNA probe from the GNR. Furthermore, the GNR also serves as a transducer for photothermal ablation. Thus, we established a novel modularity for imaging the spatiotemporal gene expression distribution in living cells and tissues. The single cell analysis capability of our techniques enables us to adopt a unique approach to study the tissue regenerative processes during normal development and diseases, and this will have a profound impact on regenerative medicine and disease treatment in future. Moreover, we applied this GNR-LNA probe to explore the endothelial cell mRNA dynamics during capillary morphogenesis. Three different types of cells were identified due to their different roles during endothelial cell capillary-like formation process. Our findings indicated that the endothelial cell behavior is directly related to the Dll4 mRNA expression, and Dll4 expression in ECs determine the cell fate. Our GNR-LNA probe enable us to investigate the correlations between Dll4 mRNA expression and cell behavior during capillary morphogenesis. Experimental results indicated that: (1) When the endothelial cells aggregate, the cells migrate with certain displacement, the Dll4 mRNA expression decreases. (2) When the endothelial cells sprout, the cells migrate with small displacement but the cell shape changes to an ellipse shape, the Dll4 mRNA expression begin to increase. (3) When the endothelial cells elongate and form cell-cell contract with adjacent cells, the Dll4 expression decreased to a certain level and keep stable until the cell activity change to another stage. Furthermore, it has been demonstrated endothelial cells compete for the leader cell position during wound healing, collective cell migration, and tip cell formation during angiogenic process. It has been demonstrated that endothelial cells compete for the tip cell formation through Notch signaling pathway. However, how the mechanical force regulates tip cell formation is still unclear, and if mechanoregulation of tip cell formation through Notch pathway still unknown. Mechanical and chemical regulations of tissue morphogenesis and angiogenesis are being investigated in both in vitro capillary-like network formation assay and in vivo mice retina angiogenesis assay. Here, we investigated the mechanoregulation of mechanotransduction of tissue morphogenesis and angiogenesis using both in vitro endothelial cell tube formation model and in vivo mice retina blood vessel development model. Our results demonstrated that (1) Notch pathway negatively regulates tip cell formation: inhibition of Notch pathway (DAPT) enhances tip cell formation, induces Dll4 and Notch1 activity, activation of Notch pathway (Jag1 peptide) inhibits tip cell formation, suppresses Dll4 and Notch1 activity. (2) Mechanical force negatively regulate tip cell formation: (a) Decrease mechanical force via Rho kinase inhibitor Y-27632, myosin II inhibitor Blebbistatin, or laser ablation, enhances tip cell formation and induces Dll4 activity through mediation of Dll4-Notch1 lateral inhibition, (b) increase mechanical force via traction force inducer Nocodazole and Calyculin A, suppresses tip cell formation and inhibits Dll4 activity through activation of Notch pathway. (3) Mechanical force negatively regulates tip cell formation partially via mediation of Notch pathway. Mechanical force is necessary for tip cell formation and negatively regulate tip/stalk selection via Dll4-Notch1 lateral inhibition. Interruption of mechanical force enhance tip cell formation via suppression of Dll4-Notch1 lateral inhibition, thus resulting the increase of Dll4 expression. Enhance of mechanical force inhibits tip cell formation via activation of Dll4-Notch1 lateral inhibition, thus resulting the decreases of Dll4 expression. All these finding wills have great significance for various biomedical applications, such as tissue engineering, cancer, and drug screening.

Magistrantūros studijų baigiamajame darbe pateikiama 2011–2012 metais Aleksandro Stulginskio universiteto Agronomijos fakulteto Agrobiotechnologijos laboratorijoje tirtų veiksnių, lemiančių sėjamosios kanapės dediferenciacijos indukciją hipokotilių ir skilčialapių kultūroje, tyrimų rezultatai. Darbo objektas – 10 dienų eksplantai auginti MS terpėje su skirtingais augimo reguliatorių kiekiais. Darbo metodai: Sėjamosios kanapės sėklos plautos po tekančiu vandeniu 1 val., 15 min. sterilintos 0,2 % kalio permanganato tirpale, 2 min. laikytos 70 % etanolio vandeniniame tirpale, 9 min. 10 % natrio hipochlorite ir 3 kartus po 5 min. mirkytos distiliuotame vandenyje. Sterilios sėklos daigintos ant MS maitinamosios terpės be augimo reguliatorių, papildytoje 10 g l-1 sacharozės ir 8 g l-1 Difco-Bacto agaru. MS terpėje su skirtingais augimo reguliatorių kiekiais auginta po 60 eksplantų. Kas keturias savaites eksplantai perkelti į šviežią tos pačios sudėties maitinamąją terpę. Vertintas kaliaus susidarymo dažnis (%), šaknų formavimosi dažnis (%), šaknų kiekis iš eksplanto (vnt.) bei ūglių formavimosi dažnis (%). Darbo rezultatai. Įvertinus sėjamosios kanapės kaliaus indukcijos tyrimų rezultatus nustatyta, kad sėjamosios kanapės izoliuoti hipokotilio audiniai nedideliu dažniu geba formuoti kalių ir terpėje be augimo reguliatorių. Naudojant vienos grupės (auksiną arba citokininą) augimo reguliatorių didžiausias kaliaus formavimosi dažnis gautas terpėje, papildytoje 0,5 mg l-¹ α... [toliau žr. visą tekstą] / The master work presents the results of factors affecting Cannabis sativa L. dedifferentiation process in vitro. Research was investigated at the Laboratory of Agrobiotechnology, Faculty of Agronomy of Aleksandras Stulginskis University in 2011 - 2012. Investigation in vitro was carried out with Cannabis sativa L. hypocotyls and cotyledons explants. Object of the work – 10 days explants grown in MS medium with different growth regulators concentrations. Method of the work – Cannabis sativa L. seeds were washed under running water for 1 h and surface sterilized with 0.2 % potassium permanganate for 15 min. sterilized and for 2 min. in 70 % ethanol, then sterilized in 10 % sodium hypochlorite for 9 min. and rinsed 3 times for 5 min. with sterile distilled water. Sterile seeds sprouted on MS nutrient medium without growth regulators, supplemented with 10 g l-1 sucrose and 8 g l-1 Difco Bacto-agar. MS medium with different growth regulators concentrations were grown 60 explants. Every four weeks, explants were transferred to the same composition fresh medium. The callus formation frequency (%), root formation frequency (%), root number of the explant (units) and the frequency of shoot formation (%) were evaluated. The results of the work – Cannabis sativa L. hypocotyls tissues with low frequency can form callus and without growth regulators in the medium. Using a single group of growth regulators (auxin or cytokinin) highest callus formation frequency obtained in the medium... [to full text]

Magistrantūros studijų baigiamajame darbe pateikiami veiksnių, lemiančių pluoštinių linų morfogenezę in vitro, tyrimų duomenys. Darbo objektas – keturios pluoštinių linų veislės 'Dangiai', 'Sartai', 'Snaigiai' ir 'Vaižgantas'. Darbo metodai. Pluoštinių linų morfogenezei somatinių audinių kultūroje tirti naudoti hipokotilio eksplantai. Eksplantai auginti MS terpėje su skirtingais augimo reguliatorių kiekiais auginimo kambaryje. Vertintas pridėtinių pumpurų formavimosi dažnis (%) ir pridėtinių ūglių kiekis iš eksplanto (vnt.). Darbo rezultatai. Egzogeninių augimo reguliatorių priedas indukcijos terpėje daugeliu atvejų skatino tirtų pluoštinių linų veislių ūglių regeneraciją, tačiau optimali šio junginio koncentracija priklausė nuo genotipo. Iš tirtų pluoštinių linų veislių didžiausia morfogenine galia pasižymėjo veislės 'Vaižgantas' izoliuoti hipokotiliai. Priklausomai nuo genotipo ir maitinamosios terpės sudėties pluoštinių linų pridėtinių pumpurų formavimosi procesas hipokotilių kultūroje vyko 2,87-94,50 % dažniu regeneruojant 1,67-6,00 ūglius iš eksplanto. Efektyviam veislės 'Dangiai' ūglių regeneravimui maitinamąją terpę tikslinga papildyti 2,0 mg l-1 BAP + 0,1 mg l-1 NAR bei eksplantus paveikti 4 °C temperatūra 24 valandas. Veislės 'Sartai' izoliuotų hipokotilių audinių antrinę diferenciaciją skatino 72 valandų 7 °C temperatūros poveikis bei citokinino 1,0 mg l-1 2iP priedas maitinamojoje terpėje. Intensyviausiai veislės 'Snaigiai' eksplantai pumpurus formavo maitinamojoje... [toliau žr. visą tekstą] / The master work presents the results of fibre flax morphogenesis in vitro affecting factors. Object of the research − four fibre flax cultivars 'Dangiai', 'Sartai', 'Snaigiai' and 'Vaižgantas'. Research methods. To study fiber flax morphogenesis of somatic tissues hypocotyls were used as explant. Explants were placed on MS medium supplemented with different combinations of growth regulators and cultivated in growth chamber. The percentage of bud regeneration and the number of shoots per explant were evaluated. Research results. Exogenous growth regulators concentration in induction media increased fiber flax shoots regeneration, and therefore specific combination of growth regulators must be designed for each genotype. Among investigated genotypes, the hypocotyls of cultivars 'Vaižgantas' manifested the best morphogenic capability. Depending on genotype and medium composition fiber flax adventitious buds regeneration frequency varied from 2.87-94.50% and the number of shoots per explant reached 1.67-6.0, respectively. Hypocotyls of cultivar 'Dangiai' showed the best response on medium supplemented by 2.0 mg l-1 BAP with 0.1 mg l-1 NAA and pretreated by 4 °C temperature 24 hours. In the presence of only cytokinins, the highest mean value of regenerated shoots was observed on medium supplemented with 1.0 mg l-1 2iP and 72 hours 7 °C temperature pretreatment for hypocotyls of 'Sartai'. Growth regulators combination 2.0 mg l-1 2iP with 0.1 mg l-1 NAA and treatment by 4 °C... [to full text]