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Developmental Maturation within the Hematopoietic SystemArora, Natasha 04 December 2014 (has links)
Stem cell biologists creating cells and tissues for therapies, disease modeling, and drug screening have observed that differentiating pluripotent stem cells (PSCs) tend to produce cells at an embryonic stage of development but have difficulty maturing into adult definitive cells. A better understanding of developmental maturation will provide insights into embryogenesis and permit more accurate disease modeling. In the hematopoietic system, primitive and definitive cells are distinguished by functional transplantation assays, well characterized cell surface antigens, and gene expression signatures. We examined the transition in vivo in transplanted murine hematopoietic stem cells (HSCs) and in vitro in human PSC (hPSC) derived red blood cells (RBCs). We found that the hematopoietic microenvironment of the recipient significantly affects the outcome of HSC transplantation. The earliest embryonic HSCs perform better in neonatal recipients, whereas more mature adult-like HSCs perform better in adult recipients. The preference may be related to different active hematopoietic niches in neonates and adults, as we observed adult HSCs homing to different tissues in neonatal and adult recipients. Additionally, we found that proliferation may enhance the neonatal engraftment potential of adult-like HSCs. Our data highlight the importance of the host environment on transplantation outcomes, and point to the neonatal transplant model as a tool to functionally examine the earliest HSCs and primitive derivatives of PSCs.
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Development of Delivery Strategies Facilitating Broad Application of Messenger RNA Tumor VaccinePhua, Kyle K.L. January 2014 (has links)
<p>Genetic modification of dendritic cells with plasmid DNA is plagued with low transfection efficiencies because DNA taken up by non-dividing dendritic cells rarely reaches the nucleus. But this difficulty can be overcome by the use of messenger RNA (mRNA), which exerts its biological function in the cytoplasm and obviates the need to enter the nucleus. Since pioneering work of Boczkwoski et al, the ex-vivo application of mRNA-transfected dendritic cells as a vaccine has been evaluated in numerous phase I trials worldwide and is still currently being actively optimized in clinical trials. </p><p> However, a major disadvantage of using mRNA-transfected DCs as a vaccine is that it requires patients to undergo at least one 4-hour leukapheresis procedure, followed by separation of the peripheral blood mononuclear cells (PBMCs), from which monocytes are isolated and cultured for a week in a defined medium with cytokines. The resulting DCs are matured after being loaded with mRNA and frozen for storage. Aliquots are subsequently thawed prior to administration to patients. This process of harvesting, culturing and loading DCs is more time- and resource-intensive than Provenge, the first FDA approved cell based tumor vaccine in 2011.Recent evidence has confirmed a lack of broad translation of Provenge due to complexity and cost of treatment. This predicates a similar fate for mRNA-transfected dendritic cell vaccine going forward. </p><p> This thesis presents alternative delivery strategies for mRNA mediated tumor vaccination. Through the application of synthetic and natural biomaterials, this thesis demonstrates two viable approaches that reduce or eliminate the need for extensive manipulation and cell culture.</p><p> The first approach is the direct in vivo delivery of mRNA encapsulated in nanoparticles for tumor vaccination. A selected number of synthetic gene carriers that have been shown to be effective for other applications are formulated with mRNA into nanoparticles and evaluated for their ability to transfect primary DCs. The best performing formulation is observed to transfect primary murine and human dendritic cells with an efficiency of 60% and 50% (based on %GFP+ cells) respectively. The in vivo transfection efficiency and expression kinetics of this formulation is subsequently evaluated and compared with naked mRNA via various routes of delivery. Following this, a proof-of-concept study is presented for a non-invasive method of mRNA tumor vaccination using intranasally administered mRNA encapsulated in nanoparticles. Results show that intranasally administered mRNA induces tumor immunity only if it is encapsulated in nanoparticles. And anti-tumor immunity is observed in mice intranasally immunized under both prophylactic as well as therapeutic models. </p><p> The second approach evaluates whole blood cells as alternative cell based mRNA carriers. A method is developed to encapsulate intact and functional mRNA in murine whole blood cells. Whole blood cells loaded with mRNA not only include erythrocytes but also T cells (CD3+), monocytes (CD11b), antigen presenting cells (MHC class II) as well as plasmacytoid DCs (CD45R-B220). Mice immunized with mRNA-loaded whole blood cells (intravenously) develop both humoral and cellular antigen-specific immune responses, and demonstrate delayed tumor onset and progression in a melanoma therapeutic immunization model (using tyrosinase related protein -2, TRP-2, as an antigen). Importantly, the therapeutic efficacy of mRNA-loaded whole blood cell vaccine formulation is found to be comparable to mRNA-transfected dendritic cell vaccine.</p><p> In conclusion, this thesis presents new methods to the delivery of mRNA tumor vaccines that reduce or eliminates the need for extensive cell manipulation and culture. Results presented in this thesis reveal viable research directions towards the development and optimization of mRNA delivery technologies that will address the problem of broad translation of mRNA tumor vaccines in the clinics.</p> / Dissertation
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Prions and platelets: a possible role for cellular prion proteinRobertson, Catherine 28 April 2005 (has links)
Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion.
The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity.
In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion.
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The Rational Design of Potent Ice Recrystallization Inhibitors for Use as Novel CryoprotectantsCapicciotti, Chantelle 07 February 2014 (has links)
The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for
hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10%
dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic
both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using
complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in
cryopreservation processes to reduce/eliminate the use of DMSO and glycerol.
Ice recrystallization during cryopreservation is a significant contributor to cellular injury and
reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently,
compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation.
This thesis focuses on the rational design of potent ice recrystallization inhibitors and on
elucidating important key structural motifs that are essential for potent IRI activity. While
particular emphasis in on the development of small molecule IRIs, exploration into structural
features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also
investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products.
Through structure-function studies the influence of (glyco)peptide length, glycosylation and
solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural
AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are
ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation.
One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization.
To understand how these novel small molecules inhibit this process, structure-function studies
were conducted on highly IRI active molecules. These results indicated that key structural
features, including the configuration of carbons bearing hydroxyl groups and the configuration of
the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity.
Finally, various IRI active small molecules were explored for their cryopreservation potential
with different cell types including a human liver cell line (HepG2), HSCs obtained from human
UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were
found to be effective cryo-additives for RBC freezing with significantly reduced glycerol
concentrations (less than 15%). This is highly significant as it could drastically decrease the
deglycerolization processing times that are required when RBCs are cryopreserved with 40%
glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel
cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.
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Effects of red blood cells and shear rate on thrombus growthMehrabadi, Marmar 12 January 2015 (has links)
Thrombosis formation upon rupture or erosion of an atherosclerotic plaque can lead to occlusion of arteries. An occlusive thrombus is the most common cause of clinical events such as angina, myocardial infarction, ischemic attacks and strokes. Occlusive thrombi can cause ischemic cardiac arrest in less than an hour. Thrombosis formation requires rapid platelet accumulation rates exceeding thrombosis lysis and embolization rates. Hemodynamics greatly affects platelet accumulation rate through affecting platelet transport to the surface of a growing thrombus. The presence of red blood cells (RBCs) in blood increases platelet transport rate by several orders of magnitude compared to transport due to Brownian motion. Margination of platelets towards the vessel walls also results in higher platelet concentration at the RBC-depleted layer relative to the bulk. In this thesis, we studied the effects of hemodynamics on thrombus growth. We investigated the effects of important flow and particle properties on margination of particles in RBC suspensions by direct numerical simulation (DNS) of cellar blood flow. We derived a scaling law for margination length. Based on this scaling law, margination length increases cubically with channel height and is independent of shear rate. Using DNS, we verified the proposed scaling law for margination length in straight channels. We also showed that rigidity and size both lead to particle margination. We show that platelet margination can be explained by RBC-enhanced shear-induced diffusion of platelets in the RBC-filled region combined with platelet trapping in the RBC-free region. A simple continuum model is introduced based on the proposed mechanism. Using an experimental correlation for effective diffusivity in blood, the continuum model can recover experimental results from the literature over a wide range of tube diameters. We created an in vitro experimental model of thrombosis with and without RBCs. Surprisingly, we found that rapid thrombus growth does not require enhanced platelet transport in the presence of RBCs at high shear. Instead, our results suggest that thrombus growth rate at high shear is dependent on the availability of vWF-A1 domains as opposed to convective transport of platelets. Finally, we obtained empirical correlations for thrombus growth and lag time based on flow parameters by using an in vitro model of thrombosis. We developed a simple model for predicting thrombus formation using the obtained empirical correlations. We demonstrated the capability of the model in predicting thrombus formation over a wide range of experimental geometries. This model may be useful for designing blood-contacting devices to avoid unwanted thrombosis.
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Prions and platelets: a possible role for cellular prion proteinRobertson, Catherine 28 April 2005 (has links)
Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion.
The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity.
In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion.
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Molecular regulation of Megakaryopoiesis: the role of Fli-1 and IFI16Johnson, Lacey Nicole, St George Clinical School, UNSW January 2006 (has links)
Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.
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Development of a microfluidic system for efficient DNA purification from large-volume blood samples /Wen, Jian. January 2007 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.
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Effect of isolated red blood cell membrane proteins on cpmplement lysis /Nuanthip Kamolvarin. January 1978 (has links) (PDF)
Thesis (M.Sc. Biochemistry)--Mahidol University, 1978. / Supported by the World Health Organization.
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Υπολογισμός γεωμετρικών διαστάσεων ερυθρών αιμοσφαιρίων με επεξεργασία ψηφιακής εικόνας σκεδασμένης ακτινοβολίαςΠάλλα, Ελένη 19 January 2010 (has links)
Η διπλωματική εργασία περιγράφει μια μέθοδο επίλυσης του προβλήματος προσδιορισμού των γεωμετρικών χαρακτηριστικών ανθρώπινων ερυθρών αιμοσφαιρίων από προσομοιωμένες εικόνες σκέδασης ΗΜ ακτινοβολίας He-Ne laser 632.8nm. Αρχικά παρουσιάζεται το ευθύ πρόβλημα σκέδασης ΗΜ ακτινοβολίας από ανθρώπινο ερυθρό αιμοσφαίριο και στη συνέχεια το αντίστροφο πρόβλημα επιλύεται με χρήση τεχνικών συμπίεσης εικόνας και τεχνητού νευρωνικού δικτύου ακτινικής συνάρτησης. Τέλος, αναπτύσσεται μια τεχνική εύρεσης των αναλογιών των ερυθρών αιμοσφαιρίων στις εικόνες σκέδασης. / This thesis describes a method of estimating the geometrical features of the human red blood cell, from a set of simulated light scattering images produced by a
He-Ne laser beam at 632.8nm. The light scattering problem by a human RBC is presented and afterwards the inverse problem is solved using image compression
techniques and a radial basis function neural network. Finally, a method of finding the ratio of RBCs in the scattering images is developed.
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