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1
Copeman, M. C. "Alterations to the extracellular matrix in neoplasia : a study in suppressed and tumorigenic hybrid cells." Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233450.
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2
Hess, Patricia M. "Role of c-Jun NH-terminal Kinase in Bcr/Abl Induced Cell Transformation: a dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/88.
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The c-Jun NH2-terminal kinase (JNK) group of kinases include ten members that are created by alternative splicing of transcripts derived from Jnk1, Jnk2 and Jnk3 genes. The JNK1 and JNK2 protein kinases are ubiquitously expressed while JNK3 is expressed in a limited number of tissues. The JNK signaling pathway is implicated in multiple physiological processes including cell transformation. There is growing evidence that JNK signaling is involved in oncogenesis. Nevertheless, the role that JNK plays in malignant transformation is still unclear. The aim of this thesis is to examine the role of JNK in malignant transformation. For this purpose, I used the Bcr/Abl oncogene as a transforming agent. Bcr/Abl is a leukemogenic oncogene that is created by reciprocal translocation between chromosome 9 and 22. The translocation breakpoint is variable and several different Bcr/Abl isoforms have been identified such as Bcr/AblP185 and Bcr/AblP210, whose expression is associated with different types of leukemia. Bcr/Abl activates the JNK signaling pathway in hematopoietic cells and increases AP-1 transcription activity. Furthermore, dominant negative approaches demonstrate that inhibition of c-Jun or JNK prevents Bcr/ Abl-induced cell transformation in vitro. These data implicate the JNK signaling pathway in Bcr/Abl transformation although the role that JNK might have in this process is unclear. Thus, I examined the importance of JNK signaling in Bcr/Abl-induced lymphoid or myeloid transformation. For this purpose I compared Bcr/AblP185- and Bcr/AblP210- induced transformation of wild-type and JNK1-deficient cells using three approaches: in vitro, in vivo and ex vivo. The results obtained with the in vitro approach suggest that both Bcr/AblP185 and Bcr/AblP210 require JNK activity to induce lymphoid transformation. While JNK1-deficiency inhibits Bcr/AblP210 oncogenic potential in lymphoid cells both in vitro and in vivo, pharmacological inhibition of JNK activity (JNK1 and/or JNK2) blocked Bcr/AblP185 induced malignant proliferation in vitro. The differential requirement for JNK observed in the two Bcr/Abl isoforms can be ascribed to the presence in Bcr/AblP210 of the Dbl domain which can activate the JNK pathway in vitro. In the case of Bcr/AblP210, JNK1 is critical for the survival of the ex vivo derived transformed lymphoblasts upon growth factor removal. This result correlates with the fact that mice reconstituted with Bcr/AblP210 transformed Jnk1-l- bone marrow showed normal malignant lymphoid expansion in the bone marrow yet they had reduced numbers of lymphoblast in the bloodstream and lacked peripheral organ infiltration. Thus JNK1 is essential for the survival of the transformed lymphoblast outside the bone marrow microenvironment in Bcr/AblP210induced lymphoid leukemia. Interestingly, while JNK1 is essential for lymphoid transformation, it is dispensable for the proliferation of transformed myeloblasts.
Taken together these results indicate that the JNK signaling pathway plays an essential role in the survival of Bcr/AblP210 lymphoblasts and that JNK-deficiency decreases the leukomogenic potential of Bcr/AblP210 in vivo. Thus, cell survival mediated by JNK may contribute to the pathogenesis of proliferative diseases.
3
CHAMBI, ROSA M. C. "Proteínas de fusão endostatina-peptídeos com atividade apoptótica: expressão e estudo de atividade antiangiogênica." reponame:Repositório Institucional do IPEN, 2009. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10166.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
4
Bardsley, David William. "Electroacoustic cell fusion." Thesis, Cardiff University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305186.
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5
Heilman, Susan Ann. "Cooperative Oncogenesis and Polyploidization in Human Cancers: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/327.
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A common phenotype observed in most cancers is chromosomal instability. This includes both structural and numerical chromosomal aberrations, which can promote carcinogenesis. The fusion gene CBFB/MYH11 is created by the structural chromosomal inversion(16)(p13.1q22), resulting in the fusion protein CBFβ-SMMHC, which blocks differentiation in hematopoietic progenitor cells. This mutation alone, however, is not sufficient for transformation, and at least one additional cooperating mutation is necessary.
The role of wildtype Cbfb in modulating the oncogenic function of the fusion protein Cbfβ-SMMHC in mice was examined. Transgenic mice expressing the fusion protein, but lacking a wild-type copy of Cbfb, were created to model the effects of these combined mutations. It was found that wild-type Cbfb is necessary for maintaining normal hematopoietic differentiation. Consequently, complete loss of wild-type Cbfb accelerates leukemogenesis in Cbfb/MYH11 mice compared to mice expressing both the fusion and wild-type proteins. While there is no evidence in human patient samples that loss of wild-type Cbfb expression cooperates with the fusion protein to cause transformation, it is apparent from these experiments that wild-type Cbfβ does play a role in maintaining genomic integrity in the presence of Cbfβ-SMMHC. Experiments have also shown that loss of Cbfb leads to accumulation of hematopoietic progenitor cells, which may acquire additional cooperating mutations.
Not unlike CBFB/MYH11, the human papillomavirus (HPV) E6 and E7 proteins are not sufficient for cellular transformation. Instead, high risk HPV E7 causes numerical chromosomal aberrations, which can lead to accumulation of additional cooperating mutations. Expression of HPV-16 E7 and subsequent downregulation of the retinoblastoma protein (Rb) has been shown to induce polyploidy in human keratinocytes. Polyploidy predisposes cells to aneuploidy and can eventually lead to transformation in HPV positive cells.
There are several possible mechanisms through which E7 may lead to polyploidization, including abrogation of the spindle assembly checkpoint, cleavage failure, abrogation of the postmitotic checkpoint, and re-replication. Rb-defective mouse and human cells were found to undergo normal mitosis and complete cytokinesis. Furthermore, DNA re-replication was not found to be a major mechanism to polyploidization in HPV-E7 cells upon microtubule disruption. Interestingly, upon prolonged mitotic arrest, cells were found to adapt to the spindle assembly checkpoint and halt in a G1-like state with 4C DNA content. This post-mitotic checkpoint is abrogated by E7-induced Rb-downregulation leading to S-phase induction and polyploidy.
This dissertation explores two examples of the multi-step pathway in human cancers. While certain genes or genetic mutations are often characteristic of specific cancers, those mutations are often not sufficient for transformation. The genetic or chromosomal abnormalities that they produce often stimulate the additional mutations necessary for oncogenesis. The studies with Cbfb/MYH11 and HPV E7 further exemplify the significance of numerical and structural chromosomal aberrations in multi-step carcinogenesis.
6
Lichius, Alexander. "Cell fusion in Neurospora crassa." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/7561.
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The primary research aims of this thesis were the identification of novel cell fusion mutants of Neurospora crassa and the subsequent functional characterization of selected candidate proteins during conidial anastomosis tube (CAT)-mediated cell fusion by means of genetic, molecular, biochemical and live-cell imaging analysis. Chapter 1 provides a general introduction of the model organism and the cell fusion processes studied during different stages of the fungal lifecycle. Chapter 2 summarizes the materials and methods used. Chapter 3 introduces the comparative genomics screen conducted between appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae and hyphal fusion in Neurospora crassa. Novel cell fusion mutants were identified in MAP kinase signalling, redox-signalling and Rho-type GTPase signalling pathways, whereas no functional overlap in the cAMP response pathway between both species could be found. Chapter 4 demonstrates how newly developed fluorescent reporters for F-actin and activated Rho GTPases in filamentous fungi lead to novel insights into the dynamic rearrangement of the F-actin cytoskeleton and cortical activation of Rho GTPases during cell symmetry breaking, polarized tip growth and cell fusion. Chapter 5 focuses on the role of the cell wall integrity (CWI) MAP kinase pathway during cell fusion, and in particular, on the function of the terminal MAP kinase MAK-1 during CAT homing and fusion pore formation. Inhibitor studies indicated that MAK-1 kinase activity is required for its own recruitment to the fusion site already during homing and for cell wall remodelling during fusion pore enlargement between interacting cells. Chapter 6 presents ultrastructural scanning electron microscope (SEM) studies which indicate that defects in hyphal attachment, extracellular matrix deposition and cell wall remodelling prematurely abort morphogenesis of the female fruitbody. These findings are put into context with defects observed in mutants of components acting in related signalling pathways which appear to regulate non-self fusion events at later stages of sexual development leading to fertilization in N. crassa. Chapter 7 provides the first evidence for a role of NADPH-oxidase (NOX)-generated reactive oxygen species (ROS) in the regulation of morphogenetic changes required for CAT-mediated cell fusion. Redox-modification of signalling proteins might be involved in cell-cell chemoattraction. Chapter 8 provides a summary of the key findings and discusses future directions.
7
Manson, Graham. "Electrofusion of cells : development of a fusion apparatus and a protocol for cell fusion." Thesis, University of Aberdeen, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297646.
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Design and construction of an apparatus to provide signal sources for electrofusion of cells is described. Experiments were performed with the apparatus on mammalian human cells and plant protoplasts to derive the best protocols to achieve alignment and fusion. Four versions of the apparatus were constructed with modifications being determined by both the results of the cell experiments and electronic experiments on circuit design. The protocols to be followed to achieve fusion of cells in different media were confirmed by experiment, and the signal on the cell was mathematically analysed. Using the results of this analysis, an improved protocol was produced for achievement of cell fusion in binary or multiple cell clusters in various suspension media, by manipulation of electrical signals. Suggestions are made for circuit construction using new integrated circuit elements and microcontrollers, with machine operation and information logging being directed by a personal computer.
8
Côté, Marceline. "Fusion and cell entry by oncogenic sheep retroviruses." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66893.
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Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumour virus (ENTV) are two highly related oncogenic retroviruses that induce a contagious cancer in sheep and goats respectively. Both JSRV and ENTV use hyaluronidase-2 (Hyal2) as a cell entry receptor, yet JSRV induces lung tumours and ENTV causes tumours in the nasal epithelium. Unlike most acutely transforming retroviruses, the genomes of JSRV and ENTV do not contain an oncogene derived from the host cell; instead, the viral envelope protein (Env) functions as an active oncogene in addition of mediating cell entry. JSRV and ENTV Env are first synthesized as precursors that are cleaved by a cellular protease into two functional subunits: the surface (SU) subunit that contains the receptor binding domain and the transmembrane (TM) subunit that mediates membrane fusion. While most of previous studies have focused on the oncogenic properties of these proteins, little was known about how they mediate membrane fusion and viral entry. The goal of my Ph.D study was to investigate the mechanisms of fusion and cell entry by JSRV and ENTV Env as well as their regulations. We found that, although most retroviruses are believed to use a pH-independent pathway for entry, JSRV requires an acidic pH for entry and fusion and that its fusogenicity was negatively regulated by its cytoplasmic tail. Unexpectedly, ENTV Env requires an unusually low pH (<4.5) for fusion activation. While an irreversible inhibitor, Bafilomycin A1, which prevents acidification in the endosomes and lysosomes inhibited entry of JSRV and ENTV, ENTV Env-mediated infection was considerably enhanced in the presence of lysosomotropic agents or leupeptin, suggesting that JSRV and ENTV likely fuse in distinct cellular compartments. Importantly, we found that SU also modulates the fusion activity of JSRV and ENTV Env, despite that TM dictates the differential pH requirements between JSRV and ENTV.Le virus JSRV (Jaagsiekte sheep retrovirus) et ENTV (enzootic nasal tumor virus) sont deux rétrovirus oncogéniques apparentés qui induisent un cancer contagieux chez le mouton et la chèvre respectivement. Le récepteur utilisé lors de l'entrée de JSRV et d'ENTV dans la cellule cible est la protéine cellulaire hyaluronidase-2 (Hyal2). Cependant, alors qu'ils reconnaissent le même récepteur à la surface de la cellule, JSRV cause la formation de tumeurs au poumon tandis qu'ENTV induit l'apparition de tumeurs nasales. À l'opposé de la plupart des rétrovirus oncogéniques causant rapidement la formation de tumeurs, les génomes du JSRV et d'ENTV ne contiennent pas d'oncogène dérivé de la cellule hôte. Étonnamment, la protéine virale d'envelope (Env) joue un rôle d'oncogène actif en plus d'assurer ses fonctions durant l'entrée virale. Les Envs du JSRV et d'ENTV sont d'abord synthétisées sous forme de précurseurs qui seront éventuellement clivés dans l'appareil de golgi par une protéase cellulaire en ses deux sous-unités fonctionnelles : la sous-unité de surface (SU), qui contient le domaine de liaison au récepteur, et la sous-unité transmembranaire (TM) qui possède l'activité fusogénique. Alors que la plupart des études sur le JSRV et l'ENTV se concentrent sur les propriétés oncogéniques d'Env, les mécanismes par lesquels Env accomplit l'entrée virale et provoque la fusion de la membrane virale et la membrane cellulaire demeurent inconnus. Le but de ce projet de doctorat était d'étudier les mécanismes d'activation de la fusion ainsi que de mieux comprendre leur régulation. Alors qu'en principe la majorité des rétrovirus entrent dans la cellule via la fusion à la surface de la cellule à pH neutre, notre étude démontre que JSRV requiert un pH acide pour l'entrée et la fusion virale. De plus, l'activité fusogénique d'Env est régulée négativement par sa queu
9
Wong, Wing-sze, and 黃詠詩. "Fusion genes in non-small cell lung cancer." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43781378.
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10
Paterson, David Archibald. "Human cytomegalovirus glycoprotein H complex and cell fusion." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271225.
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11
Wong, Wing-sze. "Fusion genes in non-small cell lung cancer." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43781378.
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12
Joshi, Supriya. "Role of membrane fusion protein Ykt6 in regulating epithelial cell-cell and cell-matrix adhesions." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3350.
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Intercellular junctions and cell-matrix adhesions play important roles in the maintenance of epithelial integrity. Assembly and remodeling of the plasma membrane complexes are regulated by membrane trafficking and fusion. This thesis is aimed to elucidate the roles of an important membrane fusion protein, Ykt6, in the regulation of epithelial cell adhesion and migration. For the first time, we show that Ykt6 is essential for assembly of adherens junctions and tight junctions in human prostate epithelial cells. We also observed that Ykt6 negatively regulates both collective epithelial cell migration and cell invasion into Matrigel. The effects of YKT6 on epithelial junctions involves expressional regulation of key junctional proteins, E-cadherin and claudin-4, whereas its effects on cell motility can be explained by antagonizing functions of junctional adhesion molecule-A. Overall, this study identifies YKT6 as a novel regulator of epithelial cell adhesions and motility.
13
Symeonides, Menelaos. "HIV-1-Induced Cell-Cell Fusion: Host Regulation And Consequences For Viral Spread." ScholarWorks @ UVM, 2016. https://scholarworks.uvm.edu/graddis/589.
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Human immunodeficiency virus type 1 (HIV-1) is a human retrovirus of the lentivirus subgroup which primarily infects T cells and macrophages, and causes acquired immune deficiency syndrome (AIDS). Since its emergence in the early 1980s, HIV-1 has caused a global pandemic which is still responsible for over one million deaths per year, primarily in sub-Saharan Africa.
HIV-1 has been the subject of intense study for over three decades, which has resulted not only in major advances in cell biology, but also in numerous drug treatments that effectively control the infection. However, cessation of treatment always results in reemergence of the infection due to the ability of HIV-1 (and other lentiviruses) to establish a persistent quiescent infection known as latency. The elimination of latently-infected cells is the primary goal of current research towards a cure for HIV-1, alongside efforts to develop vaccines, which have thus far been fruitless.
The spread of HIV-1 to susceptible target cells (which express the receptor CD4 and a co-receptor; CXCR4 or CCR5) can take place when antigen-presenting cells, such as dendritic cells, capture virus particles and then pass them on to target cells, without themselves becoming infected. Alternatively, productively infected T cells or macrophages can spread HIV-1 either by shedding virus particles to the milieu, which are then stochastically acquired by target cells, or through transient contacts between infected and uninfected cells known as virological synapses (VSs). VS-mediated cell-to-cell transmission is thought to be highly efficient due to the release of virus directly onto (or very near to) a target cell, and some evidence suggests that the VS is a privileged site which allows the virus to evade neutralizing antibodies and drugs. However, and most importantly, it is of central interest to us because the same transient cell adhesions that facilitate virus transfer can also result in the fusion of the two cells to form a syncytium, due to the presence of the viral fusogen Env and its receptor and co-receptor on either side of the VS. While T cell syncytia can be found in vivo, they remain small, and it appears that the majority of VSs resolve without fusion.
The regulation of HIV-1-induced cell-cell fusion and the fate of those syncytia are the focus of the work presented here. A family of host transmembrane proteins, the tetraspanins, which regulate cell-cell fusion in other contexts (e.g. the fusion of myoblasts to form and maintain myotubes), were found to inhibit HIV-1-induced cell-cell fusion. Our investigations have further characterized this regulation, concluding that tetraspanins allow cells to reach the fusion intermediate known as hemifusion before their ability to repress fusion takes effect. In parallel, because syncytia are nevertheless found both in infected individuals and in a humanized mouse model for HIV-1, we also became interested in whether small T cell-based syncytia were able to participate in HIV-1 spread by transmitting virus to target cells. Using a simple three dimensional in vitro culture system which closely recapitulates those in situ observations, we found that small syncytia can contact target cells and transmit virus without fusing with them. Overall, these studies further our understanding of HIV-1-induced syncytia and reveal a previously unrecognized role for these entities as active participants in HIV-1 spread.
14
Barkley, Russell. "Investigation of an Oncolytic MeV Cell-Cell Fusion Phenomenon Induced by an siRNA." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41531.
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Oncolytic measles virus is a promising cancer therapeutic in clinical trials which
possesses multiple characteristics that are advantageous over traditional therapies.
Currently, clinical oncolytic measles virus vectors are unmodified or express reporter
transgenes that benefit its therapeutic efficacy. The next phase in its development will
see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.
15
Fernandes, Jyothi. "The use of electrical charge to produce cell-cell contact prior to electrofusion." [Tampa, Fla] : University of South Florida, 2005. http://purl.fcla.edu/usf/dc/et/SFE0001251.
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16
Flear, Andrea Karen. "The cytoskeleton during muscle cell fusion in early myogenesis." Thesis, University of Oxford, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236303.
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17
Chartier-Hollis, J. M. "Cell fusion for the transference of CMS in petunia." Thesis, University of Nottingham, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303999.
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18
Hromowyk, Kimberly. "Genetic analysis of skeletal muscle cell fusion in zebrafish." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512114979019823.
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19
Santos, Denise Takehana dos. "Mapeamento topográfico metabólico de carcinomas espinocelulares de cabeça e pescoço utilizando a fusão de imagens 18 F-FDG PET - TC." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/23/23139/tde-09082005-124541/.
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O objetivo desta pesquisa foi estabelecer uma metodologia para avaliar carcinomas espinocelulares (CEC) de cabeça e pescoço, identificando e distinguindo áreas de maior atividade metabólica dentro da neoplasia, associando dados simultaneamente adquiridos, obtidos por diferentes modalidades de aquisição de imagens, combinando informações metabólicas e anatômicas num único exame. A população estudada consistiu de 17 pacientes com carcinoma espinocelular (CEC) de cabeça e pescoço pertencentes aos arquivos do Departamento de Imagem do Hospital do Câncer, São Paulo. As imagens de TC (tomografia computadorizada) e do 18 F-FDG-PET (tomografia por emissão de pósitrons) foram simultaneamente adquiridas utilizando um aparelho não dedicado. Os dados originais foram transferidos para uma estação de trabalho independente com programa de computação gráfica para o processamento dos grupos individuais e fusão em um único grupo, contendo os dados fisiológicos e metabólicos. Os achados foram definidos como positivos na presença de focos com aumento da concentração do radiofármaco em áreas não relacionadas à distribuição normal do mesmo. Em 77% dos casos (n=13), a hipercaptação foi detectada ao centro da lesão e em 23% (n=4) dos casos houve comportamento diferente, com hipercaptação excêntrica. A fusão de imagens simultaneamente adquiridas num único exame ( 18 F- FDG PET e TC) possibilitou o mapeamento topográfico metabólico das lesões estudadas e foi possível localizar áreas de maior atividade metabólica dentro do próprio 13 tumor, verificando recidivas ou metástases, possibilitando aumentar as opções quanto ao planejamento radioterápico ou cirúrgico a serem seguidos.The aim of this study is to propose a methodological approach to evaluate head and neck squamous cell carcinoma (SCCA) in order to identify and to distinguish areas of higher metabolic activity inside the lesion combining the functional metabolic and morphological data simultaneously acquired in a non dedicated PET-CT device. The study population consisted of 17 patients, with SCCA of the head and neck carcinoma. These patients were submitted to a non-dedicated 18 F- FDG- PET imaging using a system with low dose CT and Positron emission coincidence acquisition capabilities. The image acquisition was then transferred to an ENTEGRA 2 NT workstation to generate groups of individual images (metabolic and anatomical data) and image fusion (CT + PET). In those patients with anomalous concentrations of 18 F-FDG, the lesion was depicted on three planes (axial, coronal and sagittal) in CT, PET, and the image fusion at the computer screen. The findings were defined as positive in the presence of well-defined focal area of increased uptake in regions unrelated to the normal biodistribution of the tracer on visual inspection. Two examiners interpreted the images in different sessions, in order to get an agreement. Subsequently, the sites of higher metabolic activity inside the tumor were identified and classified in centric or eccentric, according to their relative location. Observing the images, we found 77.00% of the patients with the site of higher activity at the center of lesion. In 23.00% of the patients a different 15 behavior, with the tracer increased eccentrically to the lesion. This technique gave a realistic view of the functional metabolism, locating the anatomical tumor area and helping in future treatment planning.
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Bracq, Lucie. "Analysis of HIV-1 cell-to-cell transfer to macrophages." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB063/document.
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Les macrophages sont une cible particulièrement importante de l’infection par le VIH-1 et jouent un rôle crucial dans la physiopathologie de l’infection. Lorsqu’ils sont infectés, leur capacité de survie dans les tissus leur permet de jouer un rôle essentiel dans la dissémination virale et l’établissement de réservoirs viraux au niveau des différents territoires tissulaires. In vitro, les étapes précoces et tardives du cycle de réplication virale dans les macrophages ont été analysées dans le cadre de l’infection par des virus libres. Cependant, les modalités d’infection des macrophages lors d’une transmission intercellulaire reste largement inexplorées. Les travaux présentés ici ont permis d’établir un modèle de transmission intercellulaire du VIH-1 de lymphocytes T infectés vers les macrophages. Nous avons montré que les lymphocytes T infectés sont capables d’interagir étroitement avec les macrophages, conduisant ainsi à la fusion cellulaire de ces deux cellules et permettant le transfert de matériel viral dans les macrophages cibles. Ce transfert viral par fusion cellulaire, rapide et efficace, est restreint aux virus utilisant le corécepteur CCR5 et dépend de l’interaction entre l’enveloppe virale et le récepteur CD4. Les virus transférés sont alors stockés au sein de compartiment cytoplasmique des cellules fusionnées mais nous observons également des évènements précoces d’assemblage et de bourgeonnement du VIH-1 à la membrane plasmique des cellules fusionnées résultant de la fusion des membranes des lymphocytes T infectés et des macrophages cibles. Ces cellules fusionnées acquièrent alors la capacité de fusionner avec les macrophages non infectés environnants permettant la dissémination du VIH-1. L’ensemble de ces résultats met en évidence un nouveau mécanisme de transmission intercellulaire entre lymphocytes T et macrophages via un mécanisme de double fusion cellulaire dépendant de l’enveloppe virale et des récepteurs CD4 et CCR5. Ces évènements successifs de fusion entre lymphocytes T et macrophages puis entre macrophages permettent la formation de cellules géantes multinucléés capables de produire de grande quantité de virus infectieux. Ces cellules multinculées pourraient correspondre aux macrophages multinuclées observés in vivo dans les organes lymphoïdes et le système nerveux central de patients infectés par le VIH-1 ou de singes infectés par le SIV. Ce mécanisme représente donc un modèle de transmission intercellulaire original permettant la dissémination virale et la formation de macrophages réservoirs durant l’infection par le VIH-1Macrophages are important targets of HIV-1 and play crucial roles in physiopathology of infection. Because of their long time survival capacity, infected macrophages participate in virus dissemination and establishment of persistent virus reservoirs in numerous tissues. In vitro, macrophages infection and analysis of the different steps of the virus cycle have been largely documented using cell-free virus infection. However, there is a paucity in knowledge of the mechanisms that control infection and dissemination to macrophages by cell-to-cell transfer. In the work presented here, we establish a model of HIV-1 cell-to-cell transfer from infected T cells to macrophages. We observed that infected T cells are able to interact with macrophages leading to cell fusion for transfer of viral material to macrophages targets. This cell-to-cell fusion transfer, very fast and efficient, is restricted to CCR5-tropic viruses, and mediated by viral envelope-receptor interactions. Transferred viruses can then accumulate in cytoplasmic compartments of newly lymphocyte/macrophages fused cells but we also observed early viral assembly and budding events at the plasma membrane of these fused cells, resulting from the merge of viral material between infected T cells and macrophages. These cells then acquire the ability to fuse with neighboring non-infected macrophages for virus dissemination. Together, these two-sequential envelope-dependent cell fusion process lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in vivo in lymphoid organs and the central nervous system of HIV-1 infected patients and simian immunodeficiency virus-infected macaques. These mechanisms may represent an original mode of virus transmission for viral spreading and formation of macrophage virus reservoirs during HIV-1 infection
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Rodrigues, Frade João Manuel 1988. "Ploidy reduction determines the fate of reprogrammed cell fusion hybrids." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/565684.
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Fenómenos
de
fusión
celular
heterotípica
están
asociados
con
regeneración
de
tejidos
en
mamíferos.
La
reprogramación
de
células
somáticas
a
estadios
de
células
madre
progenitoras
o
pluripotentes,
a
través
de
fusión,
tiene
posibles
aplicaciones
en
la
medicina
regenerativa.
Sin
embargo,
originar
células
tetraploides
por
fusión
lleva
a
consecuencias
que
deben
ser
caracterizadas
antes
de
que
esta
estrategia
sea
aplicada
in
vivo.
En
este
trabajo,
hemos
estudiado
híbridos
reprogramados
formados
por
fusión
in
vitro
entre
células
madre
embrionarias
y
progenitores
neuronales
de
ratón.
Demostramos
que
después
de
la
fusión,
híbridos
tetraploides
reprogramados
han
podido
reducir
su
ploidía
a
través
de
una
mitosis
tripolar
reductiva.
Este
proceso
resulta
en
dos
células
diploides
y
una
tetraploide.
Los
análisis
de
cariotipo
y
de
microscopia
indican
que
la
mitosis
tripolar
ocurre
sin
perdida
de
cromosomas
y
sin
muerte
de
las
células
hijas.
Además,
los
polimorfismos
de
un
solo
nucleótido
y
la
marcación
de
cromosomas
han
mostrado
que
la
segregación
de
cada
cromosoma
durante
la
mitosis
tripolar
depende
de
su
célula
de
origen.
Así,
proponemos
que
la
segregación
de
cromosomas
puede
no
ser
aleatoria
y
así
producir
células
“hijas”
diploides
con
un
contenido
cromosómico
euploide.
Esta
observación
puede
llevar
a
nuevas
aplicaciones
de
la
reprogramación
por
fusión
celular
en
la
reparación
de
tejidos
y
a
un
mejor
entendimiento
de
cómo
la
mitosis
de
células
tetraploides
es
regulada.Heterotypic cell fusion has been implicated in tissue regeneration in mammals. Reprogramming of somatic cells to progenitor or pluripotent stem cell states by cell fusion has possible applications in regenerative medicine. However, the consequences of generating tetraploid cells need to be carefully addressed before further applying this approach in vivo. Here, we studied the fate of reprogrammed hybrids formed after fusion of mouse embryonic stem cells and neuronal progenitor cells in vitro. We showed that, after fusion, reprogrammed tetraploid hybrids could reduce their ploidy through reductive tripolar mitosis. This originated two diploid cells and one tetraploid cell. Single cell karyotype analysis and time-‐lapse live microscopy revealed that tripolar mitosis occurred with no apparent chromosome loss or subsequent daughter cell death. Furthermore, single nucleotide polymorphism genotyping and chromosome targeting revealed that chromosomes segregated in a cell-‐of-‐origin-‐dependent fashion during tripolar mitosis. We therefore propose that chromosome segregation can be non-‐random and thus produces diploid daughter cells with a defined euploid chromosomic content. Overall, this novel observation may contribute to the application of cell fusion-‐mediated reprogramming in tissue repair and leads to a better understanding of how mitosis is regulated in tetraploid cells.
22
Hsieh, Hsiang Chuan. "Checkpoint modulation of T cell immunity by novel fusion cytokines." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121154.
Full textAbstract:
Functional immunity requires a balanced T cell immune response, which entails the maintenance of de novo production (i.e. TCR repertoire diversity) and the appropriate differentiation of effector subsets at the periphery. However, numerous pathogenic changes can perturb this homeostasis. On the one hand, diminished thymopoiesis or exhausted effectors cause immune dysfunction, leading to the persistence of virally infected or cancerous cells. Unrestrained immune reaction, on the other hand, can cause significant tissue damage. The main objective of my thesis therefore was to develop novel therapeutics to modulate T cell immunity in the context of cancer and inflammatory diseases. Interleukin-7 (IL7) is critically involved in T cell development and homeostatic proliferation. In order to pharmacologically induce T cell neogenesis for immune reconstitution and cancer therapy, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7 (GIFT7). GIFT7 administration in aged mice led to cortical hyperplasia, effectively reversing tissue involution. GIFT7-mediated hypertrophic effect includes an increase in total thymic cellularity and more importantly a 4-fold increase in the number of CD4-CD8-CD44intCD25-early thymic precursors. In the periphery, GIFT7 selectively expand a CD8+subset from pre-activated T cells with a phenotype defined as CD8+CD44+CD62L+CCR7+KLRG-CD27+, hereafter TGIFT7. The adoptive transfer of OTI-derived CD8 TGIFT7 into OVA-EG7-bearing mice leads to significant tumor regression. Furthermore, the human ortholog of GIFT7 (hGIFT7) leads to a two-fold increase in total cell number after 3 days and >80% of Ki67+expression in both CD4+ and CD8+ PBMC with concurrent reduction in PD1 expression, the cardinal marker of T cell exhaustion. Therapeutically, augmented T cell immunity via GIFT7 delivery rescues mice from disseminated leukemia. On the opposite spectrum of hypofunction, self-directed T cell over-reaction also demands therapeutic intervention. We have previously shown N-terminal modified(tetra-peptide-cleaved) MCP3 possessed immune suppressive activity. In view of this, we hypothesized that a synthetic cytokine linking GMCSF to MCP3 (GMME3) as part of a single polypeptide would augment its immune plasticity. We demonstrated that GMME3 induces significant Ca++ influx, activating IL10+CD21hiCD24hi CD1.dhi subset of splenic B cells (BGMME3) capable of inhibiting antigen presentation and Th17. The adoptive transfer of BGMME3 to mice symptomatic with experimental autoimmune encephalitis attenuated disease progression. Overall, the research presented in this thesis supports the use of GMCSF-based fusion cytokine as novel immune regenerative and modulatory therapeutics to (i)augment thymopoiesis, (ii) promote effector expansion, (iii) and regulate helper polarization. Therefore, our work points to the translational potential of fusion cytokines or fusion-primed immune cells as treatment of T cell dysfunction.L'immunité fonctionnelle des lymphocytes T exige le maintien de la diversité de répertoire et la différenciation appropriée des sous-ensembles à la périphérie. Cependant, les nombreux changements pathogènes peuvent perturber cette homéostasie. D'une part, la diminution de thymopoïèse ou l'épuisement des effecteurs provoquent un dysfonctionnement immunitaire, conduisant à la persistance des cellules infectées par des virus ou des cellules cancéreuses. Une réaction immunitaire effrénée peut, d'autre part, endommager les tissus. Le principal objectif de ma thèse était donc de développer de nouvelles thérapies pour moduler l'immunité des cellules T dans le contexte du cancer et des maladies inflammatoires. L'interleukine-7 (IL7) est particulièrement importante dans le développement des cellules T et sa prolifération homéostatique. Afin d'induire pharmacologiquement la néogenèse des cellules T, pour la reconstitution immunitaire et le traitement du cancer, nous avons développé un produit biopharmaceutique basé sur la fusion de GM-CSF et IL7 (GIFT7). L'administration de GIFT7 à des souris âgées a conduit à une hyperplasie corticale, inversant efficacement l'involution des tissus. L'effet hypertrophique du GIFT7 provoque une augmentation de la cellularité thymique totale et surtout une multiplication par 4 du nombre de CD4-CD8-CD44intCD25-précurseurs thymiques. Dans la périphérie, GIFT7 provoque la prolifération sélective d'un sous-ensemble CD8+ avec un phénotype défini comme CD8+CD44+CD62L+CCR7+KLRG-CD27+ (TGIFT7). Le transfert adoptif de cellules CD8 dérivée OTI-TGIFT7 dans l'OVA-EG7 des souris porteuses conduit à une régression tumorale significative. En outre, l'orthologue humain de GIFT7(hGIFT7) conduit à une multiplication par deux du nombre de cellules total après 3 jours et > 80% de Ki67+ expression dans les deux CD4+ et CD8+ PBMC avec réduction concomitante de PD1 expression, qui est le marqueur cardinal de l'épuisement des cellules T. L'augmentation de l'immunité des cellules T, via la livraison de GIFT 7, sauve les souris de leucémie diffusée. Sur le spectre opposé d'hypofonction, la sur-réaction de l'auto-cellule T exige également une intervention thérapeutique. Nous avons montré précédemment que le N-terminal modifié (tétra-peptide clivé) MCP3 possédait une activité immunitaire suppressive. Compte tenu de cela, nous avons émis l'hypothèse qu'une cytokine GM-CSF synthétique liée au MCP3 (GMME3) dans le cadre d'un polypeptide unique permettrait d'accroître sa plasticité immunitaire. Nous avons démontré que le GMME3 induit significativement Ca++ afflux, l'activation de l'IL10+CD21hiCD24hiCD1.dhi sous-ensemble de cellules B spléniques (BGMME3) capables d'inhiber la présentation de l'antigène et Th17. Le transfert adoptif de BGMME3 a atténué la progression de la maladie auto-immune de souris présentant des symptômes d'encéphalite expérimentale. Dans l'ensemble, la recherche présentée dans cette thèse soutient l'utilisation de la fusion pour la régénération immunitaire et de la thérapeutique modulation de (i) lathymopoïèse, (ii) l'expansion effecteur, (iii) et de la polarisation d' CD4+. Nos points de travail concernent le potentiel de translation de cytokines de fusion ou de fusion-apprêtées cellules immunitaires pour le traitement de la dysfonction des cellules T.
23
Bickerton, Erica Jane. "Cellular tropism and cell-to-cell fusion properties of the infectious bronchitis virus spike glycoprotein." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35165/.
Full textAbstract:
There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells.
24
Garg, Himanshu. "Feline Immunodeficiency Virus (FIV) Envelope Glycoprotein-Mediated Cell Fusion and Apoptosis." NCSU, 2003. http://www.lib.ncsu.edu/theses/available/etd-11042003-141554/.
Full textAbstract:
Feline Immunodeficiency Virus (FIV) and Human Immunodeficiency Virus (HIV) are lentiviruses that are remarkable similar in their genomic organization, receptor usage and pathogenesis. Based on this FIV has evolved into a well-established small animal model for studying AIDS. FIV and HIV cause a progressive depletion of T cells via a still unknown mechanism though numerous studies support a role of membrane expressed HIV env glycoprotein in apoptotic killing of CD4+ T cells. HIV env glycoprotein is a heterodimer of surface expressed gp120 that binds to CD4 and a chemokine receptor and transmembrane gp41 that mediates fusion and syncytia formation. The role of the fusion process in HIV env-mediated apoptosis remains controversial even though evidence suggests that cytopathic effect of HIV is related to the fusogenic potential of env glycoprotein. Blocking HIV env receptor interactions either at the level of gp120 or gp41 blocks both syncytia formation and apoptosis. This suggests a crucial role for HIV gp41 in fusion, as well as apoptosis. The hydrophobic pre-transmembrane (pre-TM) region of HIV gp41 is important for membrane fusion and sequence analysis reveals a similar region in FIV gp41. The current study was undertaken to determine the role of different regions of FIV env in mediating fusion and apoptosis in bystander cells and to determine whether the two phenomena are related. FIV env interactions with target cells were blocked at the level of gp120 or gp41 and the effect of these on fusion and apoptosis studied. The role of FIV gp41 pre-TM region in fusion and apoptosis was also determined. Our findings support a role of FIV env in apoptotic loss of T cells and this phenomenon correlates with env-mediated fusion.
25
Hurtley, S. M. "Reconstitution of an endocytic fusion event in a cell-free system." Thesis, Open University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484412.
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Bewry, Nadine N. "STAT3 Contributes to Resistance Towards BCR-ABL Inhibitors in a Bone Marrow Microenvironment Model of Drug Resistance in Chronic Myeloid Leukemia Cells." [Tampa, Fla] : University of South Florida, 2009. http://purl.fcla.edu/usf/dc/et/SFE0002892.
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Stein, Joshua. "Cellular electrofusion utilizing corona fields and DC pulse technology." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0001980.
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Hutchinson, Lloyd M. "Glycoprotein K of herpes simplex virus (HSV), role in viral egress and HSV-induced cell-cell fusion." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0016/NQ30094.pdf.
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Achanzar, William Edward 1967. "Analysis of a gene required for membrane fusion during nematode spermiogenesis." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/282106.
Full textAbstract:
C. elegans spermatids contain large vesicles called membranous organelles (MOs) that fuse with the plasma membrane during maturation to spermatozoa. This fusion is essential since mutations in the gene fer-1 block MO-plasma membrane fusion and result in abnormal spermatozoa. To determine the function of the fer-1 gene product during sperm maturation, I have cloned and sequenced the gene and several cDNAs. fer-1 is approximately 8.6kb in length and encodes a 6.3kb sperm-specific transcript. In situ hybridization experiments have shown fer-1 expression is limited to the primary spermatocytes, the cells in which the MOs are formed. fer-1 is predicted to encode a 235kD basic integral membrane protein (FER-1) that is highly charged and rich in lysine and glutamic acid. Database searches revealed FER-1 is similar to several predicted human proteins of unknown function. Mutations have been identified for four of the eleven fer-1 alleles, all of which cause amino acid changes in this predicted protein. FER-1 contains no recognizable functional motifs other than a single transmembrane domain at the C-terminus, a feature common to viral membrane fusion proteins. Antibodies raised against FER-1 and used for immunolocalization and western blot experiments did not yield reliable results. The work presented in this dissertation gives some evidence for my hypothesis that FER-1 is a membrane fusion protein, although the membrane fusion defect observed could be an indirect result of fer-1 mutations.
30
Sottile, Francesco 1988. "Mesenchymal stem cells generate distinc functional hybrids via cell fusion or entosis." Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/565598.
Full textAbstract:
Homotypic and heterotypic cell-cell fusion are key processes during development and tissue regeneration. On the other hand, aberrant heterotypic cell fusion between cancer and normal cells can contribute to tumor initiation and metastasis. Additionally, a form of cell-in-cell structure called entosis has been observed in several human tumors. Here we investigate cell-cell interaction between mouse mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs). MSCs have a great potential for regenerative medicine, but they are also involved in cancer progression. Here we report that MSCs can either fuse forming thereby heterokaryons, or be invaded by ESCs through entosis. Importantly we show that hetero-to-synkaryon transition occurs through a mitotic cell division and not by nuclear membrane fusion. Moreover, we also observe that the ROCK-actin/myosin pathway is required for both fusion and entosis in ESCs but only for entosis in MSCs. Overall, we show that MSCs can undergo fusion or entosis in culture by generating distinct functional cellular entities. Therefore, we conclude that both cellular processes should be explored for possible therapeutic application of MSCs.Las fusiones célula-célula homotípica y heterotípica son procesos clave durante el desarrollo y la regeneración de tejidos. Por otro lado, la fusión celular heterotípica aberrante entre células tumorales y células normales puede contribuir a la tumorigénesis y a la metástasis. Además, una forma estructural donde una célula esta dentro de la otra llamada entosis se ha observado en varios tumores humanos. Aquí estudiamos la interacción célula-célula entre las células madre mesenquimales (MSCs) de ratón y las células madre embrionarias (ESCs). Las MSCs no solo tienen un gran potencial para la medicina regenerativa, sino que también están involucradas en la progresión del cáncer. Aquí mostramos que las MSCs pueden fusionarse formando de este modo heterokaryons, o ser invadidas por las ESCs a través de entosis. Es importante destacar que la transición desde el heterokaryon al synkaryon se produce a través de una división celular mitótica y no por la fusión de la membrana nuclear. Por otra parte, también se observa que se requiere de la vía ROCK-actina/miosina tanto para la fusión como para la entosis en ESCs, pero solo para entosis en MSCs. En definitiva, se muestra que las MSCs pueden someterse a la fusión o entosis en cultivo mediante la generación de distintas entidades celulares funcionales. Por tanto, concluimos que ambos procesos celulares deben ser explorados para una posible aplicación terapéutica de las MSCs.
31
Baker, Nicole. "Muscle Stem Cell Fate is Directed by the Mitochondrial Fusion Protein OPA1." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/41974.
Full textAbstract:
During aging there is a decline in (MuSCs) and muscle regeneration, though the underlying reason is unknown. Interestingly, mitochondrial fragmentation is a common feature in aging, however, how this impacts MuSC function and maintenance has not been investigated. To address the effect of mitochondrial fragmentation in MuSCs, we generated a knockout mouse model using the Pax7CreERT2 inducible system to target deletion of the mitochondrial fusion protein Opa1 specifically within MuSCs (Opa1-KO). Analysis of MuSC function following muscle injury revealed a defect in the regenerative potential of Opa1-KO MuSCs. Moreover, following injury there was a substantial decrease in the number of MuSC in Opa1-KO animals with a concomitant increase in the number of committing cells, illustrating that loss of Opa1 drives MuSC towards commitment at the expense of self-renewal. Furthermore, loss of Opa1 in MuSCs alters the quiescence state, priming MuSCs for activation, as indicated by a reduction in quiescence-related genes, increased EdU incorporation, and enhanced cell cycle kinetics. To address the impact of mitochondrial dysfunction on muscle stem cell capacity, we generated a model of chronic Opa1 loss. Analysis of muscle stem cell function 3 months after Opa1 ablation revealed mitochondrial dysfunction and a defect in proliferation upon activation, leading to failed muscle regeneration. These data are the first to demonstrate a novel role for mitochondrial structure in the regulation of MuSC maintenance and regenerative capacity.
32
Petrany, Michael J. "Consequences of Cell Fusion and Multinucleation for Skeletal Muscle Development and Disease." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595847866440328.
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Henderson, Rosalind. "Streptavidin-cytokine fusion proteins for use as adjuvants in cancer vaccines." Thesis, University of Hull, 2008. http://hydra.hull.ac.uk/resources/hull:1606.
Full textAbstract:
The work presented in this thesis describes the production and characterisation of recombinant streptavidin-cytokine fusion proteins. Immunoadjuvants can augment the weak or non-existent antitumour response of a patient with cancer to immunotherapy. The response of breast cancer patients to cancer vaccines directed against tumour associated antigens, such as HER2/neu, is often weak and it has been reported that additional immunostimulation, such as that provided by an immunoadjuvant, is required in order to achieve effective therapeutic results. One approach to the development of novel immunoadjuvants is the production of fusion proteins which are designed to perform the dual functions of general activation of the immune system as well as directing the immune response specifically towards a TH1-type response, which is widely regarded as the optimal patient response for cancer immunotherapy.Streptavidin is a bacterial protein and an immunogenic molecule that induces an unspecific immune response. The cytokines IL-2 and IL-18 both promote TH1-type responses; IL-2 is already in clinical use as a cancer therapy, while investigation of the role of IL-18 as an immunotherapy is ongoing.A strategy was devised for the production of novel recombinant fusion proteins designed for use as immunoadjuvants; these proteins comprised an N-terminal truncated streptavidin core protein sequence and a C-terminal cytokine, specifically either IL-2 or IL-18, separated by a short polypeptide linker region. Molecular cloning techniques were used to generate DNA expression constructs encoding the recombinant fusion proteins which were expressed in an inducible bacterial system using plasmid expression vector pCR(R)T7/NT-TOPO(R). The expressed recombinant proteins were found to accumulate within insoluble bacterial inclusion bodies and protocols were developed and optimised for the isolation and solubilization of these proteins. Protein solubilization required the use of buffers at high pH (pH 12.5) which resulted in disrupting protein structural integrity; a pulsed dilution method was subsequently employed to achieve refolding of the proteins prior to further analysis.Characterization of fusion proteins STV/IL-2 (streptavidin-IL-2) and STV/IL-18 (streptavidin-IL-18) was conducted using native and dissociating PAGE and Western analysis. Antibody binding studies provided preliminary confirmation of the identity of the STV/IL-2 fusion protein. Similar studies to characterize STV/IL-18 were initially encouraging but proved inconclusive and further analysis of this protein is required.These initial investigations have validated this approach using the expression systems established here for the production of recombinant streptavidin-cytokine fusion proteins. A number of issues to be addressed have been highlighted regarding problems encountered with protein yields, solubilisation and maintenance of structural integrity. It is therefore concluded that further modification and optimisation of the expression system and protein isolation procedures employed is necessary to provide an appropriate system for the production of these fusion proteins; this will subsequently permit the investigation of their potential for use in therapeutic applications.
34
Stein, Kathryn Kreimborg. "Sperm proteins involved in sperm-egg fusion : a cell biological and proteomic approach /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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35
Leung, Sze-Yui Horasis, and 梁思睿. "Fibronectin: role in viral cell association, fusion and entry of influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48329708.
Full textAbstract:
The influenza A viral hemagglutinin (HA) protein binds to sialic acid (SA) groups of cellular surface glycoproteins to achieve viral attachment and entry. The SA binding specificity of HA is one of the major determinants for controlling viral tropism and host specificity.
Fibronectin (FN) is a ubiquitinious glycoprotein secreted on cell surface, either circulating in plasma, or as one of the best characterized components of the extra cellular matrix. With its binding properties towards different types of molecules and pathogens, it has been utilized by different bacterial and viral pathogens for binding, entry, propagation and pathogenesis. The binding affinity and region of plasma FN to influenza A viral glycoprotein was identified in early 1980s. Evidence also suggests the binding is SA associated. FN associates with different viral pathogens. However, evidence of FN direct involvement in influenza A pathogenesis remains unknown.
The objective of this thesis is to test the involvement of cellular FN in influenza A viral infection. To perform the study, FN siRNA and anti-FN antibody were applied. This study demonstrated possible involvement of FN in the replication of human H1N1 and highly pathogenic avian H5N1 viruses. It also discovered that FN is very important for the replication of H1N1 virus, but not H5N1 virus. Interestingly, the result suggested that FN does not affect the initial virus-host binding, but it has an effect on post-attachment events.
Key amino acid positions controlling the SA binding specificity of seasonal human or avian influenza A viruses have been identified in the HA. In this thesis, reverse genetics and mutagenic work identified that viruses with a α2,3-linked SA (SA α2,3) binding preference were not inhibited by anti-FN antibody, while viruses with a α2,6-linked SA (SA α2,6) specificity were severely inhibited. This surprising finding of SA binding preference related FN involvement in post-attachment event led to the further investigation on the structural involvement of FN and viral entry pathway analysis.
The 9th and 10th of type III repeating units of FN form the cell-binding domain of the protein for cell attachment. From site specific antibody inhibitory studies, the cell binding region of FN near the synergy adhesion site(SAS) and Arg-Gly-Asp-Ser(RGDS) cell adhesion signal was identified to be important for the replication of viruses that have a α2,6 SA binding preference, but it was also found to be independent of α5β1 integrin receptor.
After attaching to a host cell, the virus was internalized in an endosome via clathrin- or caveolin- mediated endocytosis. By application of pathway inhibitors, the FN association with viral entry pathway was evaluated. Though this study failed to identify a single specific FN mediated viral entry pathway, this pathway study indicated the possibility of FN various involvement in influenza viral entry. The study indeed indicated that viruses have difference SA binding preferences are different in their choices in viral entry pathways.
This thesis did not only introduce cellular FN as a novel host factor, but also identified possible target and brought new light in the control of influenza A viral infection.published_or_final_version
Public Health
Doctoral
Doctor of Philosophy
36
Wagenaar, Timothy Robert. "Regulation of infected cell fusion by the vaccinia virus A56 and K2 proteins." College Park, Md.: University of Maryland, 2008. http://hdl.handle.net/1903/8044.
Full textAbstract:
Thesis (Ph. D.) -- University of Maryland, College Park, 2008.Thesis research directed by: Dept. of Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
37
Girardi, Francesco. "TGFbeta signalling pathway in muscle regeneration : an important regulator of muscle cell fusion." Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS114.
Full textAbstract:
La régénération musculaire s’appuie sur une réserve de cellules souches résidant dans le muscle appelées cellules satellites (MuSCs). Les MuSCs sont quiescentes et peuvent s’activer à la suite d’une blessure du muscle afin de former des progéniteurs amplificateurs (myoblastes) qui se différencieront et fusionneront pour former de nouvelles myofibres. Durant ce processus, un réseau complexe de voies de signalisation est impliqué, parmi lequel la signalisation du facteur de croissance transformant bêta (TGFβ) joue un rôle fondamental. Précédents rapports ont proposé de nombreuses fonctions pour la signalisation TGFβ dans les cellules musculaires, comme leur quiescence, activation et différenciation, mais l’impact de TGFβ sur la fusion de myoblastes n’a jamais été étudié. Dans cette étude, nous avons montré que cette signalisation réduit la fusion des cellules musculaires indépendamment de leur différenciation. Au contraire, l’inhibition de la signalisation TGFβ accroît la fusion cellulaire et favorise les ramifications entre myotubes. Une pharmaco-modulation de la voie in vivo perturbe la régénération musculaire après blessure. Une addition exogène de la protéine TGFβ conduit à une perte de fonction du muscle, tandis que l’inhibition de la voie induit la formation de myotubes géants. Les analyses transcriptomiques et fonctionnelles ont montré que TGFβ agit sur la dynamique de l’actine afin de réduire la diffusion cellulaire à travers une modulation des protrusions à base d’actine. Nos résultats ont donc révélé une voie de signalisation qui limite la fusion de myoblastes et ajoutent un nouveau niveau de compréhension sur la régulation moléculaire de la myogenèseMuscle regeneration relies on a pool of muscle-resident stem cells called satellite cells (MuSCs). MuSCs are quiescent and can activate following muscle injury to give rise to transient amplifying progenitors (myoblasts) that will differentiate and finally fuse together to form new myofibers. During this process, a complex network of signalling pathways is involved, among which, Transforming Growth Factor beta (TGFβ) signalling cascade plays a fundamental role. Previous reports proposed several functions for TGFβ signalling in muscle cells including quiescence, activation and differentiation. However, the impact of TGFβ on myoblast fusion has never been investigated. In this study, we show that TGFβ signalling reduces muscle cell fusion independently of the differentiation step. In contrast, inhibition of TGFβ signalling enhances cell fusion and promotes branching between myotubes. Pharmacological modulation of the pathway in vivo perturbs muscle regeneration after injury. Exogenous addition of TGFβ protein results in a loss of muscle function while inhibition of the TGFβ pathway induces the formation of giant myofibres. Transcriptome analyses and functional assays revealed that TGFβ acts on actin dynamics to reduce cell spreading through modulation of actin-based protrusions. Together our results reveal a signalling pathway that limits mammalian myoblast fusion and add a new level of understanding to the molecular regulation of myogenesis
38
Beard, David Andrew. "Cell-selective expression of a glutamine synthetase fusion gene in embryonic chick retina /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487862972135908.
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Takahashi, Tsuyoshi. "Clinicopathologic Features of Non-Small Cell Lung Cancer with EML4-ALK Fusion Gene." Kyoto University, 2010. http://hdl.handle.net/2433/120559.
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Marshall, Philip. "Expression of measles fusion protein in insect and human cells using Eukaryotic expression vectors." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61265.
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Measles virus is an animal enveloped virus that is a member of the genus morbillivirus in the paramyxoviridae family. Its envelope contains two surface glycoproteins H and F which are required for viral attachment and entry respectively. Virus penetration occurs via a process which involves fusion of the viral membrane with the plasma membrane at the cell surface. Replication of the virus thus follows and leads to giant cell (syncytia) formation.The infectivity of measles virus is dependent upon a host proteolytic cleavage of the F$ sb0$ glycoprotein into two active subunits F$ sb1$ and F$ sb2$. This cleavage was later shown to expose a hydrophobic sequence at a NH$ sb2$ terminal of the F$ sb1$ which is directly involved in cell fusion and virus penetration.
In order to increase our knowledge concerning cell mediated fusion events we have expressed the fusion glycoprotein of measles virus in insect and human cells by using recombinant baculo- and adenoviruses respectively. Analysis by SDS-PAGE demonstrated that our protein was first synthesized as a 60 Kd protein and cleaved subsequently into its two respective subunits F$ sb1$ and F$ sb2$ of 40 Kd and 20 Kd respectively. Hemolysis assays confirmed the biological activity of this protein in both systems. However, the fusion protein was unable to fuse insect cells.
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Marques, Sandra Eugénia Leite. "Expressão em Escherichia coli de antigénios do Cell fusing agent virus (Flaviviridae: Flavivirus) como proteína de fusão." Master's thesis, Faculdade de Ciências Médicas. UNL, 2012. http://hdl.handle.net/10362/8531.
Full textAbstract:
RESUMO: O Cell Fusing Agent Vírus (CFAV), considerado como o primeiro “flavivírus
específicos de insectos” (ISF), parece estar exclusivamente adaptado aos seus
hospedeiros, não replicando em células de vertebrados. Apesar de ter sido identificado
há mais de três décadas (1975), a verdade é que muito pouco se conhece sobre a sua
biologia. Dado o seu parentesco filogenético com alguns outros flavivírus encontrados
naturalmente em mosquitos de diferentes géneros colhidos em diferentes regiões do
globo, este vírus poderá ser usado como modelo para o estudo de ISF. No entanto,
necessitam do desenvolvimento de ferramentas básicas, tais como clones moleculares
ou baterias de soros contendo anticorpos que reconheçam uma ou mais proteínas
codificadas pelo genoma viral, produzidas, por exemplo, a partir de antigénios virais
produzidos de forma recombinante.
Com este trabalho pretendeu-se a optimização de protocolos que permitiram a
expressão e purificação parcial de quatro proteínas [duas proteínas estruturais (C e E) e duas não estruturais (NS3hel e NS5B)] do CFAV em E. coli, todas elas produzidas
como proteínas de fusão com “caudas” (tags) de hexahistidina nos seus extremos
carboxilo.
Para a expansão do CFAV foram utilizadas células Aedes albopictus (C6/36).
Após a realização da extracção do RNA viral e a obtenção de cDNA, procedeu-se
amplificação, por RT-PCR, das regiões codificantes das proteínas C, E, NS3hel e
NS5B, utilizando primers específicos. Os quatro fragmentos de DNA foram
independentemente inseridos no vector pJTE1.2/blunt usando E. coli NovaBlue como
hospedeira de clonagem e, posteriormente, inseridos em vectores de expressão pET-28b
e pET-29b usando E. coli BL21(DE3)pLysS e Rosetta(DE3)pLysS como hospedeiras de
expressão.
Após da indução, expressão e purificação das proteínas recombinantes C, E, NS3hel e NS5B, foi confirmada a autenticidade destas proteínas produzidas através do método Western Blot com um anticorpo anti-histidina. --------- ABSTRACT: The Cell Fusing Agent virus (CFAV) considered as the first "insect- specific
flavivirus" (ISF) and seems to be uniquely adapted to their hosts, not replicating in
vertebrate cells. Although it has been known for more than three decades (1975), the
truth is very little is known about its biology. Given its close phylogenetic relationship with other flavivirus naturally circulating in various genera of mosquitoes collected from different regions of the globe, this virus could be used as a model for the study of ISF. However, such studies require the development of experimental basic tools, such as molecular clones or serum batteries containing antibodies that recognize one or more proteins encoded by the viral genome, produced, for example, from viral antigens recombinant produced.
In this work, we carried out the optimization of protocols that allowed the
expression and partial purification of four proteins [two structural proteins (C and E)
and two nonstructural proteins (NS3hel and NS5B)] CFAV in E. coli as fusion protein
for c-terminal hexahistidine tags.
For the expansion of the CFAV we used Aedes albopictus (C6/36) cells. After completion of the viral RNA extraction and cDNA obtained, amplification of the coding
regions of the C, E, NS5B and NS3hel proteins was carried out by RT-PCR using
specific primers. The four DNA fragments were independently inserted into the vector
pJTE1.2/blunt using E. coli NovaBlue as cloning host and then inserted into expression vectors pET-28b and pET-29b using E. coli BL21(DE3)pLysS and Rosetta(DE3)pLysS
as expression host.
After induction, expression and purification of recombinant C, E, NS3hel and NS5B proteins Western Blot analyses with an anti-histidine antibody confirmed the
authenticity of these proteins produced.
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Wu, Melissa P. "Enhancing Myoblast Fusion for Therapy of Muscular Dystrophies." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10740.
Full textAbstract:
Skeletal muscle is a major organ comprising 30-40% of the human body mass. The coordination of processes resulting in mature muscle requires many genes, and their loss can result in debilitating muscle disorders. Of the strategies being developed to cure muscle diseases, enhancement of the natural process of muscle cell fusion in existing or introduced myogenic cells has great therapeutic potential. In this work, we determined whether a drug that stimulates proliferation and fusion of myoblasts could alleviate murine Duchenne muscular dystrophy. We also studied the necessity of a gene that is upregulated in early fusing human myoblast cultures and its role in muscle disease development.
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Skalsky, Yitzchak. "Study of the transcription factor TFE3 and its fusion partners in renal cell carcinoma." Thesis, Institute of Cancer Research (University Of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393639.
Full text
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Al-Torki, Reem. "Mapping of B-cell epitopes on the fusion protein of human respiratory syncytial virus." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415976.
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Cheung, Ngai. "Structural and functional characterization of EEN/EndophilinA2, a fusion partner in acute leukemia /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31540284.
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West, Megan C. "Visualization of the ribbon synapse using Ribeye a-mCherry fusion protein." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1311798467.
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Drewlo, Sascha [Verfasser]. "Molecular mechanisms involved in physiological cell-cell fusion: interactions between Syncytin-1 and its receptor as a model system / Sascha Drewlo." Köln : Zentralbibliothek der Deutschen Sporthochschule, 2006. http://d-nb.info/1071861204/34.
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Rankin, Alasdair Menzies. "An investigation of CD28/B7 family binding interactions and costimulation, using immunoglobulin fusion proteins." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360469.
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Hummel, Kimberly Brown. "Alteration of the measles virus glycoproteins as a mechanism to reduce cell fusion during persistence." Diss., Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/25597.
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Villafranca, Locher Maria Cristina. "Fusion of bovine fibroblasts to mouse embryonic stem cells: a model to study nuclear reprogramming." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/82864.
Full textAbstract:
The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates.
In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells.
Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome.Ph. D.