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.

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.

<p>Metastasis is a feared aspect of cancer and little is known about the underlying mechanisms. It is proposed that metastasis is caused by cell fusion between tumour and immune active phagocyte cells, for example macrophages. Such hybrid cells could then develop immortality and chemo tactic mobility. In two different systems it was examined whether it is possible to detect variation in cancer cells that would explain an initial fusion between tumour cells and leukocyte cells. Both systems included use of STR markers. Human colon carcinoma cells, which originally had been grown in nude mice, were investigated with mouse specific primers. These showed no trace of mouse DNA, which they most probably would have if cell fusion had occurred. Human breast cancer cells grown in nude mice, that had received injection of stem cell from male blood, showed no presence of Y-chromosomes. Blood, which was analyzed from one of the mice, showed a weak presence of something else than just mouse DNA. The result was however vague and hard to evaluate, and tries to reproduce the positive outcome failed. No evidence, which indicated that cell fusion occurred, was possible to demonstrate. On the other hand, there are previous studies that show how metastases can express macrophage specific properties, which gives all reason for further investigations.</p>

From previous studies it has been demonstrated that the fusion of tumor cells with antigen-presenting cells generates hybrids that are known to induce anti-tumor immunity. With the advancement of scientific research and medicine, the need to produce cell-cell hybrids for cancer immunotherapy and for various other applications is substantial. Among the many methods used to generate these hybrid cells, electrofusion is a technique that is more widely used and recognized as a method to efficiently produce hybrids. Electrofusion requires two steps. In the first step, cells are brought into close adjacent contact either by a mechanical method like centrifugation or by dieletrophoresis using alternating current (AC). The second step includes the reversible breakdown and fusion of cell membranes induced by high voltage direct current (DC) pulses. The goal of this investigation was to study the use of electrical charge to bring cells into close contact with one another in the cell contact stage prior to delivering high voltage fusion pulses. The possibility of achieving considerable cell-cell contact was tested in two separate electrical systems. In the first system B16 murine melanoma cancer cells were subjected to a range of direct current (DC) voltages between 4 V/cm and 40 V/cm. With the use of DC from a small power source the response of the cells was tested in multiple fusion chambers consisting of two or four electrodes. The configurations of the chambers were varied by changing the distance between the electrodes, the thickness, material and type of coating on the electrodes. In the second system the movement of cells in the presence of corona charge was studied. B16 cells in a culture dish were confined by a circular grounded electrode and subjected to corona discharge for known periods of time. Application of corona charge (positive or negative) facilitated the contact of cells in the annular region between the two circular electrodes. After series of tests, final designs for fusion chambers to be used with DC and with corona were developed. Cell contact achieved with the DC fusion chamber was not substantial enough to produce a significant amount of fusion yield. The fusion chamber designed to be used with corona on the other hand produced exceptional cell contact results consequentially generating fusion yields as high as 40%.

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.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 93-105).

No description available.

The muscle hypertrophy of lambs expressing the Callipyge phenotype is possibly linked to characteristics of their muscle satellite cells. Therefore, characteristics (proliferation, fusion %, and protein accretion) of cultured satellite cells isolated from the longissimus muscle of Callipyge (n = 3) and normal (n = 3) lambs were compared in this study. In the first experiment, we tested whether or not the lll proliferation rates differ for satellite cells isolated from Callipyge or normal sheep when cultured in the presence of different serum types (horse, normal lamb, or Callipyge lamb). The average population doubling time (PDT, h) during log phase growth was calculated for cells from each animal grown in each serum type. Population doubling time was not affected (P > .1) by the interaction of satellite cell type with serum type, or by satellite cell type. Unexpectedly, PDT was longer (P < .05) for satellite cells grown in Callipyge serum (22 h) than for cells grown in normal sheep serum (20 h) or horse serum (18 h). These results suggest that muscle hypertrophy of Callipyge lambs is not linked to intrinsic differences in satellite cell proliferation, although hypertrophy may be associated with a decreased proliferation induced by a factor in Callipyge serum. In the second experiment, we tested whether cell fusion, or protein accretion differ for cultured satellite cells isolated from Callipyge or normal sheep. DNA and protein were determined at 24, 48, 72, and 96 h after satellite cell cultures were induced to differentiate. Fusion percentage was determined in a Giemsa stained plate after 72 h in differentiation medium (Dulbecco's Modified Eagle Medium containing 1.5% of horse serum). Callipyge cultures tended (P = .14) to have higher fusion% than normal cultures exhibited, suggesting that muscle hypertrophy of Callipyge lambs may be linked to an increased tendency of satellite cells to fuse. Protein content (μg/well) and protein:DNA ratio (ng of protein/ng of DNA) were not affected by satellite cell type (P = .80 and P = .79, respectively). Thus, there was no evidence for a link between increased protein accretion and Callipyge hypertrophy.

Les sarcomes à « génétique complexe » sont des tumeurs rares du tissu mésenchymateux. Ces tumeurs sont caractérisées par un nombre important de réarrangements chromosomiques et possèdent un génome complexe dans lequel aucune altération n’avait été retrouvée de manière spécifique et récurrente. Le but de ma thèse était donc d’identifier les conséquences de cette complexité chromosomique et de comprendre comment celle-ci pouvait être générée. Dans un premier temps, le transcriptome de 112 sarcomes à « génétique complexe » a été analysé. La recherche et la validation de transcrits chimériques a conduit l’identification de réarrangements fréquents au niveau du gènes TRIO. Ces transcrits permettent la formation soit d’une protéine tronquée et seraient des évènements issus du réarrangement global du génome de ces tumeurs. Dans un deuxième temps, nous avons alors recherché l’origine de la formation de ces altérations, en s’intéressant particulièrement à la fusion cellulaire comme mécanisme initiateur. Ce processus physiologique est observé dans des cellules mésenchymateuses comme les macrophages et les myoblastes et peut être détourné afin de permettre le développement et l’évolution tumorale. J’ai alors étudié les conséquences génomiques et phénotypiques de la fusion de fibroblastes à différents stades d’immortalisation ou à différentes phases du cycle cellulaire. Mes travaux ont alors permis de montrer que des mécanismes de fusion cellulaire conduisent à la formation d’altérations génétiques similaires à celles des sarcomes à « génétique complexe » et contribueraient à l’initiation et à la progression de ces tumeurs. / Sarcomas with a complex genetics are rare tumours from mesenchymal tissue. They are characterized by massive chromosomal without any recurrent and specific alteration. The objective of my thesis was to identify the consequences of this chromosomal complexity and mechanisms explaining how this could be generated. First, transcriptome of 112 sarcomas with a complex genetics have been analysed. Chimeric transcripts detection and validation permitted the identification of frequent rearrangements in TRIO gene. These transcripts lead to the formation of a truncated protein and they would originate from a global rearrangement of the tumour genomes. Second, we have sought the origin of these alterations, with a particular interest for the cell fusion as an initiator mechanism. This physiological process is observed in mesenchymal cells like macrophages and myoblasts and it can be hijacked to drive tumour inception and evolution. I consequently studied both genomic and phenotypic consequences of hybrids from fibroblasts at different immortalization steps or in the different cell cycle phases. This work permitted to demonstrate that cell fusion mechanism leads to the initiation of genetic alterations that mimics the ones in sarcomas with complex genetics and would contribute to their tumour initiation and progression.