Mit der vorliegenden Arbeit sollte nachgewiesen werden, dass bei in vitro kultivierten kaninen dermalen Fibroblasten einer Hunderasse nach einer bestimmte Kultivierungszeit replikative Seneszenz entsteht und dass das replikative Vermögen dieser Zellen in der Zellkultur abhängig vom Alter des Spendertieres ist. Dreißig Beagle aus zwei Versuchstieranstalten wurden als Hautspender genutzt. Diese Tiere wurden in die drei Altersgruppen jung, adult und alt unterteilt. Mit Hilfe einer Hautstanze wurde bei allen Tieren im Bereich der rechten Skapula ein Hautstück gewonnen. Diese Hautstücken wurden in Zellkulturflaschen verbracht. Die aus diesem Explantat auswandernden Zellen stellten die Grundlage für die Primärkultur dar. Die Zellen wurden in regelmäßigen Abständen subkultiviert. Dabei wurden immer die Gesamtzellzahl und die Vitalität der Kulturen bestimmt. Diese Werte bildeten die Grundlage für die Berechnung der Parameter des replikativen Vermögens der Zellen. Auf Grundlage der erstellten Wachstumskurven konnte die Generationszeit der Zellen berechnet werden. Parallel zur Kultivierung der Zellen erfolgte die morphologische Betrachtung der Zellen mit Hilfe eines Phasenkontrastmikroskops und histologischen Färbungen, die unter dem Lichtmikroskop näher beurteilt wurden. / This thesis aims to demonstrate that, after a certain period of time, replicative senescence develops in canine dermal fibroblasts of a certain dog breed when cultured in vitro. It is also shown that the replicative capacity of these cells is related to the age of the donor animal. Thirty Beagles from two experimental facilities were used as skin donors. The animals were divided in three age groups: young, adult and old. Skin samples from the right scapula were taken from all animals by means of a punch biopsy and transferred to cell culture vessels. The primary culture was based on the cells emigrating from these explants. The cells were subcultured at regular intervals, at which the total number of cells and the vitality of the cultures were also determined. Based on these parameters, the replicative capacity of the cells was calculated and growth curves were created, which were then used to calculate the generation times of the cells. Parallel to cultivation, the cells underwent morphological dissection using a phase contrast microscope on the one hand and a light-optical microscope with histological staining on the other hand.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-05-04T16:56:07Z No. of bitstreams: 1 DIS_PATRICIA_LAZZAROTTO_BELLICANTA_COMPLETO.pdf: 1443666 bytes, checksum: 536295a41c399cfa2da6b360bf54a344 (MD5) / Made available in DSpace on 2017-05-04T16:56:07Z (GMT). No. of bitstreams: 1 DIS_PATRICIA_LAZZAROTTO_BELLICANTA_COMPLETO.pdf: 1443666 bytes, checksum: 536295a41c399cfa2da6b360bf54a344 (MD5) Previous issue date: 2016-04-26 / Introduction: The inherent characteristics of plasticity of human fibroblast cells makes it an important tool for evaluating the effects of microgravity at a cellular level. This study analyzed the behavior of human skin fragment fibroblasts in a simulated microgravity environment. Methods: Human fibroblast cells in the 8th and 17th passage, cultured under standard incubator conditions at 37? C with 5% CO2, were submitted to simulated microgravity in a 3D-clinostat for a period of 24h and 40h. After exposure, both passage cells were analyzed and compared with the control group (1G) in population doubling assays, tests of passage and microscopic analysis, as well as PCR analysis for detection of variations in the gene expression related to the cell cycle (p21, p16). Results: Before microgravity exposure, cells belonging to the 17th passage presented characteristics of cells in an apoptotic state. After 24h and 40h of microgravity, the cells of both groups showed themselves to be more confluent and elongated. PCR analysis demonstrated that p21 expression was decreased while p16 increased. In addition, PCR analysis showed a difference in expression of p21 and p16 genes between the 24h and 40h samples. Discussion: The present research showed cells to be more confluent and elongated after microgravity exposure, a characteristic of cells with fewer passages, suggesting alterations in their cytoskeleton. This result was confirmed by PCR analysis where a decrease in p21 expression was demonstrated. This result corroborates previous findings that among 588 genes tested, the p21 gene presented a negative expression. Conversely, the p16 gene showed a positive expression. Since both the p21 and p16 genes are related to the cell cycle, these results suggest the hypothesis of important changes having occurred in the cellular cytoskeleton and, consequently, a probable alteration in the production of cell cycle regulatory proteins (cyclins). Furthermore, RT-PCR analysis demonstrated a difference in p21 and p16 gene expression between the 24h and 40h samples, indicating the need for a more detailed comparison between the exposure times. Also pluripotency markers were found, Oct4 and Nanog, which suggests alterations in plasticity levels. / Introdu??o: As caracter?sticas da plasticidade inerentes das c?lulas de fibroblastos humanos os tornam uma ferramenta importante para avaliar os efeitos da microgravidade em um n?vel celular. Este estudo analisou o comportamento dos fibroblastos fragmento de pele humanas em um ambiente de microgravidade simulada. M?todos: c?lulas de fibroblasto humanos em 8a e 17a passagem, cultivadas em condi??es normais em incubadora a 37? C com 5% de CO2, foram submetidos ? microgravidade simulada em um clinostato-3D por um per?odo de 24h e 40h. Ap?s a exposi??o, as c?lulas de cada passagem foram analisadas e comparadas com o grupo de controle (1G) ensaios de prolifera??o celular (population doubling), ensaios de passagem e an?lise microsc?pica, bem como a an?lise de PCR para a detec??o de varia??es na express?o de genes relacionados com o ciclo celular (p21, p16). Resultado: Antes da exposi??o microgravidade, c?lulas pertencentes ? passagem 17? apresentaram caracter?sticas de c?lulas num estado apopt?tico. Depois de 24h e 40h de microgravidade, as c?lulas de ambos os grupos se mostraram mais confluentes e alongadas. A an?lise de PCR demonstrou que a express?o de p21 foi diminu?da enquanto p16 aumentou. Al?m disso, a an?lise PCR mostrou a diferen?a na express?o de genes p21 e p16 entre as amostras 24h e 40h. Discuss?o: A presente pesquisa mostrou que as c?lulas de 17? passagem tornaram- se mais confluentes e alongadas ap?s a exposi??o microgravidade, uma caracter?stica das c?lulas com menor n?mero de passagens, sugerindo altera??es no seu citoesqueleto . Este resultado foi confirmado por an?lise de PCR, onde foi demonstrado uma diminui??o na express?o de p21. Esse resultado confirma descobertas anteriores de que entre 588 genes testados, o gene p21 apresentou uma express?o negativa. Por outro lado, o gene p16 mostrou uma express?o positiva. Uma vez que ambos os genes P21 e P16 est?o relacionadas com o ciclo celular, estes resultados sugerem a hip?tese de altera??es importantes tenham ocorrido no citoesqueleto celular e, consequentemente, uma prov?vel altera??o na produ??o de prote?nas reguladoras do ciclo celular (ciclinas). Al?m disso, a an?lise de RT-PCR demonstrou uma diferen?a na express?o do gene p21 e p16 entre as amostras 24h e 40h, indicando a necessidade de uma compara??o mais detalhada entre os tempos de exposi??o. Foram tamb?m percebidas altera??es no n?vel de plasticidade dos fibroblastos de 17? passagem e 8? passagem, devido a express?o de marcadores de pluripot?ncia Oct4, Nanog.

Mit der vorliegenden Arbeit sollte nachgewiesen werden, dass bei in vitro kultivierten kaninen dermalen Fibroblasten einer Hunderasse nach einer bestimmte Kultivierungszeit replikative Seneszenz entsteht und dass das replikative Vermögen dieser Zellen in der Zellkultur abhängig vom Alter des Spendertieres ist. Dreißig Beagle aus zwei Versuchstieranstalten wurden als Hautspender genutzt. Diese Tiere wurden in die drei Altersgruppen jung, adult und alt unterteilt. Mit Hilfe einer Hautstanze wurde bei allen Tieren im Bereich der rechten Skapula ein Hautstück gewonnen. Diese Hautstücken wurden in Zellkulturflaschen verbracht. Die aus diesem Explantat auswandernden Zellen stellten die Grundlage für die Primärkultur dar. Die Zellen wurden in regelmäßigen Abständen subkultiviert. Dabei wurden immer die Gesamtzellzahl und die Vitalität der Kulturen bestimmt. Diese Werte bildeten die Grundlage für die Berechnung der Parameter des replikativen Vermögens der Zellen. Auf Grundlage der erstellten Wachstumskurven konnte die Generationszeit der Zellen berechnet werden. Parallel zur Kultivierung der Zellen erfolgte die morphologische Betrachtung der Zellen mit Hilfe eines Phasenkontrastmikroskops und histologischen Färbungen, die unter dem Lichtmikroskop näher beurteilt wurden. / This thesis aims to demonstrate that, after a certain period of time, replicative senescence develops in canine dermal fibroblasts of a certain dog breed when cultured in vitro. It is also shown that the replicative capacity of these cells is related to the age of the donor animal. Thirty Beagles from two experimental facilities were used as skin donors. The animals were divided in three age groups: young, adult and old. Skin samples from the right scapula were taken from all animals by means of a punch biopsy and transferred to cell culture vessels. The primary culture was based on the cells emigrating from these explants. The cells were subcultured at regular intervals, at which the total number of cells and the vitality of the cultures were also determined. Based on these parameters, the replicative capacity of the cells was calculated and growth curves were created, which were then used to calculate the generation times of the cells. Parallel to cultivation, the cells underwent morphological dissection using a phase contrast microscope on the one hand and a light-optical microscope with histological staining on the other hand.

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.

This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers