Cianobactérias são microrganismos capazes de produzir substâncias com potencial biológico e biotecnológico. Tais substâncias podem acarretar em malefícios, causados pelas toxinas, ou ter em sua composição substâncias benéficas como carotenoides, lipídeos e enzimas, de interesse biotecnológico. Assim, esse trabalho teve como objetivo avaliar a toxicidade e prospectar componentes presentes na biomassa global da cianobactéria Microcystis aeruginosa BB005. A toxicidade foi avaliada através de bioensaios com Daphnia magna, e a biomassa seca foi caracterizada por análises dos ésteres metílicos de ácidos graxos (EMAGs), determinação de carotenoides, obtenção do perfil proteico por eletroforese e hidrólise ácida de carboidratos. Também foi empregada uma metodologia de separação adsortiva por bolhas a fim de separar o pigmento ficocianina e enzimas de interesse biotecnológico (e.g. fosfatase alcalina e lipase) utilizando o sobrenadante de um cultivo. Apesar de ter sido possível quantificar a produção de toxinas pela cepa BB005, os ensaios ecotoxicológicos não permitiram estabelecer uma correlação entre a concentração de toxina e a imobilidade dos organismos. O perfil de EMAGs apresentou preponderância de PUFAs (ω-ácidos graxos) sendo que os linolenatos corresponderam a 25% do total. O teor de carotenoides encontrado durante a purificação dos lipídeos transesterificados e expresso como β-caroteno foi de 6,66 mg/g de biomassa seca. A eletroforese mostrou pelo menos 15 bandas distintas de proteínas e o perfil cromatográfico dos hidrolisados de carboidratos mostrou maior concentração do monossacarídeo glucose e de um dissacarídeo de glucose, possivelmente maltose. A separação de proteínas por espumação apresentou uma correlação direta entre fluxo de nitrogênio e a fração de espuma coletada. O fator de enriquecimento para a ficocianina foi de 41,69 utilizando fluxo de 20 mL/min de N2. Para a enzima fosfatase alcalina, os melhores resultados foram obtidos para o fluxo de 120 mL/min de N2, que apresentou fator de enriquecimento e de purificação de 5,25 e 2,85, respectivamente. Portanto, este trabalho demonstrou a importância de investigações sobre os componentes oriundos de M. aeruginosa, sobretudo dos EMAGs incomuns, a quantidade expressiva de carotenos e ainda da separação da ficobilina e de enzimas chave nos processos bioquímicos utilizando o processo por espumação. / Cyanobacteria are microorganisms able to produce substances with high biological and biotechnological potential. These substances may cause disorders because of the toxins or may be composed by beneficial substances such as carotenoids, lipids and enzymes of technological interest. Hence, the scope of the current research was centered in the toxicity evaluation or prospect the substances from the Microcystis aeruginosa cyanobacterium biomass – BB 005. Daphnia magna was used in the toxicity bioassays and the cyanobacterium dry biomass was characterized by the transesterified lipids analysis, carotenoid determination, electrophoretic profile for proteins and acid hydrolysis of polysaccharides. The foam fractionation methodology was performed to separate the phycocyanin pigment and enzymes (e.g. alkaline phosphatase and lipase), using the supernatant from a liquid culture of the cyanobacterium. Although it was possible to quantify the production of toxins by BB005 strain, the ecotoxicological tests have no establish a correlation between toxin concentration and the immobility of organism bodies.The methyl esters profile in which there was a large amount of PUFAs (ω-fatty acids) with 25% of linolenates. The carotenoid content found during the lipids transesterificaction, expressed as β – carotene, was 6.6 mg/g of dry mass. The electrophoresis indicated at least 15 distinct bands of proteins and the chromatographic profile from the native polymeric carbohydrates indicated higher concentration of monosaccharide glucose and a glucose disaccharide, possible maltose. Proteins separation through foaming showed a direct correlation between the nitrogen flow and the collected foam. The phycocianin enrichment factor was high, 41.69, at a 20 mL/min N 2 flow. While in the case of alkaline phosphatase the best result was attained in the 120 mL/min of N2 flow which lead to enrichment and purification factors equal to 5.25 and 2.85 respectively. Therefore, this research has demonstrated the importance of the investigation on compounds from the M. aeruginosa entire biomass, especially the methyl esters from less common and highly insaturated fatty acids, an expressive content of carotenoids and, furthermore, the separation of phycobillin and key- enzymes for biochemical processes, that were feasibly and primarily demonstrated in this experiment by the use of foaming procedure. / 5000

abstract: A mule deer herd exists on the northern rim of the Grand Canyon, located on the North Kaibab Plateau. Historical references to this indigenous mule deer herd presented reports of periodic population irruption and collapse. Partially funded by the Arizona Game and Fish Department and the Arizona Deer Association, examination of herd nutritional and metabolic status from the Fall 2005 - Spring 2008 was completed at the request of AzGFD and ADA. Habitat analysis included forage micro-histological, protein, and caloric content plus whole blood and plasma assays gauging herd metabolic response. Modelling was completed using best management practices wildlife energy demand calculations and principal component analysis. Forage quality analysis and modelling suggest a sufficient amount of nitrogen (N) available (DPI) to the deer for protein synthesis. Energy analysis (MEI) of forage suggest caloric deficiencies are widely prevalent on the north Kaibab plateau. Principal component analysis integrates forage and metabolic results providing a linear regression model describing the dynamics of forage utilization, energy availability, and forage nitrogen supply with metabolic demand and response of the mule deer herd. Most of the plasma and blood metabolic indicators suggest baseline values for the North Kaibab mule deer. Albumin values are in agreement with albumin values for mule deer in the Southwest. I suggest that the agreed values become a standard for mule deer in the Southwestern U.S. As excess dietary N is converted to a caloric resource, a continual state of under-nutrition exists for the deer upon entering the N. Kaibab winter range. The population is exceeding the nutritional resource plane that the winter habitat provides. Management recommendations include implementation of multiple small-scale habitat rehabilitation efforts over time, including invasive juniper (Juniperous osteosperma) and piñon (Pinus edulis) management, prescribed burning to control big sage (Artemesia tridentata) populations, and reseeding treated areas with a seed mix of native shrubs, grasses and forbs. I recommended that the population size of the North Kaibab deer herd is maintained at the current size with natural selection controlling growth, or the population be artificially reduced through increased hunting opportunities. / Dissertation/Thesis / Ph.D. Environmental Design and Planning 2014

INTRODUÇÃO: A resposta do osteossarcoma (OS) à quimioterapia (QT) préoperatória é atualmente o indicador mais sensível para o prognóstico de sobrevida dos pacientes diagnosticados com OS. Esta resposta é avaliada mediante a porcentagem de necrose encontrada pelo patologista após a extração da peça na cirurgia, utilizando- se o índice de necrose tumoral de Huvos, no qual a necrose é expressada percentualmente. Existem estudos correlacionando os valores da fosfatase alcalina (FA) e da desidrogenase lática (DHL) com a sobrevida do paciente. Neste trabalho foi pesquisada a relação que existe entre os valores sanguíneos, pré e pós QT, de FA e DHL, com a porcentagem de necrose tumoral encontrada na peça cirúrgica após a realização da QT pré-operatória. MÉTODOS: Foram estudados 647 prontuários de pacientes tratados pelo Grupo de Oncologia Ortopédica do Instituto de Ortopedia e Traumatologia do Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, no período de 1990 até o inicio de 2013, com diagnóstico anatomopatológico de OS. Destes, 510 foram excluídos por não apresentarem dados completos para a análise posterior. Foram incluídos um total de 137 prontuários. Os valores da FA e da DHL dos pacientes incluídos foram obtidos da realização do estadiamento, antes da QT pré-operatória e dos valores reportados após a finalização da QT pré-operatória. Também foi coletado o grau de necrose tumoral de cada peça extraída na cirurgia de cada paciente. Classificamos os resultados da FA e DHL obtidos em dois grupos. No grupo I os pacientes com valores normais de FA e DHL, no grupo II, os pacientes com valores acima do limite de normalidade. A classificação utilizada para agrupar os valores de sorológicos em investigação foi adaptado do trabalho realizado por Bramer et. al. Foi reportada a porcentagem de necrose tumoral descrita pelos patologistas, obtida das peças extraídas nas cirurgias realizadas pós QT. Esta porcentagem foi classificada de acordo com a classificação de Huvos. Foram calculadas as correlações da necrose da peça cirúrgica (Huvos) com as enzimas pré, pós e alteração (pós-pré) QT e também entre as enzimas, com uso de correlações de Spearman para verificar a existência de correlação entre elas. RESULTADOS: Tanto a FA como a DHL diminuíram nos pacientes estudados, quando comparados os valores pré QT e pós QT. A média da diferença da FA obtida pré QT e pós QT foi de 795,12 U/L sendo o valor pré QT maior ao valor pós QT. A diferença entre os valores da DHL pré QT e pós QT foi em média de 437,40 U/L, sendo o valor pré QT maior ao valor pós QT. Não houve correlação estatisticamente significativa entre o índice de Huvos e os valores de FA e DHL. A ausência da relação foi observada tanto com os valores pré-quimioterapia, quanto com os valores pós-quimioterapia, não obtendo correlação com a alteração das enzimas após a quimioterapia (p < 0,05). CONCLUSÃO: A medição de FA e da DHL não são fatores preditivos sobre a resposta tumoral à quimioterapia préoperatória em pacientes portadores de osteossarcoma / BACKGROUND AND AIMS: The osteosarcoma´s (OS) response to pre surgical chemotherapy (CT) is actually the most sensible predictor for life prognosis in patients diagnosed with OS. This response is evaluated by the chemotherapy-induced necrosis percentage found by the pathologist after the removal of the surgical piece, using the Huvos tumor necrosis (TN) index, in which the necrosis is expressed in percentage. There are studies that correlate the alkaline phosphatase (AP) and the lactate deshydrogenase (LDH) with the patient\'s life prognosis. In this study we researched the relationship between the serum levels of pre and post CT of AP and LDH and the percentage of TN found in the surgical piece after the pre surgical CT. METHODS: This is a retrospective study in which we studied 647 medical history files with anatomopathologic diagnosis of OS, treated by the Orthopedic oncology group of the Orthopedic and Traumatology Institute of the Hospital das Clinicas of the Medical School of the University of São Paulo between 1990 and 2013. Out of these, 510 were excluded for not having complete data for posterior analyses. We included 137 files in the study. The AP and the LDH values were obtained before and after pre surgical CT. We also obtained the degree of chemotherapy-induces TN obtained from the surgical piece. We classified the AP and the LDH into two groups. In the first group the patients with normal AP and LDH values and in the second group those with values above normal limit. This classification was adapted from the study published by Bramer et al. We classified the percentage of TN obtained from the removed surgical pieces after CT, using the Huvos index. We calculated the correlation between the TN with the pre, post and pre-post difference CT enzymes, using the Spearman correlation. RESULTS: The AP and the LDH decreased when comparing the pre CT values to the post CT values. The mean of the difference was of 795.12 U/L, the pre CT value being higher than the post CT. The difference between the LDH values pre and post CT was of 437.40 U/L, being the pre CT higher than the post CT. There was no statistically significant correlation between the Huvos index and the AP and LDH values. This was observed with the pre CT as well as with the post CT values (p < 0,05). CONCLUSIONS: The values of AP and LDH are not predictors for the tumor\'s chemotherapy-induced necrosis to pre surgical CT in patients with osteosarcoma

The purpose of the study was to elucidate the mechanisms by which polyunsaturated fatty acids (PUFAs) prevent bone loss. MG-63 human osteoblasts and MC3T3-E1 murine osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) as well as oestrogen (E2) and parathyroid hormone (PTH) and the effects thereof tested on a variety of biological parameters characteristic of osteoblasts. These parameters included prostaglandin E2 (PGE2) synthesis, proliferation, differentiation to mature mineralising osteoblasts as well as osteoprotegerin (OPG) and receptor activator of nuclear factor êB ligand (RANKL) secretion. Results showed that AA stimulates PGE2 production significantly in both cell lines. Stimulated PGE2 production by MC3T3-E1 cells however, was significantly higher, which might be attributed to auto-amplification by PGE2 itself in this cell line. Pre-incubation of the MG-63 cells with cyclo-oxygenase (COX)-blockers inhibited PGE2 production significantly, suggesting that both COX enzymes were involved in PGE2 synthesis. The number of functional osteoblasts is important for bone formation therefore in vitro osteoblastic cell proliferation was investigated. In contrast to the hormones E2 and PTH, both AA and DHA inhibited proliferation significantly. The AA-mediated anti-proliferative effect is possibly independent of PGE2 production, as PGE2 per se had little effect on proliferation. DHA inhibited proliferation of MG-63 cells more severely, which might be attributed to the osteosarcoma nature of the MG-63 cells. The anti-proliferative effect of these PUFAs might be attributed to modulation of cell cycle progression or anti-mitotic effects of PUFA peroxidation products. Morphological studies showed apoptotic cells after DHA exposure in MG-63 cells. There is a reciprocal relationship between reduced proliferation and the subsequent induction of cell differentiation in vitro. High basal levels of alkaline phosphatase (ALP) activity, a marker of the mature mineralising osteoblastic phenotype, were detected in MC3T3-E1 cells. Long-term exposure to AA inhibited ALP activity in these cells. This process might be PGE2-mediated. Exposure to PUFAs, however, did not compromise the ability of the MC3T3-E1 cells to differentiate to mature mineralising osteoblasts. In contrast with MC3T3-E1 cells, MG-63 cells demonstrated low basal ALP activity and were unable to differentiate to mature mineralising osteoblasts. In the absence of osteogenic-inducing supplements, PUFAs induced adipocyte-like features that might be due to the expression of high levels of PPARã in this cell line. Lipid-filled vacuoles were absent in the MC3T3-E1 cells suggesting that the MC3T3-E1 cell line may not express PPARã mRNA. The study furthermore demonstrated that PUFAs are able to modulate OPG and RANKL secretion in osteoblasts. AA inhibited OPG secretion dose-dependently in both cell lines, this could be PGE2-mediated. AA dose-dependently stimulated soluble RANKL (sRANKL) secretion in MC3T3-E1 cells thereby affecting the OPG/RANKL ratio in a negative way, supporting various reports that AA and PGE2 do cause bone resorption. No sRANKL could be detected after exposing the MC3T3-E1 cells to DHA suggesting that DHA could be protective to bone. In conclusion, contrary to in vivo evidence, this in vitro study could not indisputably demonstrate protective effects of PUFAs on the osteoblastic cell lines tested. / Thesis (PhD (Physiology))--University of Pretoria, 2006. / Physiology / unrestricted

As integrinas são proteínas de membrana envolvidas em diversos processos biológicos como embriogênese, inflamação e agregação plaquetária. A alteração de seu padrão de expressão está relacionada com processos patológicos como trombose e câncer, fazendo das integrinas ótimos indicadores da progressão destas doenças. Assim, a integrina &#945;2&#946;1 pode ser considerada como indicadora de angiogênese, sendo expressa nas células endoteliais durante o crescimento de novos vasos. Já a ausência da &#945;IIb&#946;3 em plaquetas está relacionada com a doença de Glanzmann, caracterizada por uma agregação plaquetária deficiente, e sua presença em outros tipos celulares está relacionada com processos de metástase e invasão de tecidos. Estas integrinas contêm antagonistas naturais nos venenos de serpentes da família VIPERlDAE: As RGD-disintegrinas bloqueiam a integrina &#945;IIb&#946;3 e as ECD-disintegrinas bloqueiam a integrina &#945;2&#946;1. Estas últimas formam parte de metaloproteinases com múltiplos domínios de aproximadamente 50 kDa. Interessados em desenvolver marcadores moleculares para estas integrinas, direcionamos nossos objetivos para: (1) a elucidação da região mínima das ECD-disintegrinas com atividade biológica e (2) construção de biomarcadores para a integrina &#945;2&#946;1 utilizando a região elucidada, e para a integrina &#945;IIb&#946;3 utilizando uma RGD-disintegrina. Neste trabalho conseguimos determinar uma região de 49 resíduos na porção C-terminal do domínio ECD-disintegrina da jararagina que reage com um anticorpo neutralizante das atividades hemorrágica e de ligação ao colágeno da proteína. Esta região foi subclonada e expressa em fusão com a fosfatase alcalina de E. colí, mas não teve funcionalidade como marcador. No entanto, a RGD-disintegrina eristostatina em fusão com a fosfatase alcalina mostrou bifuncionalidade, apresentando tanto atividade disintegrina como atividade catalítica. Além disso, verificamos sua seletividade pela integrina &#945;IIb&#946;3 e padronizamos um ensaio direto de dot blot para a presença dessa integrina em plaquetas, com um único passo de incubação. / Integrins are membrane proteins involved in biological processes such as embriogenesis, inflammation and platelet aggregation. The alteration of their expression pattem is related to pathological disorders such as thrombosis and cancer, making integrins suitable indicators of the progression of these diseases. Thus, the &#945;2&#946;1 integrin, which is expressed in endothelial cells during capillary growth, may be an indicator of angiogenesis. The absence of &#945;IIb&#946;3 integrin in platelets is related to Glanzmann disease, characterized by defective platelet aggregation and its expression in other cellular types is related with metastasis and tissue-invasion processes. These integrins contain natural antagonists in venoms from the VIPERIDAE snake family: RGD-disintegrins, which block the &#945;IIb&#946;3 integrin and ECD-disintegrins, which block the &#945;2&#946;1 integrin. The latter forming part of multidomain metalloproteinases of approximately 50 kDa. In order to develop molecular markers for integrins, we have directed our objectives at: (1) determining the minimal region of ECD-disintegrins with biological activity and (2) engineering biomarkers for the &#945;2&#946;1 integrin using the previously determined minimal region and for the &#945;IIb&#946;3 using an RGD-disintegrin. In this work we have determined a 49-residue region located in the C-terminus of jararhagin ECD-disintegrin domain, which reacts with a neutralizing antibody of hemorrhagic and binding to collagen activities of the protein. This region was subcloned and expressed in fusion with E. coli alkaline phosphatase, but did not present a marker activity. On the other hand, the RGD-disintegrin eristostatin fused to alkaline phosphatase showed bifunctionality, presenting both, disintegrin and catalytic activities. Furthermore, we have characterized its selectivity for the &#945;IIb&#946;3 integrin and we have standardized a direct dot blot assay with one-step incubation for the presence of this integrin in platelets.

Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures (REPs) are a type of endodontic treatment aimed at replacing damaged tooth structures, including dentin and root structures, as well as cells of the pulp-dentin complex. Double antibiotic paste (DAP) has been shown to be efficacious in achieving disinfection of the root canal system while minimizing cytotoxicity to dental pulp stem cells (DPSCs). Hydroxyapatite (HA) is an extracellular, mineralized component of bone that has shown much promise as a scaffold in the field of regenerative medicine. Objective: The objective of this study was to evaluate the effects of HA in a DAP loaded methylcellulose (MC) carrier on the differentiation and mineral deposition of DPSC over time. Materials and Methods: DPSCs were plated in 24-well plates with culture media. The following day, semi-permeable 0.1 m chambers were inserted into the wells to separate the reservoirs and permit delivery of medicaments. 100 L treatment paste composed of MC with 1% DAP and either 0.5% or 1.0% nano-HA was added, followed by additional culture media. After 3 days of treatment, medicaments were removed and DPSCs were cultured for an additional 9 days with replacement of media every 3-4 days. At Day 12, DPSCs were evaluated for alkaline phosphatase (ALP) activity using a biochemical assay and mineral deposition using an Alizarin Red S Ca2+ staining assay (4 wells/group). Comparisons between groups were performed using one-way analysis of variance (ANOVA) with a 5% significance level used for all tests. Results: A trend towards increased ALP and mineral deposition activity was noted among the groups with HA added to DAP with MC. Although these trends were not statistically significant, a trend towards increased ALP and mineral deposition was observed after 3-day medicament exposure. The results were similar to previous findings using 7-day medicament treatments. Conclusion: The addition of HA showed a trend towards improved differentiation and mineral deposition of DPSCs compared to DAP with MC. Although additional studies are required, these results showed suggest that even with a shortened treatment time, increased differentiation and mineral deposition of DPSCs may be possible. This study provides additional support that low concentration DAP in a MC carrier has potential application in regenerative endodontic procedures. The novel addition of HA may provide additional osteogenic potential.

El objetivo de este estudio fue evaluar los factores asociados a la impactación del tercer molar inferior en pacientes atendidos en una clínica universitaria de Chiclayo durante el año 2023. En este estudio se incluyó 120 participantes que contaban con al menos un tercer molar inferior impactado visible en una radiografía panorámica, donde se evaluó el grado de impactación según Pell & Gregory y el espacio retromolar; además, se tomó una muestra sanguínea donde se determinó el grupo sanguíneo y el nivel de fosfatasa alcalina en sangre. Los resultados mostraron una impactación de los niveles A (47.5%), B (40.8%) y C (11.7%), mayor prevalencia de impactación en el sexo femenino y no se encontró una correlación positiva entre la fosfatasa alcalina, grupo sanguíneo, espacio retromolar y la impactación del tercer molar mandibular. Se concluye que no existe relación entre la fosfatasa alcalina, el grupo sanguíneo, el espacio retromolar y la impactación del tercer molar inferior. / The objective of this study was to evaluate the factors associated with the impaction of the lower third molar in patients treated at a university clinic in Chiclayo during the year 2023. This study included 120 participants who had at least one impacted lower third molar visible in a panoramic radiograph, where the degree of impaction according to Pell & Gregory and the retromolar space were evaluated; In addition, a blood sample was taken where the blood group and the level of alkaline phosphatase in the blood were determined. The results showed an mpaction of levels A (47.5%), B (40.8%) and C (11.7%), a higher prevalence of impaction in the female sex and no positive correlation was found between alkaline phosphatase, blood group, space retromolar and impaction of the mandibular third molar. It is concluded that there is no relationship between alkaline phosphatase, blood group, retromolar space and impaction of the lower third molar.

O objetivo deste estudo foi analisar o metabolismo ósseo de pacientes com proteinúria glomerular sem uso prévio de drogas que afetassem esse metabolismo. Dezessete pacientes foram estudados com biópsia óssea para análise histomorfométrica e fragmentos ósseos foram obtidos para cultura de célula (n=13) na qual nós avaliamos proliferação de osteoblasto. A comparação dos achados histomorfométricos a controles de literatura demonstrou uma diminuição da remodelação óssea e comprometimento de sua microarquitetura. Corroborando com esse resultado houve diminuição da proliferação dos osteoblastos dos pacientes quando comparados a controles (n=5) doadores de órgãos. Análise bioquímica revelou correlação negativa da 25(OH)D3 com a proteinúria e positiva com a proliferação dos osteoblastos em cultura / The objective of this study was to analyze bone metabolism in proteinuria glomerular patients not having previously used drugs affecting bone metabolism. Seventeen patients were studied with histomorphometric analysis of bone biopsies and bone fragments were obtained for cell culture (n = 13), in which we evaluated osteoblastic proliferation. Comparing patients to controls of literature indicate reduced bone remodeling and altered bone microarchitecture. In corroboration, mean osteoblast proliferation was lower in patient samples when compared with those for normal osteoblasts obtained from age-matched, gender-matched donor organs (n = 5). Concentrations of 25-hydroxyvitamin-D3 correlated negatively with proteinuria and positively with osteoblast proliferation in culture

Die Basis für den therapeutischen Einsatz elektrischer Felder bei der Behandlung von Knochenbrüchen liegt in der Existenz von belastungsabhängigen elektrischen Potentialen im Knochen. Die zellulären Wirkungsmechanismen sind jedoch unverstanden. Um zu untersuchen, ob und wie elektrische Felder Wachstum und Differenzierung von Knochenzellen beeinflussen, wurden humane Osteosarcomazellen (HOS TE85) und aus Minischweinen isolierte primäre Osteoblasten mit elektrischen Feldern im Frequenzbereich von 0.1 Hz bis 100 kHz befeldet und verschiedene zelluläre Parameter zeitabhängig gemessen. Das elektrische Feld wurde mittels platinierten Platinelektroden appliziert, um ausreichend hohe Feldstärken bei gleichzeitig guter Kontrolle über die im Gewebe erzeugte Wellenform zu gewährleisten. Beim Einsatz von Elektroden müssen jedoch elektrochemische Reaktionen an der Elektrodenoberfläche berücksichtigt werden. Daher wurden verschiedene Elektroden für die in vivo- und in vitro-Stimulation mit elektrischen und numerischen Methoden untersucht und verglichen. Das führte einerseits zur Erarbeitung verschiedener Kriterien zur Auswahl geeigneter Elektrodenmaterialien und -geometrien. Andererseits konnten detaillierte Angaben zur effektiven Feldstärke und Feldverlauf in der Zellebene sowie zu elektrochemisch bedingten Nebenwirkungen gemacht werden. Es konnte gezeigt werden, dass elektrische Felder das Signalsystem und den Stoffwechsel von Knochenzellen im gesamten untersuchten Frequenzbereich beeinflussen. Die Richtung des Nettoeffekts war jedoch im oberen und im unteren Frequenzbereich unterschiedlich. Im Frequenzbereich über 1 kHz fanden wir immer eine Erhöhung des Wachstums und der Konzentration der "second messenger" und eine Herabsetzung der Konzentration der reaktiven Sauerstoffverbindungen (ROS). Bei Frequenzen unter 1 kHz wurde dagegen eine Erhöhung der Aktivität der alkalischen Phosphatase und des oxidativen Stress beobachtet. Signifikante Effekte traten jedoch nur bei Feldstärken oberhalb 100 V/m auf. Hinweise auf bevorzugte Frequenzen oder Wellenformen, die für "Fenstereffekte" sprechen würden, konnten nicht gefunden werden. Da alle zellulären Parameter an einem einheitlichen System gemessen wurden, ließen sich die Ergebnisse in ein Modell der feldinduzierten Signaltransduktion integrieren. Prinzipiell muss man von einem gemeinsamen Effekt mehrerer Signalwege ausgehen. Dabei kann die Erhöhung der intrazellulären Kalziumkonzentration als ein genereller Mechanismus der Feldwirkung angesehen werden. Dafür sind sowohl der Influx als auch die Freisetzung von Kalzium aus intrazellulären Speichern verantwortlich. Es konnte weiterhin gezeigt werden, dass auch cGMP und Prostaglandin E2, jedoch nicht cAMP an der Vermittlung der Feldwirkung beteiligt ist. Außerdem wurde die Translokation der mitogen-aktivierten Proteinkinase (MAPK) beeinflusst. Trotzdem sind die beobachteten Effekte relativ schwach im Vergleich zu publizierten klinischen Erfolgen. Es muss daher in vivo Verstärkungsmechanismen geben, die durch Untersuchungen an Zellkulturen nicht erfasst werden können. / The therapeutic use of electric fields in the treatment bone fracture healing is based on the existence of different load dependent electric potentials in bone. However, the cellular mechanisms of field action are still not understood. We investigated the effect of electric fields on proliferation and differentiation of human osteosarcoma cells (HOS TE85) and primary osteoblasts isolated from mini pigs. The cells were exposed to electric fields (0.1 Hz - 100 kHz) and various cellular parameters were measured. The field was applied using platinized platinum electrodes ensuring sufficiently strong electric fields and a well controlled wave form in the tissue. Nevertheless, in this case electrochemical reactions which occur at the electrode surface have to be taken into account. That’s why various electrodes for in vivo and in vitro stimulation are characterized by electrical and numerical methods leading not only to criteria for choosing electrode materials and geometries, but also providing detailed knowledge about the electric field induced in the tissue and electrochemically induced side effects. We found that cellular signaling as well as proliferation and differentiation of bone cells are influenced by electric fields in the whole investigated frequency range. Proliferation and the second messenger concentrations are increased at frequencies above 1 kHz, whereas below 1 kHz the alkaline phosphatase activity is increased. Significant effects were found only for electric fields above 100 V/m, but we could not find any hints on preferred frequencies or wave forms ruling out "window effects". Because all cellular parameters were measured within one experimental system, the results can be summarized in a signal transduction model for the action of electric fields. Generally, a common action of multiple signaling pathways has to be assumed. The increase of the intracellular calcium concentration can be considered as a general mechanism of field action. This increase is caused by calcium release from intracellular calcium stores as well as influx of extracellular calcium. Additionally, the concentration of cGMP and prostaglandin E2, but not that of cAMP is increased by the electric field. Furthermore, the translocation of the mitogen activated protein kinase (MAPK) is influenced. Nevertheless, the field effects found are weak compared to those published in clinical studies. We suppose that there are amplifying mechanisms in vivo which could not be seen during in vitro investigations.