O eixo GH/IGF-I (hormônio do crescimento - fatores de crescimento insulina-símile) é um sistema de mediadores de crescimento, receptores e proteínas de ligação que controlam o crescimento somático e tecidual em muitas espécies e programas de exercícios estão relacionados a esta função anabólica por meio da ação deste eixo. Partindo deste pressuposto, o objetivo do presente estudo foi analisar a cinética das concentrações séricas de IGF-I, IGFBP-3 e ALS, das citocinas IL-6, IL-10 e TNF-? e comparar com o desempenho físico e a composição corporal de nadadores adolescentes nas diferentes fases de uma temporada de treinamento. A amostra foi composta por 9 nadadores adolescentes do sexo masculino com idade entre 16 e 19 anos, que faziam parte de uma equipe de natação universitária da cidade de Ribeirão Preto. Os níveis de IGF-I, IGFBP-3, ALS, IL-6, IL-10 e TNF-? foram determinados na fase básica, específica e final do polimento. A fim de comparar a cinética do sistema IGF-I/IGFBP-3-ALS e das citocinas com o desempenho físico e a composição corporal dos atletas, também foram avaliadas a resistência aeróbia em nado livre, a aptidão anaeróbia em nado atado, a massa corporal, o percentual de gordura e a massa magra nos diferentes momentos da temporada. Para a análise da cinética do sistema IGF-I/IGFBP-3-ALS e das citocinas nas diferentes fases do treinamento e antes e após a sessão de treino padronizada foram utilizados os testes não-paramétricos de Friedman e Wilcoxon, respectivamente, adotando-se um nível de significância de 0,05. A correlação entre duas variáveis foi analisada por meio do coeficiente de correlação de Spearman. Resultados: o IGF-I é sensível aos efeitos agudos e crônicos do treinamento, apresentando um comportamento em duas fases ao longo da temporada - uma fase catabólica (fase específica) e uma fase anabólica (polimento). O IGFBP-3 mostrou-se sensível apenas aos efeitos crônicos do treinamento, não sendo possível identificar um comportamento diferenciado intra-fase (pré x pós). A ALS manteve-se constante, mostrando que não foi afetada pelos efeitos agudos ou crônicos do treinamento. A IL-10 mostrou-se sensível aos efeitos agudos e crônicos do treinamento, aumentando significativamente durante a fase de polimento. A IL-6 não apresentou variação significante em resposta a uma sessão de treinamento (efeito agudo), apesar disso, apresentou uma correlação negativa com o IGF-I na fase específica de treinamento. O TNF-? apresentou concentrações mais estáveis ao longo da temporada. A composição corporal e a condição cardiorrespiratória dos nadadores não foram alteradas ao longo da temporada. O Pico de Força e a Força Média acompanharam a variação do IGF-I e do IGFBP-3, ou seja, diminuíram durante a fase específica e apresentaram uma elevação significativa durante o polimento. Conclusão: o IGF-I e a IGFBP-3 podem ser considerados um dos sensíveis marcadores de estado de treinamento, podendo orientar treinadores e atletas a dosar a intensidade de treinamento, especialmente de jovens que se encontram na puberdade. Foi possível observar algumas faces de interação com as citocinas, onde a soma das ações das citocinas provavelmente explicariam as mudanças nas concentrações séricas de IGF-I e IGFBP-3. / The GH/IGF-I axis is a system of growth mediators, receptors, and binding proteins that regulate somatic and tissue growth; and it has been shown that exercise programs are related to the anabolic function of this axis. The aim of this study was to analyse the kinetics of serum IGF-I, IGFBP-3, ALS, IL-6, IL-10 and TNF-? concentration in adolescent swimmers at different stages of a training season, and compare them with physical performance parameters and body composition of the athletes. Nine male athletes, aged 16 to 19 years and who trained regularly throughout the season were included in this study. Serum IGF-I, IGFBP-3, ALS, IL- 6, IL-10, and TNF-? concentrations were recorded before and after (pre x post) standardized training sessions during the different stages of a training season (extensive x intensive x tapering). Aerobic endurance in freestyle, anaerobic fitness in tied swimming (Peak Force and Average Force), body mass, fat percentage, and lean body mass were also analysed at the different stages of training in order to compare the behaviour of the IGF-I/IGFBP/ALS system with the physical performance and body composition of the athletes. Variations in the cytokines and IGF-I/IGFBP-3/ALS system before and after a standardized training session, and at the different stages of training were analysed by the Wilcoxon and Friedman nonparametric tests, respectively. The correlation between the two variables was analysed by the Spearman\'s correlation coefficient. Significance was considered at P<0.05. Results: IGF-I was sensitive to the acute and chronic effects of training, exhibiting biphasic behaviour throughout the season. The catabolic phase was characterized by a reduction in serum IGF-I levels during the intensive stage while the anabolic phase was marked by an increase in posttraining serum IGF-I levels during the tapering stage. IGFBP-3 was only sensitive to the chronic effects of training, with a reduction in post-training serum levels during the intensive stage and an increase during the tapering stage. No difference was observed in pre- or posttraining IGFBP-3 levels at the different stages. ALS and TNF-? remained estable throughout the training season. IL-10 was sensitive to the acute and chronic effects of training, increasing significantly during the tapering phase. IL-6 showed no variation in response to a training session (acute effect); nevertheless, it had a negative correlation to IGF-I at the intensive phase. The body composition and cardiorespiratory function of the swimmers remained unaltered throughout the season. Peak Force and Average Force followed IGF-I and IGFBP-3 variations, with a decrease during the intensive stage and a significant increase during the tapering stage. The body composition and cardiorespiratory condition of the swimmers did not vary significantly throughout the season, exhibiting behaviour independent of IGF-I or IGFBP-3. Conclusion: Serum IGF-I and IGFPB-3 concentrations have proven to be sensitive markers of training status and, thus, may be used as guides for coaches and athletes in the challenging task of modulating training intensity in young athletes. The cytokine interactions observed suggest that the combined effects of these cytokines may be responsible for the serum IGF-I and IGFBP-3 variations recorded.

Os hormônios de crescimento, principalmente os do eixo GH/IGF-I são responsáveis pelo crescimento tecidual e estrutural desde o nascimento. O GH produzido na hipófise é um hormônio com funções metabólicas e anabólicas e é o principal estimulador da síntese e liberação do IGF-I no fígado que tem suas ações endócrinas, parácrinas e autócrinas mediadas pelas IGFBPs. O exercício físico está intimamente ligado à função anabólica, estimulando a secreção e a ação dos hormônios do eixo GH/IGF-I.Existe a hipótese de haver um comportamento bifásico do eixo durante uma temporada de treinamento,caracterizado por uma fase catabólica, seguida de uma fase anabólica dependendo das fases do treinamento, porém vários estudos têm resultados controversos. O objetivo desse projeto foi investigar o impacto de uma temporada de treinamento em atletas de voleibol sobre o eixo GH/IGF-I e IGFBP-3 e sua relação com desempenho em testes físicos. A amostra foi composta por 10 jogadores de Voleibol categoria adulto da equipe de Franca-SP que foram analisados no início(A1), durante(A2) e ao final(A3) de 15 semanas de treinamento. Foram analisados dados antropométricos, altura de salto e potência de membros inferiores no Squat Jump (SJ), Counter Moviment Jump(CMJ)e Drop Jump 40 cm (DJ40), Índice de Força Reativa (IFR), Razão de Utilização Excêntrica (RUE) e concentrações deIGF-I e IGFBP-3. Para as análises estatísticas foram utilziadas ANOVA de medidas repetidas, Magnitude de Efeito (ES) e Probabilidade Quantitativa de Chances (QC). Os resultados mostram redução dos valores de Massa Coporal Total (MCT), Percentual de Gordura Coporal (%GC), Massa Magra (MM) e Massa Gorda (MG), com menor valor em A3, os resultados dos saltos apresentaram aumento linear com diferença estatísitca (p < 0,05) no DJ40 em A3. A sessão de treino não teve influência sobre as concentrações de IGF-I e IGFBP-3, indicando que a intensidade de disputa dessa modalide não é capaz de alterar as concentrações desses hormônios. Não foi verificada diferença estatísitca (p < 0,05) entre as coletas durante o período de treinamento, mas, as análises de ES e QC indicam tendência de aumento do IGF-I em A3. O comportamento bifásico do eixo GH/IGF-I não foi observado nesse estudo, possivelmente devido a forma de planejamento do período de treinamento, contudo, o IGF-I apresentou maiores concentrações em A3 coincidindo com os maiores resultados de altura de salto. Com esses resultados foi possível inferir que a concentração de IGF-I está correlacionada positivamente com o desempenho físico de atletas de voleibol e que a redução ou a incapacidade de aumento de IGF-I pode ser um sinal de alerta para atletas e treinadores. Ainda assim, são necessários novos estudos para investigar se o treinamento terá efeitos semelhantes durante longos períodos de treinamento, períodos de treinamento com maior intensidade, diferentes fases durante o período de preparação ou competição produzirão respostas hormonais semelhantes / Growth hormones, especially the GH/IGF-I axis is responsible for tissue and structural growth from birth. GH produced in the pituitary gland is a hormone with metabolic and anabolic functions and is the main stimulator for the synthesis and release of IGF-I in the liver that has its endocrine, paracrine and autocrine actions mediated by IGFBPs. Physical exercise is closely linked to anabolic function, stimulating the secretion and action of the hormones of the GH/IGF-I axis. There is a hypothesis of a biphasic behavior of the axis during a training season, characterized by a catabolic phase, followed by an anabolic phase depending on the training phases, but several studies have controversial results. The objective of this project was to investigate the impact of a training season on volleyball athletes on the GH/IGF-I axis and IGFBP-3 and its relation to performance in physical tests. The sample consisted of 10 adult category Volleyball players from the Franca-SP team who were analyzed at baseline (A1), during (A2) and at the end (A3) of 15 weeks of training. Anthropometric data, jump height and power of lower body in Squat Jump (SJ), Counter Movement Jump (CMJ) e Drop Jump 40 cm (DJ40), Reactive Force Index (RFI), Eccentric Usability Ratio (EUR) and IGF-I and IGFBP-3 concentrations were analyzed. Statistical analyzes were performed using repeated measures ANOVA, Effect Size test (ES) and Probability of Quantitative Chances(QC). The results show a reduction in Total Body Mass (TBM) values, Percentage of Body Fat (%BF), Lean Mass (LM) and Fat Mass (FM), with a lower value in A3, the jump results showed a linear increase with a statistical difference (p <0.05) in DJ40 in A3. The training session had no influence on the concentrations of IGF-I and IGFBP-3, indicating that the intensity of contention of this modality is not able to alter the concentrations of these hormones. There was no statistically significant difference (p <0.05) between the collections during the training period, but the ES and QC analyzes indicated an upward trend in IGF-I in A3. The biphasic behavior of the GH/IGF-I axis was not observed in this study, possibly due to the planning of the training period, however, IGF-I presented higher concentrations in A3 coinciding with higher jump height results. With these results it was possible to infer that the concentration of IGF-I is positively correlated with the physical performance of volleyball athletes and that the reduction or inability to increase IGF-I may be a warning signal for athletes and coaches. Still, further studies are needed to investigate whether training will have similar effects during long periods of training, more intense training periods, different phases during the preparation or competition period will produce similar hormonal responses.

In den letzten Jahren wurde gezeigt, dass die Serumkonzentration des insulinähnlichen Wachstumsfaktorbindungsproteins-2 (IGFBP-2) bei Krebserkrankungen, die mit dem Verlust des Tumorsuppressorgens PTEN einhergehen, erhöht ist und daher möglicherweise einen Marker für den PTEN-Status und die Aktivität des PI3K/AKT/mTOR-Signalweges darstellt. Schmid et al. haben 2014 einen Patienten mit PTEN-Hamartom-Tumor-Syndrom (PHTS) mit einer heterozygoten PTEN-Keimbahndeletion und massiver Lipomatose beschrieben, bei dem erhöhte IGFBP-2 Serumspiegel gemessen wurden. Ziel dieser Arbeit war es zu analysieren, ob PTEN-defiziente Lipomzellen des Patienten im Vergleich zu Kontrollfettzellen mehr IGFBP-2 produzieren, sowie den Einfluss verschiedener pharmakologischer Inhibitoren des AKT/PI3K/mTOR - und des MAPK- Signalwegs auf die IGFBP-2 Produktion zu untersuchen. In der PTEN-defizienten Lipomzellkultur, gewonnen aus reseziertem Lipomgewebe des Patienten, wurden vergleichbare Mengen an IGFBP-2 wie in den nicht PTEN-defizienten Kontrollzellen gefunden. Die pharmakologische Hemmung der PI3K und AKT bewirkten eine signifikante Senkung der IGFBP-2 Expression und Sekretion, wohingegen sich bei Hemmung der MEK und des mTORC1 keine Effekte zeigten. Diese Ergebnisse weisen darauf hin, dass eine heterozygote PTEN-Deletion in Lipomzellen nicht zu einer erhöhten IGFBP-2 Produktion führt und daher die erhöhten Serumspiegel des Patienten nicht darauf zurückzuführen sind. Des Weiteren bestätigen die in vitro Ergebnisse die klinische Beobachtung, dass unter der Therapie mit dem mTORC1-Inhibitor Rapamycin die IGFBP-2 Serumspiegel des Patienten nicht zurückgingen. Möglicherweise stellt IGFBP-2 jedoch einen geeigneten Verlaufsmarker für eine Therapie mit PI3K- oder AKT-Inhibitoren dar.

Das Mammakarzinom gehört zu den häufigsten Krebserkrankungen der Frau. Die Prognose einer Brustkrebserkrankung richtet sich nach den Stadien der TNM-Klassifikation, dabei ist die Wahrscheinlichkeit eines fortgeschrittenen Stadiums bei später Diagnosestellung erhöht. Daher ist insbesondere die Früherkennung der Erkrankung Gegenstand klinischer Forschung. Das Risiko, an Brustkrebs zu erkranken, liegt in der Normalbevölkerung bei 7-10%. Eine individuelle Erhöhung des Risikos findet sich bei familiärer Belastung bezüglich Brustkrebs, sowie einer früheren Erkrankung an einer Präkanzerose wie des Carcinoma ductale in situ (DCIS), des Carcinoma lobulare in situ (CLIS) und der atypischen ductalen Hyperplasie (ADH). Besonderes Augenmerk in der Krebsforschung liegt zur Zeit auf dem Zusammenhang zwischen Tumorwachstum und der Bildung neuer Gefäße, der Angiogenese. Diese Neubildung von Kapillaren ist in hohem Maße von der Ausschüttung bestimmter Wachstumsfaktoren abhängig, von denen der vaskuläre endotheliale Wachstumsfaktor (VEGF) aufgrund seiner Endothelspezifität der mitogenen Wirkung große Bedeutung besitzt. Aber auch der in fast allen Geweben vorkommende und für viele physiologische Prozesse entscheidende Insulin-like growth factor-1 (IGF-1) und seine Bindungs-proteine (IGFBP-1 bis IGFBP-7) scheinen bei der Tumorentwicklung eine wichtige Rolle zu spielen. Zahlreiche klinische Studien beschäftigen sich daher mit dem Vergleich von Serum-oder Plasmakonzentrationen an VEGF, IGF-1 und seiner Bindungsproteine zwischen Krebspatienten und Kontrollgruppen. Dabei sind für Brustkrebspatientinnen erhöhte VEGF- und IGF-1-Konzentrationen und erniedrigte IGFBP-3-Konzentrationen beschrie-ben. Auch für Patientinnen mit einer DCIS-Erkrankung sind erhöhte VEGF-Konzentrationen gefunden worden. Diese Untersuchung beschäftigt sich mit der Wertigkeit einer Messung der genannten Wachstumsfaktoren zur regelmäßigen Untersuchung von Brustkrebsrisikopatientinnen in der Klinik. Dafür wurde ein Kollektiv von 106 Frauen untersucht, die entweder aufgrund einer familiären Belastung oder der Erkrankung an einer Präkanzerose in der Spezial-sprechstunde der Frauenklinik der Charité behandelt bzw. beraten wurden. Für einen Vergleich innerhalb der Hauptgruppen "familiäre Belastung" und "Präkanzerose" wurde eine Einteilung in Subgruppen nach Menopausenstatus, in der Gruppe der familiär belasteten Frauen zusätzlich nach Höhe des berechneten Risikos und in der Gruppe der Patientinnen mit Präkanzerose zusätzlich nach Antiöstrogenbehandlung und Familienanamnese bezüglich Brustkrebs vorgenommen. Als Kontrollgruppen dienten ein Kollektiv von Mammakarzinompatientinnen sowie ein Normalkollektiv aus 234 gesunden Frauen ohne ersichtliche Risiken. Die VEGF, IGF-1 und IGFBP-3-Konzentrationen wurden mittels eines Enzyme-linked Immunoassays (ELISA) im Serum gemessen und verglichen. Dabei fanden sich für VEGF und IGFBP-3 keine signifikanten Unterschiede zwischen den einzelnen Haupt- und Subgruppen (p>0,05). Für IGF-1 konnte eine signifikante Abhängigkeit der Serumkonzentration vom Menopausenstatus bestätigt werden (p0,05). Auch der Vergleich zwischen den VEGF-Konzentrationen von Risikopatientinnen und dem Normalkollektiv wies keinen signifikanten Unterschied aus (p>0,05). Um die intraindividuelle Konstanz der Werte über einen längeren Zeitraum zu prüfen, wurden 34 Frauen, von denen mehr als ein Serum-Wert vorlag, untersucht. Dabei konnte eine intraindividuelle Streubreite der VEGF-Werte im Mittel von 115 pg/ml (Median 118,5 pg/ml, Range 2-310 pg/ml), der IGF-1-Werte im Mittel von 36 ng/ml (Median 27,5 ng/ml, Range 0-134 ng/ml) und der IGFBP-3-Werte im Mittel von 1792 ng/ml (Median 1587 ng/ml, Range 1-4074 ng/ml) festgestellt werden. Zusammenfassend lässt sich sagen, dass aufgrund der starken interindividuellen Streuung der VEGF-, IGF-1- und IGFBP-3-Werte eine Kontrolle der Serum-konzentration nur nach Bestimmung eines individuellen Referenzwertes von klinischem Nutzen sein kann. Zur Zeit scheint die Messung von VEGF, IGF-1 und IGFBP-3 zur Kontrolle von Risikopatientinnen wenig aussagekräftig zu sein. / Breast Cancer is among the most common female malignancies. The prognosis in the case of breast cancer is made according to the status of the TNM-classification, in which the possibility of an advanced stage is higher in the case of a late diagnosis. This is the reason why the early recognition of the illness receives special attention in clinical research. The average risk among the normal population lies at 7-10%. Individually increased risk is given in the case of family history for breast cancer, having earlier suffered from precancer, a carcinomca ductale in situ (DCIS), a carcinoma lobulare in situ (CLIS) and the atypical ductal hyperplasie (ADH). In cancer research special attention is currently given to the connection between tumour growth and the creation of new blood vessels, called angiogenesis. This new creation of capillaries is highly dependent on the setting free of certain growth factors of which the vascular endothelial growth factor (VEDF) is especially important due to ist endothelspecificity. However the insulin-like growth factor-1 (IGF-1) which occurs in nearly all tissues and is decisive for many physiological processes, and its binding proteins (IGFBP-1 to IGFBP-7) appear to play an important role in tumour development. Many clinical studies therefore occupy themselves with a comparison between the serum or plasma-concentrations at VEGF, IGF-1 and its binding proteins among cancer patients and control groups. These comparisons find higher VEGF and IFG-1 concentrations and lower IGFBP-3-concentrations among breast cancer patients. For patients with a DCIS illness higher VEGF concentrations have also been found. This study looks at the possible clinical use of the measurement of the mentioned growth factors during the regular check up of breast cancer risk patients. For this purpose a collective of 106 women were examined that were being treated or advised during the consultancy of the Charité's women's clinic due to a family cancer risk or having been diagnosed with a precancer. In order to compare among the main groups "family risk" and "precancer" a separation into subgroups according to menopausal status was undertaken, within the group "family risk" an additional separation according to the calculated risk was done and the group "precancer" was further split according to anti-estrogen treament and family anamnesis with regards to breastcancer. A collective of breast cancer patients as well as a collective of 234 healthy women without distinct risks functioned as control groups. The concentrations of VEGF, IGF-1 and IGFBP-3 in the serum were measured with an enzyme linked immunoassays (ELISA) and compared. No significant differences between the separate maingroups and subgroups could be found (p>0,05). In the case of IGF-1 a significant dependency of the concentration in the serum and the menopausal status could be confirmed (p0,05). The comparison between the concentrations of VEGF of risk patients and the healthy collective also did not show any significant differences (p>0,05). In order to check the intraindividual consistency of the values over a longer period of time 34 women, for which there existed more than one serum-value, were examined. Here, intraindividual differences of the VEGF-values of 115 pg/ml (Median 118,5 pg/ml, Range 2-310 pg/ml), the IGF-value of 36 ng/ml (Median 27,5 ng/ml, Range 0-134 ng/ml) and the IGFBP-3-value of 1792 ng/ml (Median 1587 ng/ml, Range 1-4074 ng/ml) was found. In summary it can be said that, due to the high interindividual differences of the values of VEGF, IGF-1 and IGFBP-3, a control of the serum concentration can only be of clinical use if a individual reference value is measured.

Normal somatic cells have a limited proliferative capacity in vivo and in vitro, termed senescence and later, thought to contribute to molecular and cellular organismal aging. There are several studies that demonstrate the importance of the GH/IGF axis in longevity, aging and cellular senescence. One primary component of the IGF signaling involves IGFBP-3. It is well documented that IGFBP-3 levels are significantly increased in senescent human diploid fibroblasts however IGFBP-3 function is not known in this system. Interestingly, Werner syndrome fibroblasts, commonly used as a model of cellular aging, have upregulated IGFBP-3 levels in young and late passage cells compared to age matched normal fibroblasts. It is known that suppression of p38 MAPK activity in WS fibroblasts can reverse the senescence and promotes cell proliferation. As increased IGFBP-3 expression is associated with cellular senescence, and suppression of p38 MAPK can reverse senescence in WS fibroblasts, it is hypothesized that “IGFBP-3 can induce senescence, by activating the p38 MAPK signaling pathway.” Our studies demonstrate IGFBP-3 and novel IGFBP-3R can induce senescence in young fibroblasts, while suppression of IGFBP-3 in pre-senescent fibroblasts, can delay the onset of replicative senescence. We identified ROS accumulation in IGFBP-3/IGFBP-R-induced senescent cells which we speculated may be signaling p38 MAPK activation. Inhibition of ROS accumulation suppressed p38 signaling and prevented IGFBP-3/IGFBP-3R-induced senescence. To evaluate the sequence of activation we inhibited p38 activity prior to senescence induction. Interestingly, p38 inhibition prevented IGFBP-3/IGFBP-3R-induced senescence, suggesting IGFBP-3 signals ROS induction which activates p38 signaling. We next examined the significance of IGFBP-3R in IGFBP-3-induced senescence. Suppression of endogenous IGFBP-3R inhibits IGFBP-3-induced senescence. We aimed to identify a possible regulatory mechanism for IGFBP-3 upregulation. Using sequence analysis software we identified 3 possible highly conserved miRNA sequences aligned to IGFBP-3. miR-19a appeared to have the most significant downregulated expression in late passage fibroblasts compared to early passage. Furthermore, overexpression miR-19a in late passage cells, significantly decreased IGFBP-3 expression, suggesting miR-19a may silence IGFBP-3 expression in senescence. Making a direct mechanistic connection between senescence and aging is significant and unraveling how IGFBP-3/IGFBP-3R can induce senescence could prove beneficial in understanding the aging process.

Two whole genome duplications (2R) occurred early in vertebrate evolution. By using combined information from phylogenetic analyses and chromosomal location of genes, this thesis delineates the evolutionary history of two receptor-ligand systems that expanded by these large scale events. A third whole genome duplication (3R) took place in the teleost fish lineage and has also contributed to the complexity of the gene families. New members of neuropeptide Y (NPY) peptide and receptor families were generated in 2R and 3R. Evolutionary comparisons show that the ancestral teleost fish had four peptides; subsequently, differential losses of the peptide genes occurred. In zebrafish the peptides and receptors display differences in tissue distribution and have  evolved binding preferences. In the frog Silurana tropicalis three peptides and six receptors werev identified, also displaying some differences in tissue distribution and receptor-ligand preferences. The findings in these experimental animals highlight both evolutionary conservation and lineage-specific features of the NPY system. The opioid system consists of four receptors and several peptides originating from four precursors. These results show that the receptor family was formed in 2R and 3R and that 2R together with one local duplication gave rise to the peptide family. The ancestral receptor and peptide genes were located on the same chromosome, suggesting coevolution. The Hox gene clusters, important in early development, provided the first strong evidence for 2R. Several neighboring gene families were analyzed and found to have expanded in 2R and 3R. In depth analyses of insulin-like growth factor binding protein (IGFBP) and voltage-gated sodium channel (SCN) gene families illustrates the importance of local duplications in combination with whole genome duplications in the formation of gene families. These findings provide additional strong evidence for two genome duplications in early vertebrate evolution and show that these events generated many new genes that could evolve new or more specialized functions.

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Understanding how insulin-like growth factor-I (IGF-I) signaling in mammary epithelial cells may be modified or interrupted by modifications in the cellular environment may lead to 1) methods to increase the growth and proliferation of normal mammary epithelial cells for an increase in the amount of milk produced on a per animal basis or to 2) the development of medical interventions to disrupt the growth and proliferation of cancerous mammary epithelial cells. IGF-I, a signaling protein provided by stromal cells and through the bloodstream, stimulates the proliferation of mammary epithelial cells and is crucial for mammary development. Collagen I is an extracellular matrix protein (ECM) found in skin and in other connective tissues throughout the body. The guiding question in this dissertation was how IGF-I signaling and how binding protein profile were influenced by autocrine IGF-I and by collagen I. The MAC-T cell line was chosen as the cell model utilized in these investigations because it is an immortalized bovine mammary epithelial cell line known to retain hormonal responsiveness to IGF-I. It was hypothesized that the production of IGF-I by mammary epithelial cells (autocrine secretion) would alter the response of these cells to additional IGF-I by de-sensitizing the IGF-I receptor on the cell surface. The normal mammary epithelial cell does not produce IGF-I and responds to IGF-I supplied either by stromal cells (paracrine pathway) or through the bloodstream (endocrine pathway). The IGF-I secreting bovine mammary epithelial cell line was investigated for the response of the cells to autocrine IGF-I, and the response was compared to the normal, parental cell line. To examine the effect of autocrine IGF-I on the cells, IGF-I was added both to MAC-T cells and to cells transfected to secrete IGF-I (SV40-IGF-I). The cell response of the two cell lines was compared using microphysiometry, a tool that measures IGF-IR stimulation by detecting resultant extracellular acidification. It was found that the SV40-IGF-I cell line retains IGF-I receptor sensitivity, yet, unlike the parental cell line, does not proliferate in response to IGF-I. Both cell lines exhibited increased protein synthesis in response to IGF-I as measured by amino acid uptake (AIB incorporation), but the lack of a proliferation response to additional IGF-I in the SV40-IGF-I cell line suggested that the autocrine cell line exhibited an un-coupling of IGF-IR stimulation with downstream cell proliferation. Both autocrine IGF-I and added IGF-I increased the amount of IGFBP-3 secreted by the cells into growth media. Additionally, it was hypothesized that the presence of collagen I, an important ECM protein, would alter the cell production of insulin-like binding protein-3 (IGFBP-3), a protein that modulates IGF-I interaction with the IGF-I receptor (IGF-IR). The literature reports that surface substrate can affect the phenotypic expression of cells, presumably via interaction with integrins, the cell surface receptors that connect cells to ECM proteins and that are responsible for cell adhesion and for cell migration. It was hypothesized that the MAC-T cells would interact with a collagen I surface (possibly via the a2b1 integrin) and that the stimulation of this transmembrane signaling molecule would in turn impact the IGF-I signaling pathway. Comparison studies on tissue culture plastic, collagen I BIOCOAT, and collagen I gel were performed. It was found that collagen I gel increased IGFBP-3 secretion and decreased insulin-like binding protein-2 (IGFBP-2) secretion in MAC-T cells. The collagen I BIOCOAT did not induce this response. Additional studies were performed to determine if there were differences in IGF-IR phosphorylation, exogenous IGF-I utilization, and IGFBP mRNA production by cells cultured on the three different substrates. IGF-IR phosphorylation was only evident following the addition of IGF-I to MAC-T cells on all three substrates. Measurement of residual IGF-I present in the cultured media of cells on all three substrates by radioimmunoassay did not reveal any differences in the amount of IGF-I present. Northern blot analysis revealed that the addition of IGF-I caused an increase in detected IGFBP-3 mRNA and a decrease in detected IGFBP-2 mRNA across all three surfaces. As measured by ligand blot analysis, cells cultured on all three surfaces showed an increase in IGFBP-3 protein in the media with IGF-I addition, and the collagen I gel showed more IGFBP-3 protein than the other two surfaces. However, cells cultured on collagen I gel showed a decrease in IGFBP-2 protein expression compared to cells cultured on tissue culture both with and without the addition of IGF-I. Cells cultured on tissue culture plastic and on collagen I BIOCOAT did not show a decrease in IGFBP-2 to correspond with the decreased IGFBP-2 mRNA detected in the presence of IGF-I on all three substrates. DNA assays to detect cell proliferation revealed no differences in cell DNA content in the absence of exogenous IGF-I and revealed similar increases in response to IGF-I addition on all three substrates. In conclusion, it was found that autocrine IGF-I un-couples increased IGF-IR stimulation by exogenous IGF-I from a downstream cell proliferation response. IGFBP-3 inhibits the ability of IGF-I to interact with the IGF-IR in MAC-T cells and inhibits subsequent cell proliferation. Collagen I gel increases IGFBP-3 secretion and decreases IGFBP-2 secretion by MAC-T cells. The relevance of this work is that it adds to the body of knowledge in understanding cellular function in mammary epithelial cells. It is known that the growth and the maintenance of living tissue are dependent on an intricate system of intercellular and intracellular responses which are orchestrated by the movement and secretion of proteins and other molecules. Goals of understanding mammary epithelial cell function include having the means to find ways to increase cell functionality via bioengineering and having the means to find ways to restore cells to normal function in disease processes such as cancer. / Ph. D.