Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 60,000 annual diagnoses in the U.S. and approximately 500,000 worldwide. About 90% of these individuals receive radiation therapy, and salivary hypofunction and xerostomia occur in 60-85% of these patients due to irreversible damage to the salivary glands. Current preventative and palliative care fail to improve quality of life, accentuating the need for regenerative therapies. Stem/progenitor-cell based therapies have been proposed to regenerate the irradiated glands; however, the identity of stem and progenitor cells in the adult salivary glands has remained somewhat elusive. Moreover, it is unclear how salivary progenitors respond to radiation and whether they can be stimulated to effectively reinstate salivary function. The second chapter of the present study describes the development of a label-retaining assay in salivary glands using EdU. The label-retaining cells (LRCs) identified in murine salivary glands have proliferative potential in vitro and expressed markers of putative salivary progenitors, such as Keratin 5, Keratin 14, and c-Kit. Interestingly, LRCs were still present 30 days following radiation, when chronic loss of saliva is evident. The significance of these findings lies in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining salivary gland homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. In the following chapter, we show that a unique population of murine salivary gland LRCs undergo compensatory proliferation in response to radiation. The initiation of compensatory proliferation is tightly associated with inactivation of the kinase aPKCζ and increased nuclear localization of YAP. This part of the study provides novel insights into the regulation of function of salivary gland progenitors, which can be utilized for the development of therapeutic agents to treat salivary hypofunction. Finally, the last chapter describes how the mechanisms found to initiate compensatory proliferation in acinar LRCs as a response to radiation are involved in the regeneration of salivary glands with IGF-1. Administration of IGF-1 post-radiation restores salivary function in mice, but the mechanisms of regeneration are still unknown. Here, we show that IGF-1 requires aPKCζ to restore saliva production. Further, IGF-1 inhibits nuclear translocation of Yap in an aPKCζ-dependent fashion. We propose that a tightly regulated balance in the levels of aPKCζ and Yap in acinar LRCs has to be maintained in order to restore function following radiation. In conclusion, the findings from this study provide new knowledge in regards to the regulation of function of salivary progenitors during a state of injury (by radiation) and during regeneration (with IGF), and offer potential targets of study for the development of new therapeutics for salivary gland dysfunction. Future studies will determine whether aPKCζ and Yap can be effectively targeted in salivary progenitors to restore salivary function in head and neck cancer patients who receive radiation therapy.

Advanced melanoma poses a major therapeutic challenge, and despite the development of recent promising therapeutic agents, resistance to treatment remains a problem. Until recently, despite low response rates, alkylating agents dacarbazine and temozolomide (TMZ) were the standard of care for the treatment of advanced melanoma. The cytotoxic effects of these agents relies upon the formation of alkylated base lesions such as O⁶-methylguanine (O⁶MeG), which is repaired by a protein implicated in TMZ resistance called O⁶-methylguanine-DNA methyltransferase (MGMT). Failure to resolve such alkylated bases results in DNA replication-associated double-strand breaks (DSBs). The type 1 insulin-like growth factor receptor (IGF-1R) mediates a number of characteristics common to cancers, including proliferation and survival, and evidence suggests it may also contribute to resistance to many anticancer agents. The aims of this study were to test whether melanoma cells could be sensitized to TMZ by small molecule IGF1R inhibitors, and to explore the mechanism of any chemosensitization. This study found an association between basal IGF1R phosphorylation and in vitro TMZ resistance in seven MGMT-proficient melanoma cell lines, suggesting that IGF1R activation may be linked with TMZ resistance. Furthermore, IGF1R inhibition caused dose-dependent sensitization of melanoma cells to TMZ, regardless of BRAF mutation status. This reduction in cell survival was not accompanied by an increase in apoptosis, but rather Chk2 activation and an accumulation of cells in the G2/M phase of the cell cycle, suggesting a possible effect of IGF1R inhibition on DNA repair. IGF1R depletion was found to increase MGMT protein levels and activity, but this effect was not seen in IGF1R inhibited cells. In addition, IGF1R inhibition was not epistatic with MGMT inhibition, and IGF1R inhibition reduced survival of TMZ treated MGMT-null cells. This suggested that TMZ sensitization by IGF1R inhibition was independent of MGMT. IGF1R inhibition did however cause an increase in the accumulation of TMZ-induced RPA foci, and delay in resolution of RAD51 foci. Together with the finding that IGF1R inhibition reduced survival in PARP inhibited melanoma cells, these results suggested that IGF1R inhibition influenced DSB repair by homologous recombination. Finally, the combination of IGF1R inhibition with TMZ was tested in a mouse model and was found to be tolerable. TMZ or IGF-1R inhibitor alone caused minor reduction in melanoma xenograft growth rates (rate reduction by 13% and 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from other treatment groups (p<0.05). The findings of this study suggest IGF1R inhibition as a possible option in overcoming alkylating drug resistance in melanoma.

Die Osteoporose des Mannes ist eine unterdiagnostizierte Erkrankung. Die Zahl der betroffenen Männer in Deutschland wird auf 0,8 bis 1 Million geschätzt, aber die reale Inzidenz ist wahrscheinlich höher. Die Osteoporose des Mannes ist Schätzungen zufolge in bis zu 60% mit Begleiterkrankungen assoziiert. Hypophysäre Störungen wie Hypogonadismus und Wachstumshormonmangel sind starke Risikofaktoren für die Entwicklung einer Osteoporose. Es gibt in der Literatur nur wenige Daten über Hypophysenerkrankungen und Osteoporose, abgesehen davon, dass bei bestehendem Wachstumshormonmangel die Assoziation der Osteopenie / Osteoporose gesichert ist. Für die nicht diagnostizierten Fälle von GH-Defizienz kann jedoch die Manifestation einer Osteoporose ein erstes und führendes Symptom sein. Es gibt Evidenzen dafür, dass IGF-1 im Serum in gesunden Populationen positiv mit der Knochendichte korreliert. Das Ziel dieser Studie ist, eine große Anzahl an Patienten mit Osteoporose auf die Inzidenz niedriger IGF-1-Spiegel hin zu untersuchen. / The osteoporosis of the man is an unterdiagnosed disease. The number of the affected men in Germany is estimated at 0.8 to 1 millions, but the real Inzidenz is probably higher. The osteoporosis of the man is associated according to estimates in up to 60% with accompanying illnesses. Hypophysis disturbances like Hypogonadism and growth hormone lack are strong risk factors for the development of an osteoporosis. There are in the literature only few data about hypophysis illnesses and osteoporosis, apart from the fact that with existing growth hormone lack the association of the Osteopenie / osteoporosis is protected. Nevertheless, for the not diagnosed cases of GH-Defizienz the manifestation of an osteoporosis can be the first and leading symptom. There is evidence for the fact that IGF-1 correlates in the Serum in healthy populations positively with the osseous density. The purpose of this study is, a great number to patient with osteoporosis examine for the Inzidenz of low IGF-1-levels.

We evaluated the efficacy of treating eccentric muscle damage with IGF-1 PEGylated to a fibrin biomatrix. With one injection, delivered one hour after the induction of eccentric muscle damage we saw an attenuation of force loss early in recovery, maintenance of muscle weight, and progression to the repair/regeneration of the damaged fibers at a greater speed and magnitude in the first week of recovery. As opposed to introducing an unbound bolus of IGF-1, we believe the ability of the PEGylated-fibrin to stabilize and sustain delivery of the molecule results in significantly better recovery. Coupling IGF-1, which has multiple beneficial effects in tissue repair, with this system of delivery provides a simple and easy to administer treatment for eccentric muscle damage. With this form of damage being the most prevalent of all skeletal muscle damage types, since it is underlies all muscle strain, a simple and effective treatment is important for increasing functional recovery after injury. / text

Master of Science / Department of Human Nutrition / Weiqun Wang / Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in mouse skin cancer models. This study is to investigate the underlying mechanisms by which exercise might impact IGF-1 pathway regulated by p53 and p53-related proteins in mouse skin epidermis. Female SENCAR mice were pair fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min for 60 min daily. Animals were topically treated with TPA or vehicle control 2 hours before sacrifice and the target proteins in the epidermis were assessed by immunohistochemistry and Western blotting. Under TPA or vehicle treatment, MDM2 was significantly reduced in exercised mice compared with sedentary control. Meanwhile, p53 was significantly increased. In addition, p53 transcription target proteins p21, IGFBP-3, and PTEN were elevated in response to exercise. An interaction between exercise and TPA was observed on the decrease of MDM2 and increase of p53, but not p53 down-regulated proteins. Taken together, exercise appears to activate p53 by reducing MDM2 suppression, resulting in enhanced expression of p21, IGFBP-3 and PTEN that might further induce a negative regulation of IGF-1 pathway and therefore contribute to the observed cancer prevention by exercise in this mouse skin cancer model.

Made available in DSpace on 2014-06-11T19:27:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-26Bitstream added on 2014-06-13T18:31:21Z : No. of bitstreams: 1 tapparo_af_me_sjrp.pdf: 812527 bytes, checksum: aac6f17be2c6552511dcaa2c2bd79c32 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Atualmente, as culturas celulares são consideradas uma das ferramentas mais importantes na virologia. Os meios de cultura utilizados na manutenção destes sistemas não oferecem capacidade às células de adsorver e se multiplicar em matrizes plásticas sem a adição suplementar de soro fetal bovino (SFB). Entretanto, o uso do SFB não é aconselhável devido às variações encontradas entre os lotes, o alto grau de proteína animal e a possibilidade da presença de agentes infecciosos. Além dos aspectos sanitários, existe o aspecto ético em relação à obtenção deste produto biológico. Com o aumento dos ensaios in vitro a quantidade estimada de produção de soro fetal bovino no mundo é de aproximadamente 500.000 litros/ano, isto significa, mais de 1.000.000 de fetos bovinos sacrificados para obtenção de SFB. O objetivo deste estudo foi cultivar as células CER (chicken embryo related) em diferentes meios de cultura: M-VSFM, 293- SFMII, VP-SFM, VP-SFM AGT, Glasgow, Leibovitz 15, adicionados de 2mg/ml ou 5mg/ml de IGF-1 (Insulin-like Growth Factor) e verificar a possibilidade das estirpes virais Lukert e Farangher de se adaptarem neste sistema. Nesta cultura a expressão da fibronectina e da laminina (LB1 e LB2) foram dectadas por imunofluorescência e imunoperoxidase indireta, respectivamente. As monocamadas de células CER foram infectadas pela estirpe Lukert e Farangher, sendo a m.o.i (multiplicidade de infecção) utilizada de 1.0. Após três passagens, o RNA viral foi extraído para determinar as substituições genéticas. A região hipervariável do gene VP2 foi amplificada por RT/PCR. Para confirmar as mutações, os produtos da PCR foram seqüênciados e comparados com as seqüências de VDIB publicadas no GenBank. Os resultados demonstraram que o meio VP-SFM e 5µg/mL de IGF-1 aplicado às culturas foi o melhor para a adaptação direta do cultivo das monocamadas... / Currently, the cell culture are considered one of the tools most important in the virology. The culture medium used in the maintenance those systems do not offer capacity to the cells of attachment and if spread in plastic surfaces without the supplementary addition of foetal bovine serum (FBS). However, the use FBS is not advisable due to batch-tobatch variation in composition, the high animal protein content, and the possibility of the presence of infectious agents. Beyond the sanitary aspects, exists the ethical aspect in relation to the attainment of this biological product. With the increase of in vitro assays the amount of FBS produced in the world is estimated at approximately 500.000 litres/year, this carry in 1.000.000 bovine fetuses sacrified for FSB attainment. The aim of this study was to adapt the CER cells (chicken embryo related) on different medium: M-VSFM, 293-SFMII, VP-SFM, VP-SFM AGT, Glasgow, Leibovitz 15, supplemented by 2mg/ml or 5mg/ml IGF-1 (Insulin-like Growth Factor) and to verify the possibility the Lukert and Farangher viral strain adapted in this system. The expression of the fibronectin and laminin (LB1 and LB2) were detected by immunofluorescence and indirect immunoperoxidase, respectively. The CER cells monolayer had been infected by Lukert and Farangher strain, being the m.o.i (infection multiplicity) used of 1.0. After three passages, viral RNA was isolated to determine the genetic changes. The hypervariable regions of the VP2 gene was amplified by RT/PCR. To confirm the genetic changes, PCR products were sequenced and compared to the sequences of the GenBank published VDIB strain.The results had demonstrated that the medium VP-SFM and 5µg/mL IGF-1 applied to the cultures was optimum for the direct adaptation of the culture of the monolayers without addition of FBS. In relation the detection of the extracellular protein, the fibronectin was induced... (Complete abstract click electronic access below)

Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to the...

Twenty untrained men (18-25 y) were assigned to consume either a milk supplement (MILK) or a carbohydrate-electrolyte supplement (CHO) immediately following each resistance workout during a 10 wk resistance training program. Subjects trained 3 d/wk beginning with an intensity of 55% 1-RM and progressing to 97% 1-RM by wk 10. Muscle strength (1-RM), body composition (DEXA) and resting, fasted serum concentrations of total and free testosterone and IGF-1 were measured pre- and post-training. CHO tended to reduce, while MILK increased body weight (P = 0.10). All subjects significantly reduced percent body fat (1.1%) and significantly increased lean body mass (1.21 kg) as a result of the resistance training with no significant differences between treatments. However, MILK tended to increase lean body mass (P = 0.1) more than CHO (1.6 and 0.8 kg, respectively). About 39% of lean mass gain for all subjects was in the leg region, while the arms accounted for about 28% of lean gain. Resistance training also caused a similar significant 44% increase in muscle strength for the seven exercises combined for both groups. Resting total and free testosterone concentrations significantly decreased from baseline values in both groups of subjects (16.7% and 11%, respectively), while resting insulin concentrations significantly increased in all subjects (P<0.01). There were no significant changes in resting, fasted IGF-1 concentrations. In summary, dietary supplementation with a MILK or CHO beverage immediately following resistance exercise resulted in similar changes in muscle strength and hormone concentrations following a 10 wk periodized resistance training program. MILK tended to increase body weight and lean body mass more so than CHO. / Master of Science

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A arginina é um aminoácido condicionalmente essencial que participa de inúmeras reações metabólicas no organismo como, por exemplo, o ciclo da uréia, a síntese de creatina e a geração de óxido nítrico (NO). Além dessas funções a arginina é associada, com a sensibilidade à insulina, a secreção de GH e mais recentemente com a síntese protéica muscular. O objetivo deste trabalho foi investigar o efeito da suplementação via oral crônica de L-arginina sobre a síntese protéica muscular, pela via da mTOR, a fim de contribuir com as novas discussões científicas acerca deste aminoácido de ampla atuação. Métodos: Foram utilizados ratos wistar machos adultos com cerca de 200g de peso corporal divididos em quatro grupos de quatorze animais denominados na seguinte forma: Arginina Treinado (AT), Arginina Sedentário (AS), Dieta-Controle Treinado (CT) e Dieta-Controle Sedentário (CS). Ambas as dietas foram elaboradas com base das recomendações da AIN-93, sendo que a dieta enriquecida com arginina recebeu acréscimo de 2% deste aminoácido e a dieta controle recebeu um mix de aminoácidos não essenciais para garantir que ambas fossem isonitrogenadas e isocalóricas e as proporções de aminoácidos presente nas rações foi conferida por aminograma. O treinamento dos animais consistiu em exercício anaeróbio com sessões que eram compostas de 4 séries de 10 saltos com um minuto de descanso entre estas em tanque de água. Os saltos eram desempenhados com carga de 50% do peso corporal acoplado ao tórax dos animais na freqüência de 5 dias por semana por 6 semanas. A evolução da massa corporal dos animais bem como o consumo de ração foram avaliadas três vezes por semana e estimada uma média semanal. Foram realizados testes de tolerância oral à glicose (OGTT) e tolerância à insulina (ITT) no início e ao final do experimento em todos os animais para avaliar alterações na sensibilidade à insulina. Após 72hs da última sessão de treinamento os animais foram anestesiados para infusão de insulina, coleta dos músculos gastrocnêmio e plantar, fígado, sangue e eutanasiados conforme protocolo aprovado pelo CEA-USP. As análises bioquímicas foram determinações séricas de insulina, GH, IGF-1 e a proteína transportadora de IGF-1 (IGFBP-3), glicose plasmática, uréia e creatinina séricas, IGF-1 muscular e hepático por kits comerciais de tecnologia multiplex Luminex e aminograma sérico por cromatografia. As análises moleculares foram realizadas para as proteínas chaves envolvidas na via de síntese protéica muscular em sua forma total e fosforilada, sendo estas: IRS-1, Akt, mTOR, 4E-BP1 e p70S6K determinadas por método de western blotting. Resultados: Não foram encontradas diferenças estatisticamente significativas nos parâmetros avaliados com exceção da creatinina que se mostrou mais elevada nos grupos suplementados com arginina. A suplementação de arginina, nas concentrações administradas, bem como o exercício de alta intensidade pelo período determinado não foram capazes de alterar a expressão das proteínas envolvidas na regulação de síntese protéica muscular de ratos nem a sensibilidade celular à insulina. Conclusão: não houve aumento da síntese protéica muscular com a suplementação de arginina, nestas condições experimentais. / The arginine is an amino acid conditionally essential that participates in innumerous metabolic reactions in the body like, for instance, the urea cycle, the synthesis of creatine and production of nitric oxide (NO). Besides those functions the arginine is associated, with the insulin sensitivity, GH secretion and most recently with muscle protein synthesis. The aim of this work was to investigate the effect of L-arginine chronic oral supplementation on the muscle protein synthesis, through mTOR pathway, in order to contribute with new scientific discussions about this broad action amino acid. Methods: Wistar male adult rats were used with about 200g body weight distribute into four groups of fourteen animals named this way: Trained Arginine (TA), sedentary Arginine (SA), Trained Diet-Control (TC) and Sedentary Diet-control (SC). Both diets were elaborated based on the AIN-93 recommendations, considering that the enriched diet with arginine was added 2% of this amino acid and the control diet received a mix of non-essential amino acid in order to ensure that both were isonitrogenous and isocaloric and the proportions of present amino acids in the rations have been checked through aminogram. The animals training consisted of anaerobic exercise with sections composed by four jump series, with one minute rest among these in a PVC cube water. The jumps were performed with a load of 50% of their body weight attached in the animal\'s trunk, five days a week over six weeks. The animals\' body weight evolution as well as the food intake were evaluated three times a week in order to figure a weekly average. Oral glucose test tolerance (OGTT) and insulin test tolerance (ITT) have been done in the beginning and in the end of the experiment in all animals to evaluate insulin sensitive changes. The animals were anesthetized to insulin infusion, gastrocnemic and plantaris muscles, liver and blood collects 72 hrs after the last training section and afterwards sacrificed according to CEA-USP approved protocol. The biochemical analysis were blood determination of insulin, GH, IGF-1 and its binding protein 3 (IGFBP-3), glucose, urea and creatinine, and muscle and liver IGF-1 through commercial kits of multiplex Luminex technology and seric aminogram through chromatography. The molecular analysis were performed for the key proteins of the muscle protein synthesis pathway in its total and phosphorylated form: IRS-1, Akt-1, mTOR, 4E-BP1 and p70S6K determined by western blotting method. Results: Significant statistical differences were not found to all evaluated biomarkers in this experiment except for creatinine which was more elevated in groups supplemented with arginine. The arginine supplementation, in these given doses, as well as the high-intense exercise, failed in stimulate both the expression of the proteins involved in the muscle protein synthesis regulation and the insulin sensitivity in the rats in this condition. Conclusion: There hasn\'t been any increase in the muscle protein synthesis with arginine supplementation, in these experimental conditions.