The current study examined the impact of plant sterols, stanols, sterol esters, and stanol esters on (i) cholesterol-lowering efficiency, (ii) gene expression of ABCG5 and ABCG8 sterol transporters in the liver and small intestine, and (iii) colon mucosal cell proliferation in hamsters. After 5 weeks on experimental diets, plasma total cholesterol levels were reduced ( P<0.05) by stanols, sterol esters and stanol esters compared to cholesterol-control diet. Different PS analogs did not alter ABCG5 and ABCG8 mRNA levels in small intestine and liver as compared to cholesterol control. In addition, colon mucosal cell proliferation was 21.4% lower (P<0.01) in group fed 0.7% stanol esters relative to cholesterol control. Results suggest that hypocholesterolemic effects of PS analogs are not associated with changes of liver and small intestine ABCG5 and ABCG8 sterol transporters. Data also indicated that plant stanol ester may possess anticarcinogenic properties.

The cholesterol-lowering effect of corn fiber oil, obtained from the seed coats of corn kernels, has been reported previously. Corn fiber oil contains phytosteryl fatty acyl esters, ferulate phytostanyl esters, and free phytosterols. To date, however, no studies have examined the cholesterol-lowering efficacy of ferulate phytostanyl esters. Moreover, although plant stanols and sterols have been established as cholesterol-lowering agents over the past five decades, their exact mechanisms of action are not clearly understood. One of the possible mechanism is that plant sterols/stanols disrupts the normal sub-cellular cholesterol absorption by down-regulation of the influx sterol transporters such as the Niemann pick C1 like 1(NPC1L1) protein and/or up-regulation of efflux sterol transporters such as the ATP binding cassette (ABC) G5 and ABCG8 protein. Hence, the objectives of this thesis were to assess the efficacy of corn fiber oil, ferulate phytostanyl esters and their parent compounds including sitostanol and ferulic acid, on plasma cholesterol levels. Further, objectives were to investigate their impact on parameters of cholesterol kinetics and gene expression of sterol transporters to obtain insight into their role in genetic control of regulation of cholesterol flux. Results of this experiment demonstrate that the hypocholesterolemic effect of corn fiber oil is mostly due to sitostanol, while esterification of ferulic acid and sitostanol yields no apparent synergistic cholesterol lowering effect. Present data exhibited a cholesterol absorption lowering effect of corn fiber oil and sitostanol and suggest that this effect may be due to up-regulation of intestinal enterocyte efflux sterol transporters such as ABCG5 and ABCG8.

We have employed a differential detector combined with dielectrophoretic (DEP) translation in a microfluidic channel to monitor dielectric response of single cells and particularly to track phenomenon related to apoptosis. Two different cell lines were studied; Chinese hamster ovary cells (CHO) and MRC5 cells. Dielectric response was quantified by a factor called Force Index. Force Index was studied statistically to identify apoptotic subpopulations. Another direction of this work was to monitor changes in the cytoplasm conductivity following inhibition of mitochondrial ATP production by Oligomycin. To make the DEP response mostly sensitive to the cytoplasm conductivity, medium conductivity and DEP frequency were adjusted such that Clausius Mossotti factor and hence DEP response become less sensitive to cell radius. Chinese hamster ovary cells were used in this work and the impact of different concentrations of Oligomycin has been studied. We show that following exposure to Oligomycin at 8 μg/ml, cytoplasm conductivity drops. The majority of the changes takes place within one hour of exposure to the drug. Furthermore, double shell models has been used to estimate cytoplasm conductivity in a medium with conductivity of 0.42 S/m and the drop in the cytoplasm conductivity following treatment with Oligomycin was estimated to be ≈ 0.16 S/m. The magnitude of the decrease in the cytoplasm conductivity is evidence that Glycolysis is active as an energy production pathway within the cell. This approach can be used to quantify Glycolysis versus mitochondria ATP production which has an application in Warburg effect in cancer cells and monitoring bioprocesses.

In order to optimize productivity of a cell culture it is necessary to understand growth and productivity and couple these features of the culture to extracellular nutrients whose profiles can be manipulated. Also, since growth and productivity are directly affected by cell death mechanisms such as apoptosis, it is imperative to understand these mechanisms. This work describes the development of a differential equation based population balance model of apoptosis in a Chinese Hamster Ovary cell culture producing Anti-RhD monoclonal antibody (mAb). The model was verified in isolation and was then coupled to a metabolic flux model. The model distinguishes between various subpopulations at normal healthy states and at various stages of apoptosis. After finding that glucose and glutamine are not limiting nutrients for this culture, different hypotheses were explored to explain growth arrest. Initially, it was hypothesized that there is some unknown nutrient in either media or serum which is depleted, thus causing growth arrest. Accordingly a first model was developed assuming depletion of this nutrient. Subsequent experiments with different additions of media and serum showed that there is no such nutrient limitation for the media and serum conditions used in most of the experiments. Additional experiments with different culture volumes showed that cell growth was actually controlled by a compound that accumulates and causes pH deviation from its optimal range of operation. Since strong correlations were found between culture volume and growth, it was hypothesized that the compound may be carbon dioxide (CO2), which is inhibitory for growth and may accumulate due to mass transfer limitations. Following this finding, a second model was proposed to take into account the accumulation of this inhibitor, although the specific inhibiting compound could not be exactly identified. This second mathematical model of cell growth was then integrated with a metabolic flux model to provide for a link between intracellular and extracellular species balances, since the latter are the ones to be manipulated for increasing productivity. This final model formulation was then used to describe mAb productivity. The model was also able to reasonably predict all cell subpopulations, nutrients, metabolites and mAb. In an attempt to mitigate the effect of CO2 accumulation and renew the cell growth, culture perfusions were performed. Although this approach resulted in some renewal of growth, the cell concentration progressively decreased after each successive perfusion event. This suggests that irreversible cell damage occurs because of CO2 accumulation. The model was used to describe the perfusion experiments. Agreement between data and model predictions were reasonable. In addition, it was shown that operation with successive perfusions results in a significant increase in productivity and therefore it can be used for further process optimization.

In Syrian hamsters, social behavior is mediated exclusively by chemosensory cues and circulating gonadal steroid hormones. Where these two signals are processed in the brain is unknown, but the posterior bed nucleus of the stria terminalis (pBNST) has been suggested as a candidate site. Therefore, we tested male hamsters’ preference for opposite-sex odors following excitotoxic lesions of the pBNST. Lesions of the pBNST (pBNST-X) eliminated male hamsters’ preference for opposite-sex odors. Furthermore, pBNST-X males spent significantly less time investigating female odors than clean odors and significantly less time investigating female odors than control males did. Lesions of the pBNST did not change male hamsters’ investigation of male odors. The deficits observed in pBNST-X males were not due to a failure to discriminate between odors, as pBNST-X males were able to distinguish between odors. Together, these data suggest the pBNST is critical for opposite-sex odor preference in male hamsters.

A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

Abstract Non-pathological change in body weight and adiposity is one distinct adaptive feature that seasonal species undergo, and it can offer a novel way to study the mechanisms underlying body weight regulation and energy homeostasis. Changes in the expression and activity of metabolic enzymes are essential for the physiological adaptation seasonal species exhibit. AMP-activated protein kinase (AMPK) is a key regulatory enzyme that controls the energy homeostasis both on cellular and whole-body level. In this thesis, the main focus was to clarify how seasonal adaptation affects AMPK and its downstream target in lipid metabolism, acetyl-CoA carboxylase (ACC), in different metabolic tissues of two model species with diverse wintering strategies: the raccoon dog and the Djungarian hamster. In addition, the effect of periodic fasting on the raccoon dog skeletal muscle was studied. It was observed that seasonal differences in AMPK and ACC expression were evident mainly in adipose tissues of both species. AMPK was down-regulated in white adipose tissue (WAT) of the winter-adapted raccoon dog, whereas in the Djungarian hamster WAT, the abundance of AMPK increased in response to winter acclimatization. ACC expression was maintained or increased in winter in both species. The seasonal changes in AMPK and ACC expression observed, in particular, in adipose tissues reflects the wintering strategy of the species and presumably facilitates the lipid usage and/or preservation during wintertime scarcity. Raccoon dogs were quite resistant to the prolonged wintertime fast, as no changes were observed in AMPK and ACC expression levels in the WAT, liver or hypothalamus between the fasted and fed groups. Skeletal muscle function also appears to be well preserved, as there were no changes in the expression of proteins involved in insulin signaling, and the fiber type composition and muscle energy reserves were not affected. This thesis offers novel information on protein level changes in metabolic adaptation. / Tiivistelmä Useat luonnonvaraiset eläinlajit ovat fysiologisesti sopeutuneet ravinnonsaannin vuodenaikaisiin vaihteluihin. Vuodenaikaisrytmiin kytketty rasvakudoksen määrän vaihtelu ja siihen liittyvät aineenvaihdunnalliset muutokset tarjoavat mielenkiintoisen tutkimuskohteen ruumiinpainon säätelyn ja energiatasapainon ylläpidon molekulaaristen mekanismien selvittämiseen. Oleellinen osa fysiologista sopeutumista ovat muutokset energia-aineenvaihduntaa säätelevien proteiinien ekspressio- ja aktiivisuustasoissa. Yksi keskeinen elimistön energiatasapainoa kontrolloiva entsyymi on AMP-aktivoituva proteiinikinaasi (AMPK). AMPK toimii solunsisäisenä energiasensorina ja säätelee energiametaboliaa koko kehon tasolla. Tässä väitöskirjatutkimuksessa selvitettiin talviadaptaation vaikutusta AMPK:n ja sen kohdemolekyylin, rasvahappojen biosynteesiä säätelevän asetyyli-CoA karboksylaasin (ACC), ilmenemiseen ja aktiivisuuteen eri kudoksissa. Mallieläiminä käytettiin kahta eri talvehtimisstrategian omaavaa ja eri lailla ruumiinpainoaan säätelevää lajia, kääpiöhamsteria ja supikoiraa. Lisäksi tutkittiin pitkäaikaisen talvipaaston vaikutusta supikoiran luustolihakseen. Tulokset osoittivat, että molemmilla lajeilla AMPK- ja ACC-pitoisuuksissa on vuodenaikaisia eroja erityisesti rasvakudoksessa. Supikoiralla AMPK:n määrä väheni talviadaptaation seurauksena, kun taas kääpiöhamstereilla talviakklimatisaatio johti korkeampaan AMPK-pitoisuuteen rasvakudoksissa. ACC-pitoisuus puolestaan säilyi samana tai oli korkeampi talviadaptoituneilla yksilöillä. Havaitut muutokset AMPK:n ja ACC:n ilmenemisessä kuvastavat supikoiran ja kääpiöhamsterin eroja talvehtimisessa ja havainnollistavat entsyymien oleellista osaa rasvavarastojen vuodenaikaisessa säätelyssä ja käytössä, mikä on edellytys eläinten selviämiselle yli talven niukkuuden. Lisäksi havaittiin talviadaptoituneen supikoiran olevan melko resistentti 10 viikon paastolle tutkittujen parametrien suhteen. AMPK- ja ACC-pitoisuus tai aktiivisuus ei muuttunut aineenvaihdunnallisesti oleellisissa kudoksissa (rasvakudos, maksa, hypotalamus) paasto- ja kontrolliryhmän välillä. Supikoiran lihasten toimintakyky vaikuttaisi säilyvän, sillä insuliinisignalointiin liittyvien entsyymien pitoisuus, lihasten solutyyppikoostumus tai energiavarastot eivät muuttuneet paaston myötä. Tämä tutkimus tarjoaa uutta tietoa proteiinitason muutoksista osana fysiologista sopeutumista.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Neurociências. / Made available in DSpace on 2012-10-19T16:14:20Z (GMT). No. of bitstreams: 1 186401.pdf: 343770 bytes, checksum: 5e503c5374159a2c9893dc87980a482b (MD5) / Determinação dos efeitos de diferentes períodos de isolamento sobre o comportamento social do indivíduo, procurando identificar as possíveis conseqüências do isolamento sobre o comportamento social de animais jovens - brincadeira; e relacionar o comportamento agonístico adulto com a situação de privação da brincadeira na fase juvenil. O modelo do estudo escolhido foi o Hamster Dourados (Mesocricetus auratus), exposto a situações experimentais controladas dentro do laboratório (grupo controle, grupo isolado, grupo 10'e grupo 60'). A análise dos dados foi realizada através de testes estatísticos paramétricos (ANOVA) e teste de Tukey. Verificando-se que, de modo geral, no caso do sujeito, existe um tempo mínimo, maior de uma hora, necessário para interação, no sentido de minimizar os efeitos do isolamento social. Além disso, a agressividade observada esteve, em geral, associada ao sexo dos animais, e a investigação social a uma nova configuração ambiental estabelecida na situação de teste.

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-21Bitstream added on 2014-06-13T20:14:37Z : No. of bitstreams: 1 candido_nm_me_sjrp_parcial.pdf: 373311 bytes, checksum: 5a4bfe9e41e36ed412f101849f1c0cbb (MD5) Bitstreams deleted on 2015-04-01T12:51:15Z: candido_nm_me_sjrp_parcial.pdf,Bitstream added on 2015-04-01T12:51:49Z : No. of bitstreams: 1 000714222.pdf: 1858898 bytes, checksum: 83dab4894cfeb9ebe8c7af4ec08962ab (MD5) / O carcinoma oral epidermóide é a neoplasia mais frequente da cavidade bucal, representando, aproximadamente, 90% dos cânceres orais. A maioria das pessoas com câncer bucal realiza diagnóstico em estádios avançados quando o prognóstico é ruim e, consequentemente, a terapia é incerta com possibilidades de cura reduzidas. Sendo assim, o tratamento deste câncer envolve a utilização de recursos terapêuticos complexos, com resultados estéticos e fisiológicos pouco promissores. Portanto, outras terapias são almejadas a fim de reverter o quadro insatisfatório de qualidade de vida, amenizando os efeitos colaterais. Para isso, a nanoterapia aplicada ao tratamento do câncer tem-se desenvolvido rapidamente e nanopartículas, como as de óxidos de ferro, vem demonstrando vantagens sobre outros carreadores, sendo o tratamento do câncer por meio de hipertermia uma aplicação bastante visada. Portanto, o objetivo do trabalho foi investigar a citotoxicidade das nanopartículas magnéticas de ferro revestidas por polifosfato em linhagem celular de câncer de assoalho de boca (UM-SCC14A) e sua ação em carcinoma oral in vivo. As células foram incubadas com nanopartículas em diferentes concentrações (0,35; 0,7 e 1,4x10 15 partículas/mL) e, em seguida, foram realizadas técnicas de microscopia eletrônica de transmissão, microsopia de luz, microscopia de fluorescência e ensaio de viabilidade celular. Os diferentes ensaios mostraram que apenas a incubação com 1,4 x10 15 partículas desencadeou processo apoptótico significativo, refletindo alterações morfológicas e menor viabilidade celular. Sendo assim, a concentração de 0,7x10 15 partículas foi escolhida para os experimentos in vivo, já que não demonstrou caráter tóxico expressivo. Após os experimentos in vitro, trinta hamsters sírios foram divididos igualmente em cinco... / Oral squamous cell carcinoma is the most common neoplasm of the oral cavity, representing, approximately, 90% of oral cancers. Most people affected by oral cancer performs the diagnosis in advanced stages when the prognosis is poor and, therefore, therapy is uncertain with reduced chances of healing. Thus, treatment of this cancer involves the use of complex therapeutic resources, with hardly promising aesthetic and physiological results. Therefore, other therapies are desired to reverse the unsatisfactory quality of life, minimizing side effects. For this, nanotherapy applied to the treatment of cancer has developed rapidly and nanoparticles, such as iron oxides, have shown advantages over other carriers, being cancer treatment by hyperthermia quite an application newsworthy. So, the aim of this study was to investigate the cytotoxicity of iron magnetic nanoparticles coated with polyphosphate in cancer cell line from the floor of mouth (UM-SCC14A) and its action on oral carcinoma in vivo. Cells were incubated with nanoparticles at different concentrations (0.35, 0.7 and 1.4x10 15 particles/mL) and, then, were performed transmission electron microscopy, light microscopy, fluorescence microscopy and viability assay. Different tests have shown that only incubation with 1.4x10 15 particles triggered significative apoptotic process reflecting morphological changes and reduced cell viability. Thereby, the concentration of 0.7x10 15 particles has been chosen for the experiments in vivo, as demonstrated no expressive toxic character. After the in vitro experiments, thirty Syrian hamsters were divided into five groups, with a normal control group in which no tumor was induced. In the remaining animals was induced oral cancer by applying the carcinogen DMBA on the hamsters’ right cheek, resulting in the development of oral squamous cell carcinoma... (Complete abstract click electronic access below)

Orientador: Paula Rahal / Coorientador: Marília de Freitas Calmon / Banca: Patrícia Simone Leite Vilamaior / Banca: Fernando Lucas Primo / Resumo: O carcinoma oral epidermóide é a neoplasia mais frequente da cavidade bucal, representando, aproximadamente, 90% dos cânceres orais. A maioria das pessoas com câncer bucal realiza diagnóstico em estádios avançados quando o prognóstico é ruim e, consequentemente, a terapia é incerta com possibilidades de cura reduzidas. Sendo assim, o tratamento deste câncer envolve a utilização de recursos terapêuticos complexos, com resultados estéticos e fisiológicos pouco promissores. Portanto, outras terapias são almejadas a fim de reverter o quadro insatisfatório de qualidade de vida, amenizando os efeitos colaterais. Para isso, a nanoterapia aplicada ao tratamento do câncer tem-se desenvolvido rapidamente e nanopartículas, como as de óxidos de ferro, vem demonstrando vantagens sobre outros carreadores, sendo o tratamento do câncer por meio de hipertermia uma aplicação bastante visada. Portanto, o objetivo do trabalho foi investigar a citotoxicidade das nanopartículas magnéticas de ferro revestidas por polifosfato em linhagem celular de câncer de assoalho de boca (UM-SCC14A) e sua ação em carcinoma oral in vivo. As células foram incubadas com nanopartículas em diferentes concentrações (0,35; 0,7 e 1,4x10 15 partículas/mL) e, em seguida, foram realizadas técnicas de microscopia eletrônica de transmissão, microsopia de luz, microscopia de fluorescência e ensaio de viabilidade celular. Os diferentes ensaios mostraram que apenas a incubação com 1,4 x10 15 partículas desencadeou processo apoptótico significativo, refletindo alterações morfológicas e menor viabilidade celular. Sendo assim, a concentração de 0,7x10 15 partículas foi escolhida para os experimentos in vivo, já que não demonstrou caráter tóxico expressivo. Após os experimentos in vitro, trinta hamsters sírios foram divididos igualmente em cinco... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Oral squamous cell carcinoma is the most common neoplasm of the oral cavity, representing, approximately, 90% of oral cancers. Most people affected by oral cancer performs the diagnosis in advanced stages when the prognosis is poor and, therefore, therapy is uncertain with reduced chances of healing. Thus, treatment of this cancer involves the use of complex therapeutic resources, with hardly promising aesthetic and physiological results. Therefore, other therapies are desired to reverse the unsatisfactory quality of life, minimizing side effects. For this, nanotherapy applied to the treatment of cancer has developed rapidly and nanoparticles, such as iron oxides, have shown advantages over other carriers, being cancer treatment by hyperthermia quite an application newsworthy. So, the aim of this study was to investigate the cytotoxicity of iron magnetic nanoparticles coated with polyphosphate in cancer cell line from the floor of mouth (UM-SCC14A) and its action on oral carcinoma in vivo. Cells were incubated with nanoparticles at different concentrations (0.35, 0.7 and 1.4x10 15 particles/mL) and, then, were performed transmission electron microscopy, light microscopy, fluorescence microscopy and viability assay. Different tests have shown that only incubation with 1.4x10 15 particles triggered significative apoptotic process reflecting morphological changes and reduced cell viability. Thereby, the concentration of 0.7x10 15 particles has been chosen for the experiments in vivo, as demonstrated no expressive toxic character. After the in vitro experiments, thirty Syrian hamsters were divided into five groups, with a normal control group in which no tumor was induced. In the remaining animals was induced oral cancer by applying the carcinogen DMBA on the hamsters' right cheek, resulting in the development of oral squamous cell carcinoma... (Complete abstract click electronic access below) / Mestre