O objetivo desse estudo foi avaliar a citotoxicidade do alendronato de sódio (ALN), em diferentes concentrações, sobre osteoblastos em cultura celular. Foi utilizado para o experimento meio sem droga (controle) e meio contendo a droga em diferentes concentrações a saber: 0,5; 1; 5; 10; 20 e 40 mg/ml. Para o estudo foi utilizado uma linhagem de osteoblastos de rato (Osteo-1). A viabilidade celular foi inferida a partir da análise da atividade mitocondrial das células através do método da redução do MTT nos tempos experimentais de 0, 24, 48 e 72 horas. Após análise estatística (teste de Mann-Whitney, p<0,05), os resultados mostraram que, com exceção da concentração de 40mg/ml, todos os grupos apresentaram células viáveis, embora todas as concentrações de ALN tenham reduzido a atividade mitocondrial em mais de 50% em relação ao grupo controle, sugerindo que o ALN nessas concentrações estudadas foi citotóxico para os osteoblastos. / The aim of this study was to analyze the cytotoxicity of a bisphosphonate (sodium alendronate) on rat osteoblasts. The experimental groups were GI (control) no sodium alendronate, and GII, GIII, GIV, GV, GVI, and GVII with sodium alendronate at the concentrations of 0.5, 1, 5, 10, 20, and 40mg/ml, respectively. The cell viability was evaluated by MTT assay in experimental times 0, 24, 48, and 72 h. Data were statistically analyzed. Cultures treated with sodium alendronate showed cell viability percentages significantly lower (p < 0.05) than those of the control groups. In GVII, no viable cell was observed in all experimental times. It could be concluded that sodium alendronate, on direct contact with rat osteoblasts, is cytotoxic in all concentrations studied.

O sucesso do tratamento com implantes osseointegráveis dependente do processo de reparo da ferida e do potencial osteogênico das células. Com o objetivo de aumentar a formação óssea na superfície dos implantes, vários tratamentos têm sido propostos, entre eles, a terapia com laser de baixa intensidade. Neste contexto, o objetivo do presente estudo foi investigar o efeito do laser diodo de Arseneto-Gálio-Alumínio (AsGaAl) em culturas osteogênicas humanas crescidas sobre titânio. Após exposição das culturas, aos 3 e 7 dias, a uma dose de 3 J/cm2, foram avaliados os seguintes parâmetros: 1) proliferação celular aos 10 e 14 dias; 2) atividade de fosfatase alcalina (ALP) em 10, 14 e 17 dias; 3) formação de matriz mineralizada em 17 dias; 4) imunolocalização de proteínas não-colágenas e do antígeno nuclear Ki-67 por fluorescência indireta em 8, 10, 14 e 17 dias; 5) expressão de genes relacionados ao desenvolvimento do fenótipo osteoblástico aos 14 dias. Os resultados dos ensaios de proliferação celular mostraram que a radiação laser aumentou a proliferação de 10 para 14 dias, mas tanto a atividade de ALP como a formação de matriz mineralizada aparentemente não foram afetadas. A análise das culturas por epifluorescência mostrou que as culturas controle e irradiadas apresentaram comportamento distintos. Aos 14 dias, foi demonstrado que a exposição à radiação laser, na dose utilizada, promoveu aumento na expressão relativa dos genes ALP, OC, BSP, BMP-7 e OPG e redução dos níveis de expressão de Runx2 e osteopontina, mas não afetou a expressão gênica de COL I, RANK-L e ICAM. As culturas irradiadas apresentaram áreas sem células após 24 h da última irradiação, que posteriormente foram repovoadas por células mais proliferativas e menos diferenciadas. Em conclusão, os resultados indicam que a radiação do laser diodo de AsGaAl estimula a expressão do fenótipo osteoblástico de culturas osteogênicas humanas crescidas sobre titânio. / The success of treatment with osseointegrated implants depends on wound healing and the osteogenic potential of cells. Aimed at increasing bone formation onto implant surfaces, several treatments have been proposed and low-intensity laser therapy is one of them. Thus, the purpose of this study was to investigate the effect of the Gallium-Aluminum-Arsenic (GaAlAr) diode laser on human osteogenic cultures grown on titanium. After irradiation of osteogenic cultures with 3 J/cm2 of GaAlAr laser at the days 3 and 7, the following parameters were evaluated: 1) cell proliferation at 10 and 14 days; 2) alkaline phosphatase activity (ALP) at 10 days; 3) mineralized matrix formation at day 17, 4) non-collagenous bone proteins and nuclear antigen Ki-67 immunolocalization by indirect fluorescence at 8, 10, 14 and 17 days; and 5) Expressions of a panel of genes related to osteoblastic phenotype at day 14. Apparently cell proliferation, ALP activity and mineralized matrix formation were not significantly different between irradiated and control cultures in any period. Epifluorescence revealed that control and irradiated cultures had presented different behaviors. At 24 h after irradiation procedures, irradiated cultures displayed areas with no cells, which were repopulated later on by proliferative and apparently less-differentiated cells. Laser radiation increased relative gene expression of ALP, OC, BSP, BMP-7, and OPG; reduced the gene expression of Runx2 and OPN; and did not affect the expression of COL-1, RANK-L and ICAM. Altogether these results indicated that the GaAlAr laser stimulates the expression of osteoblastic phenotype of human osteogenic culture cells grown on titanium.

Estudo anterior de nosso grupo demonstrou que superfície de titânio (Ti) com nanotopografia obtida por condicionamento com H2SO4/H2O2 e funcionalizada com GDF-5 por simples adsorção promove o aumento da mineralização de culturas primárias de células osteogênicas. O presente estudo teve como objetivos avaliar: 1) os efeitos da pós-adsorção de proteínas principais do plasma- albumina, fibrinogênio e fibronectina, em superfícies de Ti controle e com nanotopografia, funcionalizadas com GDF-5 a 200 ng/mL por simples adsorção, sobre a formação de matriz mineralizada in vitro; 2) parâmetros moleculares e fenotípicos característicos da aquisição do fenótipo osteogênico in vitro sobre superfícies de Ti funcionalizadas com GDF-5 por simples adsorção ou por filmes LbL; 3) parâmetros de formação óssea adjacente a implantes de Ti com nanotopografia funcionalizada com GDF-5 pelos dois métodos, em modelo de tíbia de coelhos. Os resultados mostraram que a pós-adsorção de proteínas plasmáticas não afetou o potencial osteogênico in vitro, com exceção para o efeito inibidor da albumina, quando pós-adsorvida isoladamente. Tanto a superfície de Ti como o método de funcionalização de GDF-5 afetaram, quantitativamente, as formações de matriz mineralizada, com a maior diferenciação osteogênica para Ti com nanotopografia funcionalizada com GDF-5 por simples adsorção e a menor, para os filmes LbL, independentemente das superfícies sobre as quais eles eram montados. A atividade de ALP foi maior em culturas sobre nanotopografia de Ti, incluindo aquelas funcionalizadas com GDF-5, cujos valores, no entanto, não corresponderam, necessariamente, à maior atividade osteogênica. Apesar disso, todos os grupos exibiram expressão de marcadores de diferenciação osteoblástica, com sobre-expressão de osteopontina e osteocalcina para culturas sobre LbL. As análises microtomográfica, histológica e histomorfométrica não revelaram diferenças qualitativas e quantitativas in vivo entre nanotopografias de Ti funcionalizadas ou não com GDF-5, ainda que uma tendência à maior formação óssea tenha sido observada para as superfícies funcionalizadas e, entre essas, para os filmes LbL. Considerados conjuntamente, os resultados do presente estudo contribuem para o melhor entendimento das respostas de osteoblastos e do tecido ósseo quando se propõe a estratégia de funcionalização de superfícies de Ti com GDF-5 visando à otimização da osseointegração. / It has been demonstrated that a nanostructured titanium (Ti) surface obtained by treatment with H2SO4/H2O2 and functionalized with GDF-5 by simple adsorption promotes the enhancement of mineralized matrix formation in osteogenic cell cultures. This study aimed to evaluate: 1) the effects of post-adsorption of major plasma proteins, i.e. albumin, fibrinogen and fibronectin, on control and nanostructured Ti surfaces, functionalized with 200 ng/mL GDF-5 by simple adsorption, on mineralized matrix formation by calvarial osteogenic cell cultures; 2) molecular and phenotypic parameters characteristics of the acquisition of the osteogenic phenotype in vitro on Ti surfaces functionalized with GDF-5 by either simple adsorption or layer by layer (LbL) films; 3) parameters of bone formation adjacent to Ti implants with a nanostructured surface functionalized with GDF-5 by the two methods described in item 2, in a rabbit tibia model. The results showed that the post-adsorption of plasma proteins did not affect the osteogenic potential of cultures, except for the inhibitory effect of albumin when post-adsorbed alone. Either the Ti surface topography or the method for GDF-5 functionalization quantitatively affected mineralized matrix formation, with the higher osteogenic differentiation for nanostructured Ti functionalized with GDF-5 by simple adsorption and the lower one for LbL films, irrespective of the Ti surface topography on which they were mounted. ALP activity was higher for cultures grown on nanostructured Ti, including those functionalized with GDF-5, whose values, however, did not necessarily correspond to the higher osteogenic activity. Despite that, all groups expressed osteoblast differentiation markers, with a remarkable increase in osteopontin and osteocalcin mRNA levels for cultures grown on LbL films. The microtomographic, histologic and histomorphometric analyses revealed no qualitative or quantitative differences in vivo among the nanostructured Ti implants, yet a tendency for enhanced bone formation was observed for the functionalized surfaces and, between them, for the LbL films. Taken together, the results of the present in vitro and in vivo studies contribute to a better understanding of osteoblast and bone tissue responses to the functionalization of Ti surfaces with GDF-5 aiming to optimize osseointegration.

Lodos de esgoto são resíduos do tratamento do esgoto doméstico, considerados ricos em macronutrientes e matéria orgânica, podendo também apresentar contaminação por substâncias químicas e patógenos. Sua utilização na agricultura é uma das alternativas interessantes para sua disposição final. Entretanto, a presença de contaminantes, dentre eles microrganismos patogênicos podem limitar e orientar sua aplicação. Os vírus entéricos, dentre os quais se inclui o gênero Enterovirus, são potenciais contaminantes microbianos presentes no lodo de esgoto. Esses organismos são capazes de sobreviver por meses em águas e solos e sua presença no ambiente pode trazer prejuízos à saúde da população exposta aos mesmos. O objetivo do presente estudo foi de analisar a ocorrência de Enterovirus em amostras de lodo de esgoto de seis diferentes ETEs do Estado de São Paulo, avaliar o desempenho do método analítico para a detecção desses organismos e verificar se as concentrações médias obtidas atendem à legislação. Foram coletadas um total de 35 amostras no período de fevereiro de 2009 a dezembro de 2009. As análises dos Enterovirus foram realizadas pelo ensaio de plaqueamento em cultura de células RD segundo método EPA /625/R-092/013. Os resultados obtidos foram que Enterovirus estiveram presentes em 83 por cento das amostras analisadas, com concentrações que variaram de não detectado (ND) a 12,50 UFP/gST. As taxas de recuperação obtidas variaram de 0,20 por cento a 68,50 por cento . Conclui-se que das amostras analisadas 31 por cento atendem o estabelecido pela Resolução CONAMA 375/2006 / Sewage Sludge is a waste generated from wastewater treatment and is considered besides rich in macronutrients and organic matter, contaminated with pathogen and chemical substances. The usage of sludge in agriculture has been considered an interesting option. Nevertheless, the presence of contaminates such as pathogenic organisms can lead to usage limitations. Enteric viruses in wich are included enterovirus genera, a potential sludge contaminants. These organisms are able to survive for months in water and soil and their presence can bring public health concerns. The aim of this study was to analyse the occurrence of Enterovirus in samples of sewage sludge from six sewage plant treatment located in São Paulo State, to assess the method performance (recovery rate) in detecting these organisms and to verify the results obtained meet the standard established by the law. A total of 35 samples were collected spanning February to December 2009. The analyses were carried out according to method EPA/625/R-092/013. Enterovirus were present in 83 per cent of samples examined with concentration varied from not detected (ND) to 12.5 PFU/gTS. The recovery rate varied from 0,2 per cent to 68,50¨ per cent . This study showed that 31 per cent of analyzed samples met the standard established by Resolução CONAMA 375/2006

New medical treatments, compounds that affect human health, nutritional supplements, and other substances, are introduced to society every day. The accurate determination of the potential toxicity from these substances is of critical importance to our society. Goals of the modern toxicologist not only involve the determination of the toxic potential of new substances but also: the elucidation of mechanisms; improving existing assays; and developing new assays to study toxicity. This thesis addresses these goals in two topics fundamental to toxicology. Re-evaluating the expression of dose and susceptibility of cells in culture The exposure of cells in culture to drugs, xenobiotics, and other compounds is one of the first tools used to determine the potential for toxicity. Problems can arise when results of these experiments are translated to next-level toxicity experiments (e.g. animals and humans). I hypothesized that "dose" in cell culture can be improved by designing and reporting experiments based on dose in moles per cell. When experiments were compared on an extracellular concentration basis, a large apparent variability in toxicity was observed. However, if these same exposures were expressed as moles per cell, all experiments yielded the same toxicity. In addition to the evaluation of mole per cell, I investigated the susceptibility of various cells to 1,4-benzoquinone. I hypothesized that upon exposure to toxins that bind covalently, larger cells would require more molecules per cell of toxin versus a smaller cell to achieve identical toxicities. I found a linear correlation between cell volume(pL) and ED50 (mole per cell where 50 % cell viability is lost), supporting my hypothesis. This work could improve current cell culture protocols and allow for better and less expensive determination of toxicities. Heritability of the red blood cell storage lesion Blood transfusions are an integral part of modern medicine with 5 million people receiving blood each year in the United States. There is growing evidence that red blood cells (RBCs) stored for longer periods are less therapeutically beneficial and could even be harmful to patients. This phenomenon of diminished RBC function with increased time in storage is called the storage lesion. However, there is great variation between different donors in the severity of the storage lesion in their donated RBCs. I hypothesized that part of this variability in the RBC storage lesion is determined by heritable genetic differences. To test this hypothesis, a study using mono- and di-zygotic twins was performed to determine the heritability of adenosine triphosphate (ATP), glutathione (GSH), glutathione disulfide (GSSG) and hemolysis in stored blood. Major discoveries in this study include: GSH, GSSG, and the half-cell reduction potential (Ehc) are heritable (57 %, 51 %, and 70 %, respectively) in non-stored RBCs. In addition, ATP was found to be heritable in two different storage solutions (62 % in AS-3, 71 % in CP2D); as well as GSH, GSSG, Ehc and hemolysis (59 %, 48 %, 64 %, and 53 %, respectively). These discoveries could eventually be used to develop new genetic tests that would predict the rate of deterioration in stored blood quality on an individual basis.

The prominence of insect cell culture has grown rapidly due to its ability to produce baculovirus biopesticides and recombinant proteins using the Baculovirus Expression Vector System. A critical problem in the mass production of these products is CO2 accumulation to inhibitory levels within the bioreactor. The current research investigated the effect of elevated CO2 concentrations on insect cell growth and metabolism and the roles of oxidative stress and intracellular pH (pHi) in CO2 inhibition. Spodoptera frugiperda Sf-9 insect cells were cultured in a 3 L bioreactor (1.2 L working volume) controlled at 20% air saturation, 27oC and a pH of 6.2. The cells were exposed to a constant CO2 concentration by purging the medium with CO2 and the headspace with air. The experiments were repeated for different CO2 concentrations and samples were taken every 24 h to determine cell density, viability, metabolism and oxidative stress. The population doubling time (PDT) of Sf-9 cells increased with increasing CO2 concentration. Specifically, the PDT for 0-37, 73, 147, 183 and 220 mm Hg CO2 concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4 and 69.3 ± 35.9 h (n = 3 or 4; 95% confidence level), respectively. An 80 mL working volume shaker flask was maintained as a control and had an average PDT of 24.9 ± 3.1 h (n = 7; 95% confidence level). The viability of cells in all experiments was above 90%. The osmolality for all bioreactor experiments was observed to be 300 - 360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO2 concentration did not alter the cell specific glucose consumption rate (2.5 to 3.2 x 10-17 mol/cell-s), but slightly increased the specific lactate production rate from -3.0 x 10-19 mol/cell-s to 10.2 x 10-19mol/cell-s. Oxidative stress did not contribute to CO2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO2 concentration. The experiments conducted to determine the effect of CO2 on pHi were not successful and different experimental methods tested were well documented.

Organ transplantation has made great progress since the first successful kidney transplant in 1953 and now more than one million tissue transplants are performed in the United States every year (www.organdonor.gov/statistics-stories, 2015). However, the hope and success of organ transplants are often overshadowed by their reputation as being notoriously difficult to procure because of donor-recipient matching and availability. In addition, those that are fortunate enough to receive a transplant are burdened with a lifetime of immunosuppressants. The field of regenerative medicine is currently making exceptional progress toward making it possible for a patient to be their own donor. Cells from a patient can be collected, reprogrammed into stem cells, and then differentiated into specific cell types. This technology combined with recent advances in 3D printing provides a unique opportunity. Cells can now be accurately deposited with computerized precision allowing tissue engineering from the inside out (Gill, 2016). However, more work needs to be done as these techniques have yet to be perfected. Bioprinters can cost hundreds of thousands of dollars, and the bioink they consume costs thousands per liter. The resulting cost in development of protocols required for effective tissue printing can thus be cost-prohibitive, limiting the research to labs which can afford this exorbitant cost and in turn slowing the progress made in the eventual creation of patient derived stem cell engineered organs. The objective of my research is to develop a simple and low-cost introductory system for biological additive manufacturing (Otherwise known as 3D bioprinting). To create an easily accessible and cost-effective system several design constraints were implemented. First, the system had to use mechanical components that could be purchased “off-the-shelf” from commonly available retailers. Second, any mechanical components involved had to be easily sterilizable, modifiable, and compatible with open-source software. Third, any customized components had to be fabricated using only 3D printing and basic tools (i.e. saw, screwdriver, and wrench). Fourth, the system and any expendable materials should be financially available to underfunded school labs, in addition to being sterilizable, biocompatible, customizable, and biodegradable. Finally, all hardware and expendables had to be simple enough as to be operated by high school science students.

In the Kuehn lab, it has been shown that inducible pluripotent stems cells that have been induced to be trabecular meshwork cell-like (iPSC-TM) have a unique ability to regenerate dysfunctional trabecular meshwork (TM) cells by sharing specific unknown factors. In this thesis will discuss the novel means by which I isolate primary human Trabecular Meshwork (pTMs) and efficiently prepare cell cultures for experimentation, such as a sequencing experiment in which I studied expression changes that arose when the TM cell culture’s cell cycle control is manipulated. Previous research has shown that pTM grow atypical when 100% confluent compared to other epithelial cells creating an interesting time frame by which to observe their unique cell cycle control. Using newly isolated TM cell cultures I investigated expression of mRNA and miRNA to understand their roles in cell cycle control of these atypical cultures. With regards to the isolation of TM cell cultures were able to show that the “Crawling Out” methodology is an effective way to establish a pure TM cell line with both a low contamination rate and less passages/time. With these cultures we were able to establish 50 mRNAs and 19 miRNAs that were differential expressed in the TM cell cultures that were atypically grown. When reviewing the literature many of these expression changes were linked to carcinogenics, and the progression/prognosis of various cancer types.

Doctor of Philosophy / Benign tumours grow by expanding and displacing the surrounding tissues, while malignant tumours replace and destroy the surrounding tissues by invasion. Although there is extensive literature on mechanisms of tumour invasion and metastasis, with an emphasis on angiogenesis, adhesion, degradation of the extracellular matrix and migration, an important question not clearly addressed by the literature, but nonetheless approached in this thesis, is that of the fate of normal cells during tissue replacement by migrating invasive malignant cells. Earlier work in the laboratory where this PhD candidature was carried out, investigated the effect of osteosarcoma cells on endothelium. In contrast to the expected angiogenic effect of malignant cells for endothelium, it was found that the human osteosarcoma cell line (SAOS-2) induced apoptosis in human umbilical vein endothelial cells (HUVEC) in contact dependent manner (McEwen et al., 2003). It was suggested that apoptosis of endothelium by malignant tumour cells may facilitate tumour invasion and metastasis (McEwen et al., 2003), and one of the aims of the current study was to extend these findings to include human gingival fibroblasts (HGF) and human umbilical artery smooth muscle cells (HUASMC). The major finding of this thesis was that SAOS-2 induced a reduction in the apparent cell culture density of HGF and HUASMC in a contact-dependent manner. The SW480 colorectal carcinoma cell line did not have any clear effect upon the apparent stromal cell culture density of either HGF or HUASMC, suggesting that the effect under investigation was tumour cell line specific. Surprisingly and in contrast to the similar effect reported for endothelium (Chen et al., 2005; McEwen et al., 2003), the effect of SAOS-2 upon HGF and HUASMC was not due to stromal cell apoptosis. Apoptosis was ruled out as a possible mechanism for the reduced apparent culture density under study, by using widely accepted methods which are dependent upon intermucleosomal fragmentation of DNA, the permeability of plasma membranes to dyes in advanced apoptosis and necrosis, phosphatidylserine translocation as well as inhibitor studies blocking both caspase dependent and independent pathways. While apoptosis was not demonstrated, the possibility emerged that reduced apparent stromal cell culture density reflected fusion events rather than the simple removal of cells as had been earlier reported for HUVEC (McEwen et al., 2003). This idea was supported by reduced SAOS-2 circularity in co-culture. Confocal microscopy of cells pre-labelled with fluorescent dyes further supported this idea, with dual-labelling as evidence of cell fusion. Although occasional homotypic fusion of stromal cells was seen, heterotypic fusion of stromal cells with SAOS-2 was much more prevalent. Time lapse microscopy was performed to further characteristic cell fusion in co-cultures, and revealed multiple transient fusions between SAOS-2 and HGF. To work towards determining the biological relevance of the key observation, two stable SAOS-2 GFP clones were generated for future planned studies using human gingival explants and nude mice. Importantly, the clones were similar to native SAOS-2 with regard to alkaline phosphatise expression and reducing apparent stromal cell culture density. Transient fusions between HGF and SAOS-2, may be a mechanism for cooption of stromal cells into the malignant process, facilitating tumour invasion. Additionally, heterocellular fusion of SAOS-2 with stromal cells may facilitate immune evasion, while it seems likely that despite the absence of an identical activity in SW480 cells, other malignant tumour cells may also express similar activity.

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%).