Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, which catalyses the second committed step in cholesterol biosynthesis. The mechanism by which SPF stimulates this enzyme is not understood and the goal of these studies was to see if SPF affected cholesterol synthesis in cultured cells. Rat supernatant protein factor-like protein (SPF2) shares 77% sequence identity with human SPF. In my studies SPF2 also stimulated squalene monooxygenase in vitro and incubation of SPF2 with protein kinase A (PKA) and C increased its activity by about 2-fold, as shown earlier with SPF. GTP and GDP prevented the stimulation of squalene monooxygenase by SPF2, suggesting that binding of these nucleotides inhibits SPF2. This inhibition could be prevented by the addition of -tocopherol, although -tocopherol alone had no effect on SPF2 activity in vitro. Expression of human SPF in hepatoma cells, which lack expression of endogenous SPF, increased cholesterol synthesis by 2-fold and addition of dibuytrylcAMP, a PKA activator, to these cells yielded an additional 62% increase whereas addition of a PKA inhibitor completely blocked the ability of SPF to stimulate cholesterol synthesis. To further confirm a role for phosphorylation in the regulation of SPF, substitution of alanine for serine-289 (a putative PKA recognition site) reduced the PKA-mediated activation of SPF in vitro by 50%, as measured with microsomal squalene monooxygenase and completely blocked the ability of SPF to stimulate cholesterol synthesis in hepatoma cells. In further structure-function studies, deletion of the carboxy-terminal Golgi-dynamics domain greatly increased the ability of SPF to stimulate squalene monooxygenase in microsomes, but, paradoxically prevented SPF from stimulating cholesterol synthesis in cell culture. Addition of brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate cholesterol synthesis, supporting a role for the Golgi in SPF function. Since squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway was investigated. The substitution of 14Cmevalonate for 14C-acetate completely blocked an SPF-induced 1.5-fold increase in squalene synthesis, suggesting that SPF stimulated mevalonate synthesis at HMGCoA reductase. 2,3-Oxidosqualene synthesis from 14C-mevalonate remained elevated (1.3-fold) with mevalonate demonstrating that SPF also stimulated squalene monooxygenase in hepatoma cells. SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels in cells, indicating that SPF directly activated these enzymes. Indeed, addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and, coupled with the ability of PKA-mediated phosphorylation to regulate SPF activity, suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.

This dissertation on the theme Interaction of macrophages and the prostate cancer cell line PC-3 was focused on the initial mapping of interactions between human mac-rophages and the prostate cancer cell line PC-3. Microscopic and molecular-genetic methods have been used for analysis. The structure and ultrastructure of macrophages and PC-3 cells have been de-scribed. The description of cell morphology in interaction with each other has been de-voted to processes associated with adherence of macrophages on PC-3, eferocytosis and phagocytosis. Cultivation of PC-3 with plumbagine showed an increase of apoptotic cells. Cultivation of macrophages with PC-3 supernatant has shown that solubiling fac-tors in supernatant may have an effect on inducing cell death in macrophages. In our study, the supernatant did not affect the production of TNF alfa, IL-12, IL-10 or IL-6 by macrophages. A time lapse video was created to show physical interactions of macro-phages and invasive cell. All obtained results show that there are interactions between the cancer cells and immune system cells that have not yet been discovered.

SPF (Supernatant Protein Factor) is a cytosolic protein that stimulates at least two enzymes in the cholesterol biosynthetic pathway: squalene monooxygenase and HMGCoA reductase. The mechanism of action has not been established but may be related to lipid transfer between intracellular membranes. There are three human genes for SPF: SEC14L2 (SPF1), SEC14L3 (SPF2) and SEC14L4 (SPF3). The present study differentiates these closely related genes by evaluating their tissue-specific and relative expression levels. SPF1 mRNA was found to be most abundant in liver, mammary gland and stomach. SPF2 showed negligible expression in all tissues tested; SPF3 expression pattern was similar to that of SPF1, but at 10-50-fold lower levels than SPF1. A cDNA to SPF3 was cloned and, upon transfection into rat hepatoma cells, was shown to increase cholesterol synthesis by approximately 50%, similar to that obtained with SPF1. However, in contrast to SPF1, SPF3 did not stimulate squalene monooxygenase activity in microsomal preparations, suggesting that it acts primarily through activation of HMG-CoA reductase. SPF possesses a lipid binding domain (Sec14) and a Golgi dynamics domain (GOLD). SPF resides in the cytosol and requires phosphorylation and the presence of Golgi in order to stimulate cholesterol synthesis. To determine if SPF associates with specific subcellular structures, cellular immunofluorescence studies were carried out. A phosphorylationdefective mutant, a protein lacking the GOLD domain, and the effect of protein kinase A-mediated phosphorylation of endogenous SPF were examined. No change in the subcellular location of SPF could be detected with either the phosphorylation mutant or the native SPF after protein kinase A activation. However, removal of the GOLD domain resulted in a protein that co-localized with large vesicular structures around nucleus. Studies with rat hepatoma cells showed that the expression of the two rat SPF genes is upregulated in response to serum deprivation, and is potentiated by removal of glucose. Lipid/cholesterol availability was demonstrated to be at least one of the serum components that affected SPF transcript levels. The oxysterol receptor LXR was shown not to be involved in SPF gene regulation, implicating SREBP and/or PPARα as the principal regulators of SPF gene transcription.

Separate treatment of supernatant with dewatering of digested sludge with application of partial nitritation/Anammox process is assessed to be a cost-effective way to remove about 10-15% of influent nitrogen and, thereby, facilitate possibilities to reach required effluent requirements from the plant. The combined partial nitritation/Anammox process can be performed in two separate reactors or in one-stage. Both process options have been investigated in technical- and laboratory-scale pilot plants with moving-bed biofilm reactors (MBBR) filled with Kaldnes rings. Use of the two-stage process resulted in a very stable partial nitritation with a suitable nitrite to ammonium ratio (NAR) for the following Anammox step. Dissolved oxygen (DO) and pH value were identified as key factors for the partial nitritation process. The Anammox process could also be operated in a stable way. A high nitrite concentration, however, inhibited the process and the time for recovering the process at low nitrite concentration was about four months. Seeding of the partial nitritation reactor with Anammox bacteria (the recirculation of Anammox effluent to the nitritation reactor) turned out to be a simple and easy method to enable creation of an oxic-anoxic biofilm in one reactor. Studies have shown that such a one-stage system would be the best choice for full-scale implementation due to significantly higher nitrogen removal rates and easier operation. The partial nitritation process was found to be the rate-limiting reaction to perform the overall nitrogen removal. Measurements of conductivity and pH were suitable parameters for monitoring of the nitrogen reactions. A control and monitoring system was developed both for two-stage and one-stage technology. The system was mainly based on relationships between conductivity and inorganic nitrogen components, while in the one-stage technology measurements are used of both conductivity and pH and their relationships with inorganic nitrogen compounds. Alkalinity was an additional measured parameter suitable for process control and monitoring. Theoretically calculated values of conductivity were in good agreement with experimentally obtained results. / QC 20100819

A bifidobacteria mix (nine strains) was evaluated for its effect on the growth and survival of Escherichia coli O157:H7 (ATCC 43890) in 11% NFDM and MRS broth under the optimum growth conditions for E. coli O157:H7 growth (37¡ãC, aerobic). Preliminary experiments were conducted to obtain the growth curves of the 9 strains of bifidobacteria and E. coli O157:H7 and confirm the inhibitory effect of acidity on E. coli O157:H7 (pH of media adjusted to 3.8). Acidapted E. coli O157:H7 showed no difference in resistance toward bifidobacteria (P>0.05) when compared to the non-acid adapted one. Escherichia coli O157:H7 did not survive in the supernatant of the bifodbacteria mix collected after incubation (37¡ãC) with aerobic shaking (8 h). However, the pathogen was able to grow after the pH of the supernatant was adjusted to 6.50 (pH of fresh MRS broth). Results suggest that a high content of bifidobacteria has a strong inhibitory effect on E. coli O157:H7, in part due to the low pH. However, products from bifidobacteria may also exert inhibitory effects.

At birth, the immune system is immature and the gut microbiota influences immune maturation. Staphylococcus aureus (S. aureus) and lactobacilli are part of the neonatal gut microbiota and have seemingly opposite effects on the immune system. S. aureus is a potent immune activator and early-life colonization associates with higher immune responsiveness later in life. Lactobacilli-colonization associates with reduced allergy-risk and lower immune responsiveness. Further, lactobacilli modulate immune-activation and have probiotic features. Here, we investigated S. aureus-induced activation of human lymphocytes, including T regulatory cells (Tregs), conventional T-cells (CD4+ and CD8+), unconventional T-cells (γδ T-cells and MAIT-cells) and NK-cells from children and adults, together with the modulatory effect of lactobacilli on immune-activation. Further, early-life colonization with these bacteria was related to lymphocyte-maturation, plasma cytokine- and chemokine-levels and allergy.  S. aureus cell free supernatant (CFS) and staphylococcal enterotoxin (SE) A induced an increased percentage of FOXP3+ Tregs and of CD161+, IL-10+, IFN-γ+ and IL-17A+ Tregs (Paper I). The same pattern was observed in children with a lower degree of activation, possibly due to lower CD161-expression and poor activation of naive T-cells (Paper II). S. aureus-CFS induced IFN-γ-expression, proliferation and cytotoxic capacity in conventional and unconventional T-cells, and NK-cells. SEA, but not SEH, induced activation of unconventional T-cells and NK-cells by unknown mechanism(s) (Paper III, extended data). Lactobacilli-CFS reduced S. aureus-induced lymphocyte activation without the involvement of IL-10, Tregs or monocytes, but possibly involving lactate (Paper III). Early-life colonization with S. aureus associated with increased percentages of CD161+ and IL-10+ Tregs while lactobacilli-colonization negatively correlated with the percentage of IL-10+ Tregs later in life (Paper II). Allergic disease in childhood associated with double allergic heredity, being born wintertime and with higher plasma levels of TH2-, TH17- and TFH-related chemokines early in life. Lactobacilli-colonization associated with lower prevalence of allergy, reduced chemokine-levels and increased levels of IFN-γ in plasma (Paper IV).    This thesis provides novel insights into S. aureus- and SE-mediated activation of Tregs, unconventional T-cells and NK-cells and suggests an overall impairment of immune-responsiveness towards this bacterium in children. Further, S. aureus-colonization may influence the maturation of peripheral Tregs. Our data show that lactobacilli potently dampen lymphocyte-activation in vitro and that colonization associates with Treg-responsiveness, altered plasma cytokine- and chemokine-levels and with remaining non-allergic, thereby supporting the idea of lactobacilli as important immune-modulators. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Las patologías más importantes del páncreas exocrino, como la pancreatitis crónica (PC) o el cáncer de páncreas, representan un gran problema de salud pública en Europa. En la PC, el tejido acinar es substituido por complejos ductales. Además, es difícil mantener el fenotipo diferenciado de las células acinares en cultivo ya que sufren una transdiferenciación acinar-ductal.Las células madre embrionarias (ES) de ratón han sido utilizadas en la última década para generar in vitro células completamente diferenciadas de varios linajes celulares. No obstante, la capacidad de las células ES a diferenciarse a tipos celulares de origen endodérmico es muy limitada. El objetivo principal de este proyecto ha consistido en desarrollar estrategias para diferenciar células ES de ratón a células pancreáticas acinares con una elevada eficiencia mediante 1) la optimización de las condiciones de cultivo con tal de activar vías de señalización implicadas en el desarrollo/diferenciación pancreáticas; 2) la sobreexpresión de factores transcripcionales maestros utilizando vectores virales con el fin de recapitular específicamente un programa de diferenciación acinar; 3) la selección genética de las células comprometidas al linaje acinar con el objetivo de purificar las células acinares diferenciadas.Mediante la integración de estos abordajes, hemos conseguido aislar células que comparten características fenotípicas con células acinares inmaduras según la expresión de marcadores de diferenciación y la respuesta funcional a secretagogos. / Exocrine pancreatic diseases such as chronic pancreatitis (PC) or pancreatic cancer are major health issues in Europe. In CP, the acinar tissue is substituted by ductal complexes. In addition, it is difficult to maintain the differentiated phenotype of the acinar cells in culture as within few days an acinar-ductal transdifferentiation takes place.In the last decade, mouse embryonic stem cells (mES) have been used to generate differentiated cells of a variety of cellular lineages in vitro. However, the ability of ES cells to differentiate into endodermal lineages is limited. The main objective of this project has focused on the development of strategies to differentiate mES to pancreatic acinar cells with high efficiency by means of: 1) Optimization of cell culture conditions to activate signalling pathways involved in pancreatic differentiation/development; 2) the overexpression of master transcription factors involved in pancreas development using viral vectors in order to recapitulate specific acinar differentiation program; 3) the genetic selection of cells committed to the acinar linage in order to purify the differentiated cells.The integration of these different strategies allowed us to isolate cells that share phenotypic features with immature acinar cells according to the expression of differentiation markers and the functional response to acinar secretegogues.

Som följd av dagens ökning i global befolkning och köttproduktion är alternativa hållbara köttsubstitut mycket eftertraktat. Som basal råvara kan växter eller mikrobiella produkter utnyttjas för proteinrika hållbara livsmedelsprodukter. Detta arbete syftar till att undersöka olika proteinextraktionsmetoder som i nuläget används för att erhålla ett proteinisolat som kan ha användning i livsmedelsindustrin. Utvalda råvaror som kommer studeras och diskuteras är ärta, quinoa, mjölmask och filamentösa svampar. Allmän information för varje råvara diskuteras men fokus har lagts på att studera processer för att extrahera proteiner till ett isolat från råvarorna. Arbetet inkluderar generella tillvägagångssätt vad gäller proteinextraktion likväl som mer specifika metoder från enskilda försök att extrahera ett visst protein. Slutligen jämförs val av extraktionsmetoder och vilka råvaror som idag är i kommersiellt bruk och reflekterande tankar om varför inte vissa råvaror hunnit lika långt i utvecklingen i proteinextraktion som andra. / As a result of today's increase in global population and meat production, alternative and more sustainable meat substitutes are highly sought after. As a basic raw material, plants or microbial products can be utilized for protein-rich sustainable food products. This work aims to investigate various protein extraction methods currently used to obtain a protein isolate that can be of use in the food industry. Selected raw materials studied and discussed are pea, quinoa, flour worm and filamentous fungus. General information for each raw material is discussed, however focus has been on studying processes for extracting proteins into an isolate from the raw materials. The work includes general methods to protein extraction as well as more specific methods from individual attempts to extract a specific protein. Finally, the extraction methods are compared, the raw materials that are currently in commercial use are discussed as well as thoughts why some raw materials have not progressed as far in protein extraction as others are compared.

The aim of this Ph.D. thesis was to analyze the morphine-induced changes of frontal brain cortex protein composition in rats exposed to increasing doses of morphine (10-50 mg/kg) for prolonged period of time (10 days). The first part of this work was oriented to the analysis of the phenomenon of hypersensitization/superactivation of adenylyl cyclase (AC), which is regarded as one of the crucial molecular mechanisms causing drastic pathological consequences of drug addiction. The increase of AC activity represents a "compensatory" response and is functionally related to the desensitization of G protein response to prolonged morphine exposure of target cells. The clear desensitization of µ-OR- and δ-OR-stimulated G protein response by morphine was demonstrated in our laboratory by analysis of the dose-response curves of DAMGO and DADLE-stimulated, high-affinity [35 S] GTPγS binding in plasma membranes isolated from frontal brain cortex of rats exposed to morphine according to the same protocol as that used in my Ph.D. thesis (10-50 mg/kg, 10 days). The κ-OR-stimulated [35 S] GTPγS binding was unchanged. It has been determined the amount of all AC isoforms (AC I-IX) in plasma membranes (PM) isolated from control and morphine-treated rats which were sacrificed 24 hours since the last dose of morphine....

A escassez das fontes de fósforo e o alto consumo de energia associado à produção de fertilizantes nitrogenados serão problemas que deverão ser enfrentados no futuro. A recuperação de nutrientes das águas residuárias na forma de estruvita tem sido considerada como uma alternativa para atenuar estes problemas. Na América Latina, a produção de estruvita a partir de esgoto ainda não é uma tecnologia bem conhecida e, portanto, a finalidade deste trabalho é contribuir com uma melhor compreensão dos fenômenos envolvidos. Para isso, a pesquisa foi dividida em três etapas: 1) produção de estruvita a partir de efluente de reator anaeróbio de fluxo ascendente com manto de lodo (RAFA); 2) produção de estruvita a partir de sobrenadante de digestor anaeróbio de lodo de um processo com remoção biológica de fósforo (DALRBF) e 3) influência do cálcio na estruvita produzida na etapa 2. Nas três etapas, ajustaram-se as concentrações de magnésio, a fim de obter razões fósforo:magnésio (P:Mg) pré-estabelecidas, e o pH entre 8,00 e 10,50. Os resultados da primeira etapa mostraram que não foi possível produzir estruvita no efluente do RAFA nas condições testadas. No entanto, foram observadas remoções de fósforo e de nitrogênio, devido à formação de fosfatos de cálcio e de magnésio amorfos. Os resultados da segunda etapa comprovaram a viabilidade de produção de estruvita de sobrenadante de DALRBF e mostraram que os consumos molares dos íons fosfato (PO43-), amônio (NH4+) e magnésio (Mg2+) ou as remoções destes (%) não devem ser os únicos parâmetros para avaliar a formação de estruvita, pois outros compostos cristalizam ou precipitam e reduzem a qualidade do mineral. Para um meio com condições semelhantes às testadas nesta etapa, uma razão P:Mg 1:2 e um pH igual a 9,50 asseguram a máxima recuperação de nutrientes como estruvita com concentração mínima de impurezas, facilitando seu posterior uso como fertilizante. Os resultados da terceira etapa mostraram que uma fase amorfa de fosfato de cálcio ou de magnésio se forma na superfície da estruvita. / The shortage of the phosphorus sources and high-energy consumption associated to the nitrogen fertilizers production will be problems in the future. The nutrient recovery from wastewater as struvite has been considered as an alternative to alleviate these problems. In Latin America, production of struvite from wastewater is not yet a wellknown technology and therefore the purpose of this work is to contribute to a better understanding of the phenomena involved. This research work was performed in three phases: 1) production of struvite from upflow anaerobic sludge blanket reactor effluent; 2) production of struvite from anaerobic digester supernatant of enhanced biological phosphorus removal process (ADS-EBPR) and 3) influence of calcium in the struvite produced in the phase 2. In three phases, the magnesium concentrations were adjusted to obtain the preset phosphorus:magnesium (P:Mg) ratios and the pH was adjusted between 8,00 and 10,50. The results of the first phase showed that it is not possible to produce struvite in the upflow anaerobic sludge blanket reactor effluent in the tested conditions. However, removal of nitrogen and phosphorus was observed because amorphous calcium and magnesium phosphates were produced. The results of the second phase showed that it is possible to produce struvite in the ADS-EBPR and the molar consumptions of phosphate (PO43-), ammonia (NH4+) and magnesium (Mg2+) or removals (%) should not be the only parameters to evaluate the struvite formation, because other compounds crystallize or precipitate and reduce the quality of the mineral. In the similar conditions tested in this phase, a P:Mg ratio 1:2 and pH 9,50 assure maximum nutrients recovery as struvite with minimum impurities concentration, facilitating its subsequent use as fertilizer. The results of the third phase showed that amorphous calcium or magnesium phosphates were produced on the struvite surface.