982 resultados para Inoculation and incubation,
Resumo:
Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications.
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Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of Nε- and Nα-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.
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A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium was that incorporated into carbamoyl-phosphate, since all 14C-CO2 from bicarbonate was eliminated. The method is rapid and requires only a low pressure supply of CO2 to remove the excess substrate. The reaction is linear up to 10 min using homogenate dilutions of 1:20 to 1:200 (w/v). Rat liver activity was in the range of 89±8 nkat/g. Hyperproteic diet resulted in a significant 1.4-fold increase. The design of the method allows for the processing of multiple samples at the same time, and incubation medium manipulation is unnecessary, since the plastic incubation vial and its contents are finally counted together.
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Needle trap devices (NTDs) are a relatively new and promising tool for headspace (HS) analysis. In this study, a dynamic HS sampling procedure is evaluated for the determination of volatile organic compounds (VOCs) in whole blood samples. A full factorial design was used to evaluate the influence of the number of cycles and incubation time and it is demonstrated that the controlling factor in the process is the number of cycles. A mathematical model can be used to determine the most appropriate number of cycles required to adsorb a prefixed amount of VOCs present in the HS phase whenever quantitative adsorption is reached in each cycle. Matrix effect is of great importance when complex biological samples, such as blood, are analyzed. The evaluation of the salting out effect showed a significant improvement in the volatilization of VOCs to the HS in this type of matrices. Moreover, a 1:4 (blood:water) dilution is required to obtain quantitative recoveries of the target analytes when external calibration is used. The method developed gives detection limits in the 0.020–0.080 μg L−1 range (0.1–0.4 μg L−1 range for undiluted blood samples) with appropriate repeatability values (RSD < 15% at high level and <23% at LOQ level). Figure of merits of the method can be improved by using a smaller phase ratio (i.e., an increase in the blood volume and a decrease in the HS volume), which lead to lower detection limits, better repeatability values and greater sensibility. Twenty-eight blood samples have been evaluated with the proposed method and the results agree with those indicated in other studies. Benzene was the only target compound that gave significant differences between blood levels detected in volunteer non-smokers and smokers
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Studies addressing the biological control of Botrytis cinerea have been unsuccessful because of fails in inoculating tomato plants with the pathogen. With the aim of establishing a methodology for inoculation into stems, experiments were designed to assess: i. the aggressiveness of pathogen isolates; ii. the age at which tomato plants should be inoculated; iii. the susceptibility of tissues at different stem heights; iv. the need for a moist chamber after inoculation; and v. the effectiveness of gelatin regarding inoculum adhesion. Infection with an isolate from tomato plants that was previously inoculated into petioles and then re-isolated was successful. An isolate from strawberry plants was also aggressive, although less than that from tomato plants. Tomato plants close to flowering, at 65 days after sowing, and younger, middle and apical stem portions were more susceptible. There was positive correlation between lesion length and sporulation and between lesion length and broken stems. Lesion length and the percentage of sporulation sites were reduced by using a moist chamber and were not affected by adding gelatin to the inoculum suspension. This methodology has been adopted in studies of B. cinerea in tomato plants showing reproducible results. The obtained results may assist researchers who study the gray mold.
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The interest to develop research on the host-parasite relationship in bovine tritrichomonosis has accomplished the use of experimental models alternative to cattle. The BALB/c mouse became the most appropriate species susceptible to vaginal Tritrichomonas foetus infection requiring previous estrogenization. For the need of an experimental model without persistent estrogenization and with normal estrous cycles, the establishment and persistence of vaginal infection on BALB/c mouse with different concentrations of T. foetus in two experimental groups was evaluated. Group A was treated with 5mg of b-estradiol 3-benzoate to synchronize the estrous, 48 hours before the T. foetus vaginal inoculation, and Group B was inoculated in natural estrus. At 5-7 days after treatment, estrogenic effect decreased allowing all animals to cycle regularly during the experiment. From the first week post-infection, samples of vaginal mucus were taken from all animals during 34 weeks, in order to evaluate the course of infection and the stage of the estrus cycle. Group A showed 93.6% of infected animals, and Group B showed 38%. Different doses of T. foetus were assayed to establish the vaginal infection, with a persistence of 34 weeks. Although different behavior was observed in each subgroup belonging to either Group A or Group B, there were no significant differences among the infecting doses used. The b-estradiol 3-benzoate treatment had a favorable effect on the establishment of the infection (P<0.0001), but it did not influence its persistence (P=0.1097). According to the results, an experimental mouse model is presented, appropriate for further studies on mechanisms of pathogenicity, immune response, protective evaluation of immunogen and therapeutic effect of drugs.
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In the state Mato Grosso do Sul, Brazil, outbreaks of meningoencephalitis by BoHV-5 and polioencephalomalacia (PEM) display similar epidemiological features, suggesting that meningoencephalitis may be associated with reactivation of a latent BoHV-5 infection, during the development of PEM. To test this hypothesis, four 7-8 months old steers negative for BoHV-5 antibodies were inoculated intranasally with BoHV-5 and received amprolium from day 35 to day 105 after inoculation. Because PEM was not produced during this period, ammonium sulphate was given from day 114 to day 180 after inoculation. Two uninfected control steers received amprolium and ammonium sulphate for the same periods. All inoculated cattle developed antibodies against BoHV-5 after inoculation and the virus was isolated from nasal swabs, indicating that they were infected. Two inoculated steers had clinical signs of PEM after 118 and 146 days after virus inoculation. One was euthanized after a clinical manifestation period of seven days and had severe lesions of PEM and meningoencephalitis. BoHV-5 was isolated from the central nervous system of this animal. The other animal recovered but continued to manifest chronic signs of PEM and was euthanatized. On histological examination, the cerebral cortex, caudate nucleus and thalamus had multifocal areas of malacia and mild meningoencephalitis of the cortex. BoHV-5 was not isolated from the brain. One uninfected control steer had signs of neurological disease on day 158 and had lesions of PEM without meningoencephalitis at necropsy. The simultaneous production of PEM and diffuse meningoencephalitis, with isolation of BoHV-5, in one steer treated with ammonium sulphate, 118 days after BoHV-5 inoculation, suggests that latent BoHV-5 was reactivated in this animal submitted to experimental induction of PEM.
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The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.
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The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.
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Fish vaccination has been increasingly exploited as a tool to control pathogen infection. The production of immunoglobulin following vaccination might be affected by several factors such as management procedures, water temperature, and the presence of xenobiotics. In the present study, we aimed to investigate the kinetics of immunoglobulin production in silver catfish (Rhamdia quelen) inoculated with inactivated Aeromonas hydrophila and kept at two different water temperatures (17.4±0.4° or 21.3±0.3°C). The effect of a second antigen inoculation and exposure of fish to sublethal concentrations of the herbicides atrazine and glyphosate at 10% of the lethal concentration (LC50-96h) on specific serum antibodies were also investigated. Antibodies to A. hydrophila were detected as early as 7 days post-inoculation and increased steadily up to 35 days. The kinetics of antibody production were similar in fish kept at 17.4±0.4° and 21.3±0.3°C, and reinoculation of antigen at 21 days after priming failed to increase specific antibody levels. Intriguingly, we found that, in fish exposed to atrazine and glyphosate, the secretion of specific antibodies was higher than in non-exposed inoculated fish. These findings are important for the design of vaccines and vaccination strategies in Neotropical fish species. However, because atrazine and glyphosate are widespread contaminants of soil and water, their immune-stimulating effect could be harmful, in that fish living in herbicide-contaminated water might have increased concentrations of nonspecific antibodies that could mediate tissue injury.
Resumo:
Cajá-manga, also known as golden apple and hog-plum, is an exotic fruit native from Îles de la Société (French Polynesia), which was first introduced in Brazil in 1985. The pulp of ripe fruit was treated with the commercial enzymatic pool and its effect was evaluated in terms of yield, as well as the physical properties viscosity, turbidity and color (L* values). Response surface methodology was used and three levels were adopted for the independent variables temperature (30, 40, and 50 ºC), incubation time (30, 60 and 90 minutes) and enzyme concentration (0.01, 0.05, 0.09 v/v%). A central composite statistical design was used to guide the experimental work. The enzyme treatment highly increased both juice yield (up to 56%) and color (up to 8.6%) and strongly decreased viscosity (up to 57.4%), clarity (up to 77%) and turbidity (up to 85.5%). Incubation time was the most interacting facto, whereas temperature was the least one. Optimization analysis was carried out to reduce enzyme concentration to a minimum by superposing the contour plots of the tested properties, and the recommended ranges of the variables enzyme concentration, process temperature and incubation time were, respectively, 0.042-0.068%, 47.0-49.0 ºC and 82-90 minutes.
Resumo:
The objective of the present work was to evaluate the capacity of three isolates of Aspergillus flavus to produce aflatoxin under different culture conditions. This experiment was based on a 2³ factorial design, in which the independent variables were temperature (20-40 °C), incubation time (7-21 days), and the pH (2.0-6.0) in two different synthetic media. The optimal conditions were applied to non-aflatoxigenic isolates previously tested in coconut agar. Aflatoxin B1 was extracted directly from the synthetic cultures with chloroform. Thin Layer Chromatography (TLC) and Photographic Photometry were utilized to identify and quantify the compounds. Preliminary results showed that YES agar was an alternative medium for detecting the toxigenic potential of Aspergillus flavus in the following conditions: pH of 5.2, temperature of 25 °C, and incubation time of 11 days producing 206.05 ng.CFU-1 of aflatoxin B1. Of the 30 non-aflatoxigenic isolates, 12 presented a positive result in the optimal media and conditions tested.
Resumo:
Tempeh is a food obtained by fermentation of soybean grains by the fungus Rizophus oligosporus. It is a traditional Indonesian food that presents benefits for human health protecting against diarrhea and chronic diseases. Tempeh processing includes dehulling, cooking, inoculation, and fermentation. In this study, chemical characteristics of tempeh prepared with soybean cultivars specially developed for human consumption (BRS 216, BRS 232, BRS 257, and BRS 267) were investigated. Soybean grains and tempeh obtained from these cultivars were analyzed for oil, protein, antinutrional factors, and isoflavone content. Cultivar BRS 216 presented the highest protein content in the grains (36.81%) and in tempeh (51.99%). On average, the protein content in tempeh increased 16% in relation to that of soybean grains. Isoflavone content was higher in the grains than in tempeh with significant differences among the cultivars. However, the aglycones content increased about 50% in tempeh (49.00 mg.100 g-1 on average) compared to that of raw material (soybean grains - 21.49 mg.100 g-1, on average). The content of Kunitz trypsin inhibitor (KSTI) reduced 83% in tempeh, on average, as compared to the value found in the grains. Phytic acid content was similar in both tempeh and the grains.
Resumo:
Bacillus subtilis CBTK 106, isolated from banana wastes, produced high titres of a-amylase when banana fruit stalk was used as substrate in a solid-state fermentation system. The e¤ects of initial moisture content, particle size, cooking time and temperature, pH, incubation temperature, additional nutrients, inoculum size and incubation period on the production of a- amylase were characterised. A maximum yield of 5 345 000 U mg~1 min~1 was recorded when pretreated banana fruit stalk (autoclaved at 121 ¡C for 60 min) was used as substrate with 70% initial moisture content, 400 lm particle size, an initial pH of 7.0, a temperature of 35 ¡C, and additional nutrients (ammonium sulphate/sodium nitrate at 1.0%, beef extract/peptone at 0.5%, glucose/sucrose/starch/maltose at 0.1% and potassium chloride/sodium chloride at 1.0%) in the medium, with an inoculum-to-substrate ratio of 10% (v/w) for 24 h. The enzyme yield was 2.65-fold higher with banana fruit stalk medium compared to wheat bran
Resumo:
Angesichts der Geschichte der Entwicklungspolitik, ist diese Arbeit darauf ausgerichtet, einige Beobachtungen in Bezug auf die so genannte Entwicklung hervorzuheben; insbesondere auf die andauernde prekäre Situation und Armut in ländlichen afrikanischen Gebieten. Armut ist nach Amartya SEN – weiter präzisiert von J.L. Dubois – die Deprivation von „Fähigkeiten“, die Individuen und lokale Gemeinschaften zu ausgeschlossenen und vergessenen Akteuren des Systems machen. Das nennt Paulo Freire, das Menschen zu „Objekten“ gemacht werden. Es rechtfertigt die starke Annahme, die in dieser Studie getroffen wird, dass vielmehr die Menschen als „Subjekte“ ihrer Veränderung und Entwicklung im Mittelpunkt stehen. Die Arbeit zeigt und erklärt in historischer Chronologie, wie die Entwicklungspolitiken und unterschiedliche Beteiligte auf allen Ebenen diese Situation verursachen. Trotz alledem bleiben die Individuen und lokalen Gemeinschaften, die in Symbiose mit ihrer natürlichen Umwelt leben, die reich an verschiedenen Ressourcen und Potentialen ist, als Reaktion darauf und gleichzeitig als Überlebensstrategie zutiefst verbunden mit dem, was sie vor Ort haben, womit sie eine tiefere und intensive Beziehung besitzen, wenn man von ihrer Geschichte, ihrer Kultur und der Handlungslogik ausgeht. Für externe Akteure, die sie über das vorhandene System dominieren und beeinflussen bleiben sie „Objekte“, aber in der Vielzahl ihrer endogenen Initiativen, zeigen sie die Fähigkeit und Substanz, die beweisen, dass sie auf ihrer Ebene das eigentliche Subjekt sind, die dynamischen Akteure. Aber isolierte Initiativen auf spezifische reale Bedürfnisse bei gleichzeitiger Dominierung durch das System mit seiner Marktlogik, führt dies langfristig nur zu dem Zirkulus Vitiosus der Armut. Daher ist eine ganzheitliche Sicht entscheidend für nachhaltige Entwicklung und für die notwendige Veränderung. Es geht nicht nur um die Veränderung des Systems und die Wahl politischer Maßnahmen, sondern genau genommen um das Verhalten der Akteure auf allen Ebenen und die Art der Beziehungen zwischen ihnen allen. Es ist eine Frage des erneuten Überdenkens des Entwicklungspfades, der andere Logik, Visionen, Interessen und Strategien aller Beteiligten, unserer so genannten Akteure einschließt. Ob dies von endogenen Initiativen oder neuen gemeinsamen Projekten ausgeht: man wird in einen Prozess kollektiven Lernens eintreten, den Paul Singer und Clarita Müller-Plantenberg erläutern und entwickeln in dem Konzept der Inkubation und Solidarischen Ökonomie, die Eigeninitiative, Selbstbestimmung und Selbstverwaltung von lokalen Gemeinschaften und die Öffnung für eine Neu-Konzeptualisierung und Institutionalisierung einschließt. So ein Prozess ist nur mit einem interdisziplinären Rahmen möglich. Dieser Rahmen soll auf einer zusätzlicher Kommunikation zwischen den Akteuren und Sozialwissenschaften beruhen und mit jenen, die auf dem Feld der Technologie arbeiten. So können dann technische „Experten“ angesichts eines technischen Projektfehlers, der aufgrund von bestimmten sozialen und kulturellen Realitäten zustande kam sagen, „es ist kein Scheitern ; es war ein Schritt innerhalb eines Lernprozesse der in die technischen Projekte und Studien einbezogen werden muss“. Wir haben das Energiethema gewählt; und insbesondere, Energie für eine nachhaltige ländliche Entwicklung in Subsahara-Afrika, um den Weg von der Theorie in die Praxis zu illustrieren und experimentell auszuprobieren, den Weg von den Beobachtungen zu der Veränderung, wobei Fragen, Annahmen, Strategien und konkrete Aktionen für den Wandel behandelt werden. Wir nennen unseren experimentellen Weg: DRIEE, das heißt auf Deutsch Ländliche Entwicklung und Inkubation von Energieunternehmen. Dabei gehen wir davon aus, dass: - Energie im Allgemeinen auf der internationalen Ebene fast gleichbedeutend mit Elektrizität ist. Heute bestehen die wichtigsten Bedürfnisse nach Energie dort wo die agro-pastorale Produktion, das Kochen, die Nahrungsmittelkonservierung und Verarbeitung …etc. stattfindet. - Diese ländliche Bevölkerung zu etwa 80% der nationalen Wirtschaft ausmacht. Dass sie gleichzeitig aber nur zu weniger als 5% der Energieproduktion Zugang hat, was oft auf Licht reduziert ist und nicht einmal ihrer Produktion zugute kommen kann. - Die Projekte für Energie und Elektrizität vor allem auf die Technologischen Fragen konzentriert sind und weniger auf die Bedürfnisse. Fast die Gesamtheit der Fonds für Energie wird in Bezug auf die Investitionen Infrastruktur der Produktion und Verteilung durch die konventionellen zentralisierten Netze geplant. Angesichts dieser Analysen gehen die in dieser Arbeit vorgenommenen Studien in Gambia und Kamerun von Bestandsaufnahmen und / oder beschreibenden regionalen Analysen aus: - von Bedürfnissen, von Praktiken und lokalen Initiativen von Fragen der Energie, für einzelne Professionen, Haushalte, Gruppen, spezifische Gruppen, wie Frauen, ländliche Gemeinden mit ihren spezifischen Charakteristika. - Von Potentialen: natürliche lokale Energieressourcen, soziokulturelle Ressourcen – so z.B. die empirisch feststellbaren menschliche Ressourcen wie endogenes Wissen und praktische organisatorische Fähigkeiten gegenüber den Problemen der Energie. Dieser experimentelle Schritt von Handlungsforschung (DRIEE) in Kamerun führte zu der Gründung einer Organisation, über die und mit der wir die Logik der Inkubation und Solidarischen Ökonomie einführen. Das ist FERDEDSI, das heißt auf Deutsch „Forum für Erneuerbare Energie – Nachhaltige Entwicklung und Internationale Solidarität“. Zunächst war dies eine Energiegenossenschaft und dann (im Prozess) wurde es zu einer institutionellen Nische von mehreren Mikro Initiativen in ländlichen Gebieten. FERDEDSI ist ein Prozess der Inkubation und ein Inkubator ist also gleichzeitig ein inkubiertes Energieunternehmen aber auch ein Inkubator für lokale Organisationen. Die ersten Aktionen finden in den Departments von Noun und Ménoua in der westlichen Provinz von Kamerun statt. Während der Forschungsperiode findet akademische Austausch statt (Nord-Süd und Süd-Süd), diese ist dabei zu formalen Partnerschaften zu werden, nicht nur zwischen Universitäten sondern genauer lokale Organisationen und Universitäten. Dieser letzte Typ von Partnerschaften, die die solidarische Ökonomie ausmachen ist auch eine Innovation des Prozesses für die afrikanischen Fälle, die dem Beispiel dessen, was in Lateinamerika geschieht, folgen. So kommt es zu gegenseitiger sinnvoller Ausbildung in den internationalen Arbeitsgruppen und Seminaren der Universität.