On the functional ordering of biomembranes : Implications for drug binding

Cells are packed with membrane structures, defining the inside and outside, and the different subcellular compartments. These membranes consisting mainly of phospholipids have a variety of functions in addition to providing a permeability barrier for various compounds. These functions involve cellular signaling, where lipids can act as second messengers, or direct regulation of membrane associating proteins. The first part of this study focuses on relating some of the physicochemical properties of membrane lipids to the association of drug compounds to membranes. A fluorescence based method is described allowing for determination of the membrane association of drugs. This method was subsequently applied to a novel drug, siramesine, previously shown to have anti-cancer activity. Siramesine was found to associate with anionic lipids. Especially interesting is its strong affinity for a second messenger lipid phosphatidic acid. This is the first example of a small molecule drug compound specifically interacting with a cellular lipid. Phosphatidic acid in cells is required for the activation of many signaling pathways mediating growth and proliferation. This provides an intriguing possibility for a simple molecular mechanism of the observed anti-cancer activity of siramesine. In the second part the thermal behavior and self assembly of charged and uncharged membrane assemblies was studied. Strong inter-lamellar co-operativity was observed for multilamellar DPPC vesicles using fluorescence techniques together with calorimetry. The commonly used membrane models, large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) were found to possess different biophysical properties as interlamellar interactions of MLVs drive segregation of a pyrene labeled lipid analogue into clusters. The effect of a counter-ion lattice on the self assembly of a cationic gemini surfactant was studied. The presence of NaCl strongly influenced the thermal phase behavior of M-1 vesicles, causing formation of giant vesicles upon exceeding a phase transition temperature, followed by a subsequent transition into a more homogenous dispersion. Understanding the underlying biophysical aspects of cellular membranes is of fundamental importance as the complex picture of the structure and function of cells is evolving. Many of the cellular reactions take place on membranes and membranes are known to regulate the activity of many peripheral and intergral membrane associating proteins. From the point of view of drug design and gene technology, membranes can provide an interesting target for future development of drugs, but also a vehicle sensitive for environmental changes allowing for encapsulating drugs and targeting them to the desired site of action.

What was behind the bark? : An assessment of decay among urban Tilia, Betula and Acer trees felled as hazardous in the Helsinki City area

Old trees growing in urban environments are often felled due to symptoms of mechanical defects that could be hazardous to people and property. The decisions concerning these removals are justified by risk assessments carried out by tree care professionals. The major motivation for this study was to determine the most common profiles of potential hazard characteristics for the three most common urban tree genera in Helsinki City: Tilia, Betula and Acer, and in this way improve management practices and protection of old amenity trees. For this research, material from approximately 250 urban trees was collected in cooperation with the City of Helsinki Public Works Department during 2001 - 2004. From the total number of trees sampled, approximately 70% were defined as hazardous. The tree species had characteristic features as potential hazard profiles. For Tilia trees, hollowed heartwood with low fungal activity and advanced decay caused by Ganoderma lipsiense were the two most common profiles. In Betula spp., the primary reason for tree removal was usually lowered amenity value in terms of decline of the crown. Internal cracks, most often due to weak fork formation, were common causes of potential failure in Acer spp. Decay caused by Rigidoporus populinus often increased the risk of stem breakage in these Acer trees. Of the decay fungi observed, G. lipsiense was most often the reason for the increased risk of stem collapse. Other fungi that also caused extensive decay were R. populinus, Inonotus obliquus, Kretzschmaria deusta and Phellinus igniarius. The most common decay fungi in terms of incidence were Pholiota spp., but decay caused by these species did not have a high potential for causing stem breakage, because it rarely extended to the cambium. The various evaluations used in the study suggested contradictions in felling decisions based on trees displaying different stages of decay. For protection of old urban trees, it is crucial to develop monitoring methods so that tree care professionals could better analyse the rate of decay progression towards the sapwood and separate those trees with decreasing amounts of sound wood from those with decay that is restricted to the heartwood area.

Complement factor C4 and immunoglobulins in recurrent or chronic mucosal infections

The study assessed whether plasma concentrations of complement factors C3, C4, or immunoglobulins, serum classical pathway hemolytyic activity, or polymorphisms in the class I and II HLA genes, isotypes and gene numbers of C4, or allotypes of IgG1 and IgG3 heavy chain genes were associated with severe frequently recurring or chronic mucosal infections. According to strict clinical criteria, 188 consecutive voluntary patients without a known immunodeficiency and 198 control subjects were recruited. Frequencies of low levels in IgG1, IgG2, IgG3 and IgG4 were for the first time tested from adult general population and patients with acute rhinosinusitis. Frequently recurring intraoral herpes simplex type 1 infections, a rare form of the disease, was associated with homozygosity in HLA -A*, -B*, -C*, and -DR* genes. Frequently recurrent genital HSV-2 infections were associated with low levels of IgG1 and IgG3, present in 54% of the recruited patients. This association was partly allotype-dependent. The G3mg,G1ma/ax haplotype, together with low IgG3, was more common in patients than in control subjects who lacked antibodies against herpes simplex viruses. This is the first found immunogenetic deficiency in otherwise healthy adults that predisposes to highly frequent mucosal herpes recurrences. According to previous studies, HSV effectively evades the allotype G1ma/ax of IgG1, whereas G3mg is associated with low IgG3. Certain HLA genes were more common in patients than in control subjects. Having more than one C4A or C4B gene was associated with neuralgias caused by the virus. Low levels of IgA, IgG1, IgG2, IgG3, and IgG4 were common in the general adult population, but even more frequent in patients with chronic sinusitis. Only low IgG1 was more common chronic than in acute rhinosinusitis. Clinically, nasal polyposis and bronchial asthma were associated with complicated disease forms. The best differentiating immunologic parameters were C4A deficiency and the combination of low plasma IgG4 together with low IgG1 or IgG2, performing almost equally. The lack of C4A, IgA, and IgG4, all known to possess anti-inflammatory activity, together with a concurrently impaired immunity caused by low subclass levels, may predispose to chronic disease forms. In severe chronic adult periodontitis, any C4A or C4B deficiency combined was associated with the disease. The new quantitative analysis of C4 genes and the conventional C4 allotyping method complemented each other. Lowered levels of plasma C3 or C4 or both, and serum CH50 were found in herpes and periodontitis patients. In rhinosinusitis, there was a linear trend with the highest levels found in the order: acute > chronic rhinosinusitis > general population > blood donors with no self-reported history of rhinosinusitis. Complement is involved in the defense against the tested mucosal infections. Seemingly immunocompetent patients with chronic or recurrent mucosal infections frequently have subtle weaknesses in different arms of immunity. Their susceptibility to chronic disease forms may be caused by these. Host s subtly impaired immunity often coincides with effective immune evasion from the same arms of immunity by the disease-causing pathogens. The interpretation of low subclass levels, if no additional predisposing immunologic factors are tested, is difficult and of limited value in early diagnosis and treatment.

Synthesis of neoglycoconjugates and oligosaccharides with potential anti-Helicobacter pylori activity

The significance of carbohydrate-protein interactions in many biological phenomena is now widely acknowledged and carbohydrate based pharmaceuticals are under intensive development. The interactions between monomeric carbohydrate ligands and their receptors are usually of low affinity. To overcome this limitation natural carbohydrate ligands are often organized as multivalent structures. Therefore, artificial carbohydrate pharmaceuticals should be constructed on the same concept, as multivalent carbohydrates or glycoclusters. Infections of specific host tissues by bacteria, viruses, and fungi are among the unfavorable disease processes for which suitably designed carbohydrate inhibitors represent worthy targets. The bacterium Helicobacter pylori colonizes more than half of all people worldwide, causing gastritis, gastric ulcer, and conferring a greater risk of stomach cancer. The present medication therapy for H. pylori includes the use of antibiotics, which is associated with increasing incidence of bacterial resistance to traditional antibiotics. Therefore, the need for an alternative treatment method is urgent. In this study, four novel synthesis procedures of multivalent glycoconjugates were created. Three different scaffolds representing linear (chondroitin oligomer), cyclic (γ-cyclodextrin), and globular (dendrimer) molecules were used. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc all representing analogues of the tissue binding epitopes for H. pylori. The first synthetic method included the reductive amination of scaffold molecules modified to express primary amine groups, and in the case of dendrimer direct amination to scaffold molecule presenting 64 primary amine groups. The second method described a direct procedure for amidation of glycosylamine modified oligosaccharides to scaffold molecules presenting carboxyl groups. The final two methods that were created both included an oxime-linkage on linkers of different length. All the new synthetic procedures synthesized had the advantage of using unmodified reducing sugars as starting material making it easy to synthesize glycoconjugates of different specificity. In addition, the binding activity of an array of neoglycolipids to H. pylori was studied. Consequently, two new neolacto-based structures, Glcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer and GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer, with binding activity toward H. pylori were discovered. Interestingly, N-methyl and N-ethyl amide modification of the GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer glucuronic acid residue resulted in more effective H. pylori binding epitopes than the parent molecule.

Monitoring of Fungal Growth and Degradation of Wood

Valko- ja ruskolahosienet tunnetaan luonnossa tehokkaimpina puun ja karikkeen lignoselluloosan lahottajina. Valkolahosienet pystyvät hajottamaan kaikkia puun osia: ligniiniä, selluloosaa ja hemiselluloosaa. Selektiivisesti ligniiniä hajottavat sienet lahottavat puusta suhteessa enemmän vaikeasti hajoavaa ligniiniä kuin selluloosaa tai hemiselluloosaa, jolloin jäljelle jää valkoista ja miltei puhdasta selluloosaa. Bioteknisissä sovelluksissa juuri selektiviiviset valkolahottajat ovat kiinnostavia. Niiden avulla voidaan puuhaketta esikäsitellä esimerkiksi paperinvalmistuksessa haitallisen ligniinin poistamiseksi. Ruskolahosienet ovat huomattavia puun, puutavaran ja puisten rakenteiden lahottajia, kuten tässä työssä käytetty Gloeophyllum trabeum (saunasieni ) ja Poria (Postia) placenta (istukkakääpä). Ruskolahosienet hajottavat puusta hemiselluloosan lisäksi selluloosaa, jolloin jää jäljelle ruskea ja jauhomaiseksi mureneva ligniini. Ruskolahosienet muovaavat ligniiniä jonkin verran. Kahden ruskolahosienen G. trabeumin ja P. placentan lisäksi tutkittiin valkolahosieniä, joista Ceriporiopsis subvermispora (karstakääpä) ja harvinainen Physisporinus rivulosus -sieni (talikääpä) hajottavat ligniiniä erittäin selektiivisesti. Phanerochaete chrysosporium on kaikkialla paljon tutkittu sieni, ja Phlebia radiata valkolahosientä (rusorypykkä) on tutkittu paljon mikrobiologian osastolla. Lisäksi tutkittiin Phlebia tremellosa -sienten (hytyrypykkä) ligninolyyttisten entsyymien tuottoa ja 14C-leimatun synteettisen ligniinin (DHP) hajotusta. P. radiata ja P. tremellosa -sienten on todettu aiemmin hajottavan ligniiniä selektiivisesti. Työssä selvitettiin miten sienten kasvua voi mitata, miten vertailukelpoisia eri mittaamismenetelmillä saadut tulokset ovat ja ilmenevätkö sienten aktiivisimmat kasvuvaiheet samaan aikaan eri menetelmillä mitattuna. Tärkeimmät tulokset olivat seuraavat havainnot: (i) P. radiata ja P. tremellosa -sienikannat tuottivat ligniini- ja mangaaniperoksidaasientsyymejä (LiP ja MnP) sekä lakkaasia, ja sienistä puhdistettiin 2-3 LiP- ja P. radiatasta yksi MnP-entsyymi; (ii) P. tremellosa -sienet hajottivat leimattua synteettistä ligniiniä (DHP) yhtä hyvin kuin paljon tutkitut P. chrysosporium ja P. radiata -sienet; (iii) puu, sienen luonnollinen kasvualusta, lisäsi valkolaho- ja ruskolahosienten demetoksylaatiota [O14CH3]-leimatusta ligniinin malliyhdisteestä 14CO2:ksi ilman puuta olleeseen alustaan verrattuna; (iv) demetoksylaatio (14CO2:n tuotto) oli normaalissa ilma-atmosfäärissä useimmiten parempi happeen verrattuna; (v) hapessa paras 14CO2:n tuotto saatiin puupalakasvatuksissa, joihin oli lisätty ravinnetyppeä tai typen lisäksi glukoosia sekä valkolaho- että ruskolahosienillä; (vi) ilmassa 14CO2:n tuotto oli puulla voimakkainta valkolahosienillä ilman lisäravinteita, kun taas G. trabeum -sienellä se oli yhtä hyvä eri alustoissa; (vii) biomassan muodostuminen rihmastojen ergosterolipitoisuuksista mitattuna oli ruskolahosienillä parempi kuin valkolahosienillä; (viii) ja biomassojen huippupitoisuudet olivat 6:lla sienellä eri suuruisia ja niiden maksimimäärien ajankohdat vaihtelivat viiden viikon kasvatusten kuluessa. Mikrobiologian osastolla Viikissä eristetty ja paljon tutkittu P. radiata -valkolahosieni oli mukana kaikissa tehdyissä kokeissa. Sienen LiP-aktiivisuus ja 14CO2:n tuotto 14C-rengas-leimatusta synteettisestä ligniinistä (DHP) korreloivat erittäin hyvin. Biomassan muodostuminen ergosterolilla määritettynä tuki hyvin entsyymiaktiivisuusmittauksilla ja isotooppikasvatuksilla saatuja tuloksia.

Identification of the fungal catabolic D-galacturonate pathway

Pectin is a natural polymer consisting mainly of D-galacturonic acid monomers. Microorganisms living on decaying plant material can use D-galacturonic acid for growth. Although bacterial pathways for D-galacturonate catabolism had been described previously, no eukaryotic pathway for D-galacturonate catabolism was known at the beginning of this work. The aim of this work was to identify such a pathway. In this thesis the pathway for D-galacturonate catabolism was identified in the filamentous fungus Trichoderma reesei. The pathway consisted of four enzymes: NADPH-dependent D-galacturonate reductase (GAR1), L-galactonate dehydratase (LGD1), L-threo-3-deoxy-hexulosonate aldolase (LGA1) and NADPH-dependent glyceraldehyde reductase (GLD1). In this pathway D-galacturonate was converted to pyruvate and glycerol via L-galactonate, L-threo-3-deoxy-hexulosonate and L-glyceraldehyde. The enzyme activities of GAR1, LGD1 and LGA1 were present in crude mycelial extract only when T. reesei was grown on D-galacturonate. The activity of GLD1 was equally present on all the tested carbon sources. The corresponding genes were identified either by purifying and sequencing the enzyme or by expressing genes with homology to other similar enzymes in a heterologous host and testing the activities. The new genes that were identified were expressed in Saccharomyces cerevisiae and resulted in active enzymes. The GAR1, LGA1 and GLD1 were also produced in S. cerevisiae as active enzymes with a polyhistidine-tag, and purified and characterised. GAR1 and LGA1 catalysed reversible reactions, whereas only the forward reactions were observed for LGD1 and GLD1. When gar1, lgd1 or lga1 was deleted in T. reesei the deletion strain was unable to grow with D-galacturonate as the only carbon source, demonstrating that all the corresponding enzymes were essential for D-galacturonate catabolism and that no alternative D-galacturonate pathway exists in T. reesei. A challenge for biotechnology is to convert cheap raw materials to useful and more valuable products. Filamentous fungi are especially useful for the conversion of pectin, since they are efficient producers of pectinases. Identification of the fungal D-galacturonate pathway is of fundamental importance for the utilisation of pectin and its conversion to useful products.

Noninvasive monitoring of activity in Crohn's disease

Background: The fecal neutrophil-derived proteins calprotectin and lactoferrin have proven useful surrogate markers of intestinal inflammation. The aim of this study was to compare fecal calprotectin and lactoferrin concentrations to clinically, endoscopically, and histologically assessed Crohn’s disease (CD) activity, and to explore the suitability of these proteins as surrogate markers of mucosal healing during anti-TNFα therapy. Furthermore, we studied changes in the number and expression of effector and regulatory T cells in bowel biopsy specimens during anti-TNFα therapy. Patients and methods: Adult CD patients referred for ileocolonoscopy (n=106 for 77 patients) for various reasons were recruited (Study I). Clinical disease activity was assessed with the Crohn’s disease activity index (CDAI) and endoscopic activity with both the Crohn’s disease index of severity (CDEIS) and the simple endoscopic score for Crohn’s disease (SES-CD). Stool samples for measurements of calprotectin and lactoferrin, and blood samples for CRP were collected. For Study II, biopsy specimens were obtained from the ileum and the colon for histologic activity scoring. In prospective Study III, after baseline ileocolonoscopy, 15 patients received induction with anti-TNFα blocking agents and endoscopic, histologic, and fecal-marker responses to therapy were evaluated at 12 weeks. For detecting changes in the number and expression of effector and regulatory T cells, biopsy specimens were taken from the most severely diseased lesions in the ileum and the colon (Study IV). Results: Endoscopic scores correlated significantly with fecal calprotectin and lactoferrin (p<0.001). Both fecal markers were significantly lower in patients with endoscopically inactive than with active disease (p<0.001). In detecting endoscopically active disease, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for calprotectin ≥200 μg/g were 70%, 92%, 94%, and 61%; for lactoferrin ≥10 μg/g they were 66%, 92%, 94%, and 59%. Accordingly, the sensitivity, specificity, PPV, and NPV for CRP >5 mg/l were 48%, 91%, 91%, and 48%. Fecal markers were significantly higher in active colonic (both p<0.001) or ileocolonic (calprotectin p=0.028, lactoferrin p=0.004) than in ileal disease. In ileocolonic or colonic disease, colon histology score correlated significantly with fecal calprotectin (r=0.563) and lactoferrin (r=0.543). In patients receiving anti-TNFα therapy, median fecal calprotectin decreased from 1173 μg/g (range 88-15326) to 130 μg/g (13-1419) and lactoferrin from 105.0 μg/g (4.2-1258.9) to 2.7 μg/g (0.0-228.5), both p=0.001. The relation of ileal IL-17+ cells to CD4+ cells decreased significantly during anti-TNF treatment (p=0.047). The relation of IL-17+ cells to Foxp3+ cells was higher in the patients’ baseline specimens than in their post-treatment specimens (p=0.038). Conclusions: For evaluation of CD activity, based on endoscopic findings, more sensitive surrogate markers than CDAI and CRP were fecal calprotectin and lactoferrin. Fecal calprotectin and lactoferrin were significantly higher in endoscopically active disease than in endoscopic remission. In both ileocolonic and colonic disease, fecal markers correlated closely with histologic disease activity. In CD, these neutrophil-derived proteins thus seem to be useful surrogate markers of endoscopic activity. During anti-TNFα therapy, fecal calprotectin and lactoferrin decreased significantly. The anti-TNFα treatment was also reflected in a decreased IL-17/Foxp3 cell ratio, which may indicate improved balance between effector and regulatory T cells with treatment.

Anti-TNF treatment in juvenile idiopathic arthritis and associated uveitis : Clinical perspectives on growth, uveitis, and drug survival

Juvenile idiopathic arthritis (JIA) is a heterogeneous group of childhood chronic arthritides, associated with chronic uveitis in 20% of cases. For JIA patients responding inadequately to conventional disease-modifying anti-rheumatic drugs (DMARDs), biologic therapies, anti-tumor necrosis factor (anti-TNF) agents are available. In this retrospective multicenter study, 258 JIA-patients refractory to DMARDs and receiving biologic agents during 1999-2007 were included. Prior to initiation of anti-TNFs, growth velocity of 71 patients was delayed in 75% and normal in 25%. Those with delayed growth demonstrated a significant increase in growth velocity after initiation of anti-TNFs. Increase in growth rate was unrelated to pubertal growth spurt. No change was observed in skeletal maturation before and after anti-TNFs. The strongest predictor of change in growth velocity was growth rate prior to anti-TNFs. Change in inflammatory activity remained a significant predictor even after decrease in glucocorticoids was taken into account. In JIA-associated uveitis, impact of two first-line biologic agents, etanercept and infliximab, and second-line or third-line anti-TNF agent, adalimumab, was evaluated. In 108 refractory JIA patients receiving etanercept or infliximab, uveitis occurred in 45 (42%). Uveitis improved in 14 (31%), no change was observed in 14 (31%), and in 17 (38%) uveitis worsened. Uveitis improved more frequently (p=0.047) and frequency of annual uveitis flares was lower (p=0.015) in those on infliximab than in those on etanercept. In 20 patients taking adalimumab, 19 (95%) had previously failed etanercept and/or infliximab. In 7 patients (35%) uveitis improved, in one (5%) worsened, and in 12 (60%) no change occurred. Those with improved uveitis were younger and had shorter disease duration. Serious adverse events (AEs) or side-effects were not observed. Adalimumab was effective also in arthritis. Long-term drug survival (i.e. continuation rate on drug) with etanercept (n=105) vs. infliximab (n=104) was at 24 months 68% vs. 68%, and at 48 months 61% vs. 48% (p=0.194 in log-rank analysis). First-line anti-TNF agent was discontinued either due to inefficacy (etanercept 28% vs. infliximab 20%, p=0.445), AEs (7% vs. 22%, p=0.002), or inactive disease (10% vs. 16%, p=0.068). Females, patients with systemic JIA (sJIA), and those taking infliximab as the first therapy were at higher risk for treatment discontinuation. One-third switched to the second anti-TNF agent, which was discontinued less often than the first. In conclusion, in refractory JIA anti-TNFs induced enhanced growth velocity. Four-year treatment survival was comparable between etanercept and infliximab, and switching from first-line to second-line agent a reasonable therapeutic option. During anti-TNF treatment, one-third with JIA-associated anterior uveitis improved.

Learning Challenges in Biogas Production for Sustainability : An activity theoretical study of a network from a swine industry chain

This study is about the challenges of learning in the creation and implementation of new sustainable technologies. The system of biogas production in the Programme of Sustainable Swine Production (3S Programme) conducted by the Sadia food processing company in Santa Catarina State, Brazil, is used as a case example for exploring the challenges, possibilities and obstacles of learning in the use of biogas production as a way to increase the environmental sustainability of swine production. The aim is to contribute to the discussion about the possibilities of developing systems of biogas production for sustainability (BPfS). In the study I develop hypotheses concerning the central challenges and possibilities for developing systems of BPfS in three phases. First, I construct a model of the network of activities involved in the BP for sustainability in the case study. Next, I construct a) an idealised model of the historically evolved concepts of BPfS through an analysis of the development of forms of BP and b) a hypothesis of the current central contradictions within and between the activity systems involved in BP for sustainability in the case study. This hypothesis is further developed through two actual empirical analyses: an analysis of the actors senses in taking part in the system, and an analysis of the disturbance processes in the implementation and operation of the BP system in the 3S Programme. The historical analysis shows that BP for sustainability in the 3S Programme emerged as a feasible solution for the contradiction between environmental protection and concentration, intensification and specialisation in swine production. This contradiction created a threat to the supply of swine to the food processing company. In the food production activity, the contradiction was expressed as a contradiction between the desire of the company to become a sustainable company and the situation in the outsourced farms. For the swine producers the contradiction was expressed between the contradictory rules in which the market exerted pressure which pushed for continual increases in scale, specialisation and concentration to keep the production economically viable, while the environmental rules imposed a limit to this expansion. Although the observed disturbances in the biogas system seemed to be merely technical and localised within the farms, the analysis proposed that these disturbances were formed in and between the activity systems involved in the network of BPfS during the implementation. The disturbances observed could be explained by four contradictions: a) contradictions between the new, more expanded activity of sustainable swine production and the old activity, b) a contradiction between the concept of BP for carbon credits and BP for local use in the BPfS that was implemented, c) contradictions between the new UNFCCC1 methodology for applying for carbon credits and the small size of the farms, and d) between the technologies of biogas use and burning available in the market and the small size of the farms. The main finding of this study relates to the zone of proximal development (ZPD) of the BPfS in Sadia food production chain. The model is first developed as a general model of concepts of BPfS and further developed here to the specific case of the BPfS in the 3S Programme. The model is composed of two developmental dimensions: societal and functional integration. The dimension of societal integration refers to the level of integration with other activities outside the farm. At one extreme, biogas production is self-sufficient and highly independent and the products of BP are consumed within the farm, while at the other extreme BP is highly integrated in markets and networks of collaboration, and BP products are exchanged within the markets. The dimension of functional integration refers to the level of integration between products and production processes so that economies of scope can be achieved by combining several functions using the same utility. At one extreme, BP is specialised in only one product, which allows achieving economies of scale, while at the other extreme there is an integrated production in which several biogas products are produced in order to maximise the outcomes from the BP system. The analysis suggests that BP is moving towards a societal integration, towards the market and towards a functional integration in which several biogas products are combined. The model is a hypothesis to be further tested through interventions by collectively constructing the new proposed concept of BPfS. Another important contribution of this study refers to the concept of the learning challenge. Three central learning challenges for developing a sustainable system of BP in the 3S Programme were identified: 1) the development of cheaper and more practical technologies of burning and measuring the gas, as well as the reduction of costs of the process of certification, 2) the development of new ways of using biogas within farms, and 3) the creation of new local markets and networks for selling BP products. One general learning challenge is to find more varied and synergic ways of using BP products than solely for the production of carbon credits. Both the model of the ZPD of BPfS and the identified learning challenges could be used as learning tools to facilitate the development of biogas production systems. The proposed model of the ZPD could be used to analyse different types of agricultural activities that face a similar contradiction. The findings could be used in interventions to help actors to find their own expansive actions and developmental projects for change. Rather than proposing a standardised best concept of BPfS, the idea of these learning tools is to facilitate the analysis of local situations and to help actors to make their activities more sustainable.