CLCA2 is a p53-inducible transmembrane protein that is frequently downregulated in breast cancer. CLCA2 is a 943 amino acid type I transmembrane protein that is cleaved near amino acid 700 to produce a diffusible 100kD product. The N-terminus contains a hydrolase-like domain with well-conserved HEXXH zinc binding amino acid motif that was proposed to cleave the precursor auto-proteolytically. We investigate the auto-proteolysis of CLCA2 precursor. Using membrane extracts or purified protein from CLCA2-transfected cells, we show here that CLCA2 cleavage is catalyzed by zinc and inhibited by metal chelator EDTA. Moreover, an E165Q mutation in the metal binding site abolished processing without affecting stability or trafficking. The mutant could be cleaved by co-transfected wild type CLCA2, showing that the mutation had not caused an un-cleavable conformation and suggesting that it occurs in trans. Wild type CLCA2 was able to cleave CLCA2 E165Q mutant in vitro only after denaturation and renaturation, suggesting that a conformational shift is required for cleavage. The efficiency of cleavage increased steeply with increasing concentration of precursor, consistent with trans proteolysis but not cis or cleavage by another agent. Accordingly, CLCA2 molecules bearing different epitope tags formed a stable complex that could be co-immunoprecipitated. Cleavage appears to be specific within isoforms; CLCA1 was unable to neither cleave CLCA2 nor form a stable complex with it. Furthermore, cleavage causes a conformational shift: an N-terminal antibody that immunoprecipitates the precursor fails to precipitate the N-terminal product unless it is first denatured with ionic detergent. We found that cleavage is enhanced by p53 induction due to DNA damage, implying that the cleavage has functional consequences for stress response. Moreover, we found that HEK and MCF10A cells expressing the E165Q mutant had a higher proliferation rate than cells expressing wild type CLCA2, suggesting that the metalloprotease activity contributes to the anti-proliferative effect of CLCA2. Physiologically, CLCA2 plays an essential role in epithelial differentiation. It is induced during epithelial differentiation in immortalized human mammary epithelial cells (HMEC), and its knockdown causes epithelial to mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with Epithelial V-like Antigen 1 (EVA1) and confirmed by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 family. EVA1 resembles tight junction proteins called Junctional Adhesion Molecules (JAMs) by structure but we found by confocal analysis that EVA1 is localized the lateral interface at cell-cell junctions. Analysis of transcriptional profiles revealed that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype, and upregulated during epithelial differentiation. Like CLCA2, knockdown of EVA1 resulted in rapid EMT in immortalized HMEC. The interacting domains were delimited by deletion analysis, revealing that both the proteins interact via their transmembrane segments (TMS). The interaction was specific, as other transmembrane proteins did not interact with CLCA2 or EVA1. We also found that CLCA2 binds to ZO-1 and beta-catenin at its c-terminus but EVA1 does not. Interestingly, we found that EVA1 does interact with ZO-1 in the presence of CLCA2, indicating that these three form a complex at the cell-cell junctions that allows stabilization of belt-like adherens junctions (AJ). On the other hand CLCA2 may also stabilize adherens junctions by sequestering beta-catenin at the cell-cell junctions. These results indicate that CLCA2 plays a key role in maintaining epithelial differentiation via multiple ways. Either by binding to beta-catenin or forming a complex with EVA1 and ZO-1, it plays a pivotal role in maintaining epithelial differentiation. This explains the downregulation of both CLCA2 and EVA1 during tumor progression.

Global environmental change is a concern in society, and companies sees the importance of more environmentally- friendly practices. Consequently, the need for environmental information is becoming more and more important. A common tool for strategic environmental decisions is life cycle analysis (LCA), which is recognized for its robustness in assessing the environmental performance of products systems. This study reports on a streamlined consequential life cycle analysis (CLCA) commissioned by the organisation Ragnsells, and to analyse the strenghts, weaknesses, opportunities and threats (SWOT) for applying this approach to similar projects. The initial LCA is conducted on FAIR plastic recycling, an innovation provided by Ragnsells AB. The geographical scope of the project is delimited to Denmark in 2022. Moreover, as CLCA has been applied, the used allocation has been the system expansion method. Data have been collected from direct contact with stakeholders in Denmark, literature and CLCA databases. The results of the study indicate that demanding FAIR plastic recycling is favourable from a climate change perspective, but detrimental in terms of e.g. land change. Furthermore, the lack of available data limits the study and shows need of further research. LCA applied within the organisation can increase awareness, simplify decision-making and provide comprehensive environmental information, despite the significant time and resources needed. Furthermore, increasing research within the field presents opportunities to improve quality aspects that can lead to better environmental performance and enhances collaboration and communication. However, the need for environmental information is threatened by misleading environmental claims, and could be minimized by increasing knowledge about underlying assumptions and uncertainties in LCA studies. / Miljöförändringen erkänns alltmer som strategiskt viktigt i samhället och allt fler företag ser vikten av att ställa om till mer miljövänliga tekniker. Följaktligen ökar efterfrågan på miljöinformation snabbt. Ett vanligt verktyg för strategiska miljöbeslut är livscykelanalys (LCA), som används för att bedöma miljöprestanda hos produkter och tjänster. Denna rapport syftar till att göra en initial LCA inom organisationen Ragnsells, och att analysera styrkor, svagheter, möjligheter och hot (SWOT-analys) för att tillämpa liknande projekt. Den initiala studien är gjord på FAIR plaståtervinning, en innovation från Ragnsells AB. Projektets geografiska omfattning är avgränsad till Danmark och genomförs från tidpunkten 2022. Eftersom en konsekvens-LCA (cLCA) har tillämpats har allokeringsmetoden systemexpansion använts. Data har samlats in från direkt från kontakt med intressenter i Danmark, litteratur och CLCA-databaser. Resultaten från studien visar att krav på FAIR- plast är fördelaktigt ur ett klimatförändringsperspektiv. Däremot rekommenderas insatser för att analysera och vid behov minska negativa effekter. Dessutom tyder bristen på tillgängliga data på behovet av ytterligare analyser. Analysen visar att LCA inom organisationen kan öka allmänhetens medvetenhet, förenkla beslutsfattande och ge omfattande miljöinformation. Samtigit finns svagheter med den tid och de resurser som krävs för att genomföra en LCA. Vidare visar ökande forskning inom området möjligheter till att förbättra kvalitetsaspekter för bättre miljöprestanda samt förbättrad samverkan och kommunikation. Behovet av miljöinformation hotas dock av vilseledande miljöpåståenden, och skulle kunna minimeras genom att öka kunskapen om underliggande antaganden och osäkerheter inom LCA.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

hCLCA2 and hCLCA4 are chloride channel regulators that are expressed in normal breast epithelial cells and frequently downregulated in breast cancers. Recent investigations revealed that these two proteins may have a role in suppressing breast cancer progression. In this thesis, I will address their role in maintaining epithelial differentiating and inhibiting cell proliferation of breast epithelial cells. The epithelial to mesenchymal transition (EMT) is a developmental program in which epithelial cells downregulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In breast cancer, EMT facilitates invasion of surrounding tissues and correlates closely with cancer metastasis and relapse. We found previously that the candidate tumor suppressor hCLCA2 is a p53-inducible proliferation-inhibitor that is frequently lost in breast cancer. We show here that another member of the CLCA gene family, hCLCA4, is expressed in mammary epithelial cells and is similarly downregulated in breast tumors and in breast cancer cell lines. Like CLCA2, the gene is stress-inducible, and ectopic expression inhibits colony formation. Transcriptional profiling studies revealed that hCLCA4 and hCLCA2 together are markers for mammary epithelial differentiation, and both are downregulated by TGF beta. Moreover, knockdown of either on in immortalized cells by shRNAs caused downregulation of epithelial marker E-cadherin, while mesenchymal markers N-cadherin, vimentin, and fibronectin were upregulated, indicating an EMT program. Double knockdown of hCLCA2 and hCLCA4 enhanced the mesenchymal profile. These findings suggest that hCLCA4 and hCLCA2 play complementary but distinct roles in epithelial differentiation. Clinically, low expression of hCLCA2 and hCLCA4 signaled lower relapse-free survival in breast cancers. Cellular senescence is a program of irreversible cell cycle arrest in response to stressors such as DNA damage, ROS, telomere erosion, or oncogene activation. It is one of the primary tumor suppression mechanisms mediated by p53 and is often disabled in cancer cells. However, the downstream signaling pathway whereby p53 induces cellular senescence remains incomplete. We reported previously that hCLCA2 was a p53 inducible gene that is downregulated with breast cancer progression. We and other group noticed that hCLCA2 was induced in parallel with several types of senescence. Lentiviral transduction of CLCA2 into MCF7 cells inhibited cell proliferation and cells showed senescence phenotype. To investigate the mechanism biochemically, we used pAd-Easy to express hCLCA2 in the model breast cancer cell line CA1d. A protein expression profile of these cells over a 6 day period revealed induction of p21, p53, and the DNA damage-response pathway. To test whether hCLCA2 is required for the cellular senescence process, hCLCA2 was knocked down in HMLE. The knockdown cells (KD) and negative control were treated with a low concentration of doxorubicin, and cell proliferation was measured. The KD cells were more resistant to growth inhibition by doxorubicin. Moreover, a time course experiment showed that induction of SA beta-galactosidase, DNA damage response, and lysosomal markers IFI30 and CTSS was delayed in the knockdown cells. These results suggest that hCLCA2 plays an important role in DNA damage response and the senescence program.

Research on historical developments that lead to the establishment of global organizations for climate change has shown that the phenomenon of surface temperature is not a new topic of focus. Increased policy restrictions, brand image, fear of resource scarcity, growing market trends towards sustainability and consumer awareness are among the several external factors that have influenced the growing research in corporate transition towards carbon neutrality. The main aim of this study is to understand through data accounting of major material and energy carrier changes, how a non-production facility could transition to become a carbon-neutral facility. Therefore, an exploratory case study has been performed and conducted at Volvo CE Customer center in Eskilstuna, Sweden, with two objectives: i) to identify and quantify the customer center current footprint by mapping the main contributors to greenhouse gases emissions, and ii) to recommend specific & general measures that can mitigate the carbon footprint of the facility. Three research questions related to the facility’s current carbon footprint, measures implemented so far, and the best applied assessment method, have guided us throughout the study. The methodology has been framed to give a theoretical underpinning for understanding the project from a holistic perspective. The split of the methodology has been constructed in line with the theoretical framework that gave the foundation to the needed theories to be taken into account i.e. GHG protocol, which is the tool that has been adopted by the study to attain the desired aim, including the three scopes under the protocol which were also defined accordingly. ‘Scope 1’ has been taken into account and is a representation of direct emissions, ‘Scope 2 represents the indirect emissions, and ‘Scope 3’ (according to the GHG protocol) takes into account the rest of the indirect emissions arranged into 15 categories, from which applicable to our study were 4 categories (1, 3, 4 and 6). The results showed that during the base year (2019) the highest user within Scope 1 was diesel, followed by HVO, and under Scope 2, The results from Scope 1 and 2, together with the results of Scope 3 category, were analyzed using the attributional LCA approach recommended by the GHG protocol to calculate their contribution to the customer centers’ total carbon footprint. It was found that Scope 1 stands for 128.52 t CO₂-eq while Scope 2 stands only for 1.16 t CO₂-eq and finally Scope 3 stands for most of the emissions with 3719 t CO₂-eq. It has been found that in 2019, the customer center has saved 101.05 tonnes of GHG by implementing measures, such as switching from using Diesel to HVO and switching from the mixed electricity to the renewable ones, according to the attributional perspective presented in the GHG protocol. However, different results were found when these values were discussed and analyzed from the consequential perspective, since this perspective analyses the effects of the implemented measures on the global emission level. This concluded that implementation of conservation and efficiency measures must take priority before switching to higher priced renewables. Thus, the resulting carbon neutrality will be consequentially safer. The recommendations stated in this study also follows the same principle “Conserve before investing”. Suggestions and recommendations outlined in the study for future implementation approach carbon neutrality as a strategy and not a burden, helping the customer neutral achieve the goal in an Environment, Economic and Socially sustainable manner.

Die Durchfallerkrankung Giardiasis wird durch den Protisten Giardia duodenalis ausgelöst. Die Infektion erfolgt fäkal-oral, meist über kontaminiertes Trinkwasser. Der Parasit kolonisiert den oberen Bereich des Dünndarms und heftt sich an das Epithel, wodurch es die Krankheitsbeschwerden auslöst. Allerdings sind Details über die Mechanismen der Pathogenese unbekannt. Dazu kommt, dass der Ausgang einer Infektion fallspezifisch starken Schwankungen unterworfen ist, von selbst-limitierend bis chronisch und asymptomatischer Kolonisierung bis hin zur schweren Enteritis. Ein möglicher Pathomechanismus ist der Wegfall der Barrierefunktion des Dünndarmepithels, z.B. durch Beeinträchtigung von tight junctions oder Zelltod. In dieser Arbeit wurden Effekte von G. duodenalis auf in vitro Modellsysteme des humanen Dünndarmepithels untersucht. Dazu wurden hauptsächlich Daten über die Barrierefunktion sowohl von der weit verbreiteten Caco-2 Zelllinie, als auch über ein neu etabliertes humanes Dünndarmorganoidsystem, erhoben. Es konnte gezeigt werden, dass mehrere - mitunter in der Literatur als hochvirulent beschriebene - G. duodenalis Isolate zu keinerlei Beeinträchtigung der Barrierefunktion oder irgendeiner anderen untersuchten potenziellen Schädigung an zwei unterschiedlichen Caco-2 Zelllinien unter diversen Infektions- und Kulturbedingungen führte. Jedoch andererseits das neu entwickelte Dünndarmorganoidsystem mit pseudo-luminalem Medium TYI S 33 reproduzierbar die Zerstörung des Epithelmodells mit Zellverlust, Zelltod (apoptotisch und nicht-apoptotisch), Störung der tight junctions (Abbau und Dislokation von Claudinen und ZO-1) und den Verlust von Mikrovilli innerhalb ein bis zwei Tage nach Parasiteninfektion zeigen konnte. Zudem wurde das Auftauchen von ClCa-1-Signalen unter andauerndem Infektionsstress beobachtet, was die Differenzierung bzw. Metaplasie zu Becherzellen nahelegt, jedoch keine Wirtsreaktion auf die Gewebszerstörung zu sein scheint. / The protozoan parasite Giardia duodenalis is the etiological agent for the intestinal diarrheal disease giardiasis. Infections are acquired via the fecal-oral route, mostly via uptake of cysts from contaminated drinking water. The colonization of the hosts’ duodenum and upper jejunum and the attachment of Giardia trophozoites onto the epithelium is the cause of a variety of gastrointestinal complaints but the exact pathomechanisms are unknown. Furthermore, the outcome of Giardia infections varies greatly between individuals, ranging from self-limiting to chronic, and asymptomatic to severe enteritis. One proposed mechanism for the pathogenesis is the breakdown of intestinal barrier function, e.g. by tight junction impairment or induction of cell death. In this work, effects of G. duodenalis on in vitro models of the human small intestinal epithelium were investigated by studying mainly barrier-related properties and changes of widely used Caco-2 cells as well as newly established human small intestinal organoid-derived monolayers (ODMs). It could be shown that several isolates of G. duodenalis, some described as highly virulent, fail to induce barrier dysfunction or any other investigated pathological effect on two Caco-2 cell lines under various infection and culturing conditions. On the other side, by developing a new organoid-based model system and the use of luminal mock medium TYI-S-33, considerable epithelial disruption (including loss of cells), cell death (apoptosis and non-apoptotic), tight junction impairment (degradation and dislocation of claudins and ZO-1), and microvilli depletion reproducibly induced by G. duodenalis trophozoites between one and two days after infection could be observed. Moreover, emergence of ClCa-1 positive cells with ongoing parasite infections suggest epithelial differentiation or metaplasia towards goblet cells, which is furthermore not associated to tissue damage.

Cette thèse porte sur l’étude des impacts environnementaux de systèmes de production de bovins. Le premier objectif était d’analyser et de comparer les impacts environnementaux de systèmes de production de viande et de lait par analyse de cycle de vie (ACV) attributionnelle. Les effets de pratiques d’atténuation de ces impacts ont été évalués pour les systèmes de production de viande. Le second objectif était un développement méthodologique afin d’explorer les conséquences possibles d’une préférence accrue pour un lait produit à base d’herbe, par ACV conséquentielle. Dans un système de production de viande par le troupeau allaitant, le méthane entérique a été le principal contributeur à l’impact changement climatique, et la production de l’herbe a été la principale contributrice aux autres impacts (demande énergétique cumulée, eutrophisation, acidification, occupation du sol). L’atelier naisseur (vaches allaitantes et leurs veaux, génisses) a contribué de manière majeure aux impacts du système allaitant dans son ensemble. La pratique d’atténuation la plus efficace pour le système a été la diminution de l’âge au vêlage de 3 à 2 ans. L’utilisation de lipides riches en acides gras oméga-3 dans le régime a très peu affecté les impacts du système. L’application simultanée de plusieurs pratiques d’atténuation compatibles entre elles réduit sensiblement les impacts. L’application de pratiques telles que la réduction du gaspillage d’herbe, l’engraissement des génisses non utilisées pour le renouvellement et la diminution de l’âge au vêlage réduisent l’occupation du sol. Un usage alternatif des terres libérées tel que la plantation de forêt pour séquestrer du carbone dans la biomasse semble prometteur. L’étude de systèmes de production de lait a été centrée sur les comparaisons de systèmes à base d’herbe ou d’ensilage de maïs, d’une race spécialisée (Holstein) ou mixte (Normande) et sur l’effet du niveau de production laitière par ACV attributionnelle. Quelle que soit la méthode d’attribution des impacts aux co-produits, les impacts par kg de lait ont été plus faibles pour les systèmes à base d’ensilage de maïs et pour les Holstein, sauf pour l’eutrophisation. L’accroissement de la production de lait par vache grâce à une consommation d’énergie accrue et au vêlage à 2 ans a permis de réduire les impacts du lait et de son co-produit viande. Les conséquences de la conversion d’une exploitation laitière utilisant beaucoup de maïs ensilage vers une exploitation utilisant de l’herbe comme unique source de fourrage pour répondre à une demande de lait produit à base d’herbe en France ont été évaluées par ACV conséquentielle. Cette conversion entraîne des changements notables de l’utilisation des sols en dehors de l’exploitation, et donc un fort accroissement des impacts du système dans son ensemble et du lait produit. / This thesis addresses the environmental impacts of cattle production systems. The first objective of this thesis was to analyse and compare the environmental impacts of suckler-beef and dairy production systems using attributional life cycle assessment (ALCA). Subsequently, the effects of mitigation practices for suckler-beef production systems were assessed. The second objective addressed methodology development by exploring possible consequences due to an increase in preference for grass-based milk using consequential LCA (CLCA).For a suckler-beef production system, enteric methane fermentation was the main contributor to the climate change impact, and grassland production contributed most to other impacts (cumulative energy demand, eutrophication, acidification and land occupation). The suckler cow-calf herd substantially contributed to the impacts of the suckler-beef system. The most effective mitigation practice for the suckler-beef production system was decreasing calving age from 3 to 2 years. The use of lipids rich in omega-3 fatty acids in ruminant diets did not substantially affect the impacts of the suckler-beef production system. Simultaneous application of several compatible practices can substantially mitigate the impacts of the suckler-beef production system. The application of certain practices (e.g. reducing ungrazed grass losses, fattening heifers not used for replacement and reducing calving age) reduced land occupation. Alternative uses for the “released land”, e.g. the introduction of forest to sequester C into biomass, seems promising. For dairy production systems, the assessment focused on a grass-based vs. maize-silage-based system, dual-purpose breed (Normande) vs. specialised breed (Holstein) and the effect of increasing milk yield per cow, using the ALCA approach. Independent of co-product handling methods, the impacts per kg of milk were lower with the maize-silage-based system and with Holstein cows (except for eutrophication). Increasing milk yield per cow by increasing feed energy intake and applying more intensive management (first calving at 2 years) decreased the impacts of milk and its beef co-product. The consequences of converting a maize-silage-based to a grass-based dairy farm in France to meet the increased domestic preference for grass-based milk were assessed using the CLCA approach. This farm conversion caused land-use change outside the dairy farm and thus substantially increased the impacts of the whole production system and the milk it produced.