Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

A pesquisa tem como objetivo compreender a relação entre cultura e direitos humanos estabelecida na produção normativa relativa ao Sistema Nacional de Cultura (SNC), de modo específico entre as 52ª e 54ª legislaturas do Congresso Nacional, bem como entender os impactos dessa produção na concretização dos direitos culturais por meio das políticas públicas de cultura. Desse modo, foi realizado um levantamento das propostas legislativas aprovadas ou ainda em tramitação que tratam da institucionalização e regulamentação do Sistema Nacional de Cultura e de seus componentes principais. A análise desses marcos legais é guiada pela concepção de cultura adotada nos mesmos e pelo tratamento que dão aos direitos culturais, em especial à promoção da diversidade cultural. O sentido abrangente de cultura adotado na legislação do Sistema Nacional de Cultura é analisado de maneira relacional ao conceito proposto por Raymond Williams no âmbito dos Estudos Culturais. Com efeito, as duas perspectivas buscam construir uma ampliação democrática no conceito de cultura para reconhecê-la como direito de todos. A ampliação do conceito de cultura operada pela legislação do Sistema Nacional de Cultura potencialmente oferece mais condições para a promoção da diversidade cultural e dos direitos cultuais como um todo. / This research aims at comprehending the relation between culture and human rights established by the regulatory production of the National Culture System (CNS), specifically between the 52rd and 54rh legislatures in the Brazilian National Congress, as well as understanding the impacts of this production in the achievement of cultural rights through public cultural policies. Thus, we conducted a gathering of legislative proposals which have been approved or are still in progress referring to the institutionalization and regulation of the National Culture System and its main components. The analysis of these legal frameworks was guided by the concept of culture adopted in the System and the treatment of cultural rights, particularly for promotion of cultural diversity. The comprehensive sense of culture adopted in the National Culture System legislation is analyzed by establishing relations with the concept proposed by Raymond Williams in Cultural Studies. Both perspectives aim at democratically expanding the concept of culture in order to recognize it as a right for all. The expansion of the concept of culture operated by the National Culture System legislation adds more potential to the promotion of cultural diversity and cultural rights as a whole.

A cerebral aneurysm is a weakened area of an artery in the brain, creating an abnormal expansion. Recent research for treatment is utilizing a photopolymerizable hydrogel as a possible operation for injection in situ. This paper aimed to achieve bioconjugation of peptides on a PEGDMA polymer network (using the photoinitiator PEG-BAPO) to form a biocompatible photopolymerizable hydrogel, without compromise to any of its mechanical attributes. Achieving cell adhesion to the hydrogel surface is a critical requirement as that could drive the growth of endothelium between aneurysm and artery, to considerably enhance its sustainability and decrease the risk of inflammation. The hydrogel was synthesized by functionalizing RGD with a PEG-spacer and co-polymerize it with PEGDMA using UV-radiation to create an intertwined cross-linking network. Samples of various peptide concentrations were studied in cell culture to analyze cell adhesion, followed by mechanical tests to identify possible deviations. A subsequent study was established to create a dynamic prototype as a quantifiable replication of a hydrogel inside an aneurysm in vivo. The model was designed in SolidWorks and connected with an Ibidi sticky-Slide to roughly replicate a cerebral aneurysm connected to an artery with space to introduce a hydrogel sample.

A pesquisa tem como objetivo compreender a relação entre cultura e direitos humanos estabelecida na produção normativa relativa ao Sistema Nacional de Cultura (SNC), de modo específico entre as 52ª e 54ª legislaturas do Congresso Nacional, bem como entender os impactos dessa produção na concretização dos direitos culturais por meio das políticas públicas de cultura. Desse modo, foi realizado um levantamento das propostas legislativas aprovadas ou ainda em tramitação que tratam da institucionalização e regulamentação do Sistema Nacional de Cultura e de seus componentes principais. A análise desses marcos legais é guiada pela concepção de cultura adotada nos mesmos e pelo tratamento que dão aos direitos culturais, em especial à promoção da diversidade cultural. O sentido abrangente de cultura adotado na legislação do Sistema Nacional de Cultura é analisado de maneira relacional ao conceito proposto por Raymond Williams no âmbito dos Estudos Culturais. Com efeito, as duas perspectivas buscam construir uma ampliação democrática no conceito de cultura para reconhecê-la como direito de todos. A ampliação do conceito de cultura operada pela legislação do Sistema Nacional de Cultura potencialmente oferece mais condições para a promoção da diversidade cultural e dos direitos cultuais como um todo. / This research aims at comprehending the relation between culture and human rights established by the regulatory production of the National Culture System (CNS), specifically between the 52rd and 54rh legislatures in the Brazilian National Congress, as well as understanding the impacts of this production in the achievement of cultural rights through public cultural policies. Thus, we conducted a gathering of legislative proposals which have been approved or are still in progress referring to the institutionalization and regulation of the National Culture System and its main components. The analysis of these legal frameworks was guided by the concept of culture adopted in the System and the treatment of cultural rights, particularly for promotion of cultural diversity. The comprehensive sense of culture adopted in the National Culture System legislation is analyzed by establishing relations with the concept proposed by Raymond Williams in Cultural Studies. Both perspectives aim at democratically expanding the concept of culture in order to recognize it as a right for all. The expansion of the concept of culture operated by the National Culture System legislation adds more potential to the promotion of cultural diversity and cultural rights as a whole.

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.

Agriculture is one of the most carbon-intensive sectors in today’s world. With demand for food soaring to feed an ever-increasing population, many methods are employed to produce food, ranging from open-field to greenhouse systems. However, due to their large environmental footprint, urban agriculture methods such as soilless culture systems are increasingly becoming a preferred method for food production. A case study has been performed at Hollbium in Stockholm, Sweden for the purpose of evaluating potential environmental impacts of Hollbium Loop, a vertical hydroponic system used for food production. This is achieved by carrying out a stand-alone assessment of Hollbium Loop, to identify potential environmental hotspots over the product’s lifetime of 10 years. Furthermore, Hollbium Loop’s system is compared to the production of lettuce in a horizontal open-field, a greenhouse farming system and nutrient film technique located in Spain, to identify potential trade-offs. Attributional life cycle assessment (ALCA) has been employed as a methodology with an explicit focus on eight impact categories at a midpoint level. The results of the study indicate that hotspots of environmental impacts in Hollbium Loop’s life cycle are due to the use of an internal combustion engine vehicle for maintenance, production of electronic components and the consumption of electricity in the use phase. In contrast, the results from the comparative scenarios indicated that Hollbium Loop from a life cycle perspective had the highest impacts under all impact categories in relation to the other three systems of food production. When the distance for transportation within the maintenance phase is reduced to a reasonable figure, Hollbium Loop no longer had the highest impacts within global warming and fossil resource scarcity, showing that the distance traversed plays a key role in mitigating the impacts. Other aspects and assumptions are also analyzed further using a sensitivity analysis, which shows that there is scope for decreasing Hollbium Loop’s environmental footprint. It is therefore recommended that Hollbium puts its efforts into optimizing the distance and modes of transport utilized and increasing the outer frame thickness of the Loop to make it sturdy and robust. Finally, it is also advised to increase the Loop’s vertical length to increase the yield per cycle, thereby reducing the overall impacts per kilogram of lettuce produced. / Jordbruket är en av de mest kolintensiva sektorerna i dagens värld. Med en efterfrågan på livsmedel som stiger för att föda en ständigt ökande befolkning, används många metoder för att producera livsmedel. Dessa metoder omfattar allt från öppna fält till växthussystem. På grund av deras stora miljöpåverkan blir emellertid stadsnära jordbruksmetoder, såsom system för hydroponiska odlingar (odlingar utanför jorden) en alltmer föredragen metod för livsmedelsproduktion. En fallstudie har genomförts hos Hollbium i Stockholm, Sverige med syfte att genomföra en utvärdering av den potentiella miljöpåverkan för Hollbium Loop, ett vertikalt hydroponiskt system som används för livsmedelsproduktion. Detta uppnås genom att först utföra en fristående bedömning av Hollbium Loop och på så sätt identifiera potentiella riskområden under produktens livslängd på tio år. Dessutom jämförs Hollbium Loops system med produktionen av sallad i ett horisontellt öppet fält, ett växthusodlingssystem och en näringsfilmsteknik i Spanien för att identifiera potentiella avvägningar. Attributiv livscykelanalys (ALCA) har använts som metod med ett tydligt fokus på åtta karakteriseringsfaktorer för mittpunkt. Resultaten i studien indikerar att riskområden för miljöpåverkan i Hollbium Loops livscykel beror på en användning av fordon med förbränningsmotor i underhållsfasen, produktion av elektroniska komponenter och elförbrukning i användningsfasen. Däremot visade resultaten från de jämförande scenarierna att Hollbium Loop ur ett livscykelperspektiv har den högsta miljöpåverkan i alla påverkanskategorier i jämförelse med de tre andra systemen för livsmedelsproduktion. När avståndet för transport inom underhållsfasen reduceras till en rimlig nivå så hade Hollbium Loop inte längre den högsta påverkan inom global uppvärmning och knappa fossila resurser, vilket visar att det avståndet spelar en nyckelroll för att mildra effekterna. Andra aspekter och antaganden analyseras också vidare med hjälp av en känslighetsanalys, som således visar att det finns utrymme för att minska Hollbium Loops miljöavtryck. Det rekommenderas därför att Hollbium satsar på att optimera avståndet och de transportsätt som används, men att även öka tjockleken av den yttre ramen för att göra den stabil och robust. Slutligen rekommenderas det även att öka systemets vertikala längd för att öka avkastning och därmed minska de totala effekterna per kilo producerad sallad.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-26T10:38:54Z No. of bitstreams: 2 Tese - José Tiago das Neves Neto - 2015.pdf: 1371547 bytes, checksum: a079c3c4597fc85eeef5c725389bcc04 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-26T10:39:33Z (GMT) No. of bitstreams: 2 Tese - José Tiago das Neves Neto - 2015.pdf: 1371547 bytes, checksum: a079c3c4597fc85eeef5c725389bcc04 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-26T10:39:33Z (GMT). No. of bitstreams: 2 Tese - José Tiago das Neves Neto - 2015.pdf: 1371547 bytes, checksum: a079c3c4597fc85eeef5c725389bcc04 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-04-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The manipulation of ruminal fermentation is a valuable tool aiming at increasing productivity and efficiency by using resources for animal feed. There are different kinds of tools, such as homogeneous mixing of feed, appropriate trough management, correct feed formulation, and the use of compounds (additives) aimed at assisting rumen fermentation process. The use of antibiotics in foods for animals as growth promoters is facing a social barrier, because of the waste and bacterial resistance that these products can be generating. For that reason the use of antibiotics for this end or at the diet of ruminants has been banned from the EU since January 2006. Therefore, some technologies have been developed to replace these products, and the use of vegetable extracts in ruminants nutrition as ruminal fermentation modulators have been largely studied. Some plants are rich in active compounds with antimicrobial power that makes them useful for animal nutrition. The crude extract of Pterodon Emarginatus Vogel presents sesquiterpenes and diterpenes in its composition, and β-caryophyllene is the most representative compound. Due to the limitations of using unknown products in animal nutrition, the techniques of in vitro digestibility and metabolic studies with animals cannulated in the rumen are largely used at the first stages of research. The objective of this study was to evaluate different dosages of Pterodon Emarginatus Vogel crude extract in vitro ruminal fermentation through a system of continuous culture of double flow. The treatments corresponded to four doses: 0%; 0.6%, 0.12%; and 0.24% of Pterodon Emarginatus Vogel crude extract in dry matter (DM) of the provided diet and a treatment with Sodium Monensin (30 ppm). The experimental design was entirely in randomized blocks with four replications and five treatments. The in vitro test showed a difference of (P<0.05) in the DM and OM digestibility between the treatments 0.24% of Pterodon Emarginatus Vogel crude extract and monensin. There was no difference (P>0.05) in the digestibility of NDF and ADF. The addition of the Pterodon Emarginatus Vogel extract increased the digestibility of DM and OM when compared with Sodium Monensin without effects on NDF and ADF. The treatments affected (P<0.05) acetic acid concentration, and the concentration 0.24% showed a decrease when compared with control treatment. The same happened with the relation between acetic and propionic acids, where 0.24% treatment produced a lower ratio compared with control and 0.06% treatments. The extract of Pterodon emarginatus Vogel has potential to modulate the ruminal fermentation because of its effect on ruminal microorganisms, and it is dose-dependent. Thus it can be used as an additive to manipulate ruminal fermentation and probably as a substitute of the sodic monensin. However, more studies must be carried out to answer the questions about the use of this substances, to clarify the effects of this products on the ruminal microorganisms, its effects on animal metabolism, parameters of in vivo ruminal fermentation, quantify the dosages to be used and quantify animal performance gain. The Culture System in Continuous Flow revealed to be an important alternative method and it showed accuracy for ruminal digestibility studies. / A manipulação da fermentação ruminal é valiosa ferramenta na busca do aumento da produtividade e da eficiência na utilização dos recursos destinados à alimentação animal. Existem diversas ferramentas como a mistura uniforme da ração, o manejo adequado de cocho, a formulação correta da ração e a utilização de compostos que visam auxiliar o processo fermentativo do rúmen denominados aditivos. O uso de antibióticos como promotores de crescimento em alimentos para animais tem enfrentado resistência, devido aos resíduos e a resistência bacteriana que estes produtos podem gerar. Por isso, o uso de antibióticos em sistemas de produção de ruminantes foi proibido na União Europeia em janeiro de 2006. Desta forma, algumas tecnologias têm sido desenvolvidas para substituir estes produtos e dentre estes, os extratos vegetais vêm sendo amplamente estudados na nutrição de ruminantes como moduladores da fermentação microbiana ruminal. Estas plantas são ricas em compostos ativos com poder antimicrobiano tornando-as úteis na nutrição animal como aditivos. O óleo bruto de sucupira apresenta, em sua composição sesquiterpenos e diterpenos sendo, o β-cariofileno, o composto de maior concentração. Diante das limitações da aplicação de produtos desconhecidos na alimentação animal, as técnicas de digestibilidade in vitro e estudos metabólicos com animais canulados no rúmen são recomendados nas etapas iniciais da pesquisa. Com o presente trabalho objetivou-se avaliar diferentes doses do óleo bruto de Sucupira sobre a fermentação microbiana ruminal in vitro através de um sistema de cultura contínua de duplo fluxo. Os tratamentos foram constituídos de quatro doses: 0%; 0,06%; 0,12%; 0,24% na matéria seca (MS) da dieta fornecida do extrato bruto de Sucupira e um tratamento com Monensina Sódica (30 ppm). O delineamento experimental realizado foi em blocos inteiramente casualizados com quatro repetições e cinco tratamentos. No ensaio in vitro, houve diferença (P<0,05) na digestibilidade da MS e MO entre o tratamento 0,24% do extrato de sucupira e os tratamentos controle e monensina. Não se observou diferenças (P>0,05) na digestibilidade da FDN e FDA. A adição do extrato de Pterodon emarginatus Vogel (Sucupira) aumentou a digestibilidade da MS e MO comparados com a Monensina Sódica, sem efeitos na DFN e DFA. A concentração do ácido acético foi afetada (P<0,05) pelos tratamentos, observando uma diminuição da concentração para o tratamento 0,24% comparado com o tratamento controle. O mesmo ocorreu com a relação entre os ácidos acético e propriônico, onde o tratamento 0,24% ocasionou uma menor relação comparado com os tratamentos controle e 0,06%. O extrato de Pterodon emarginatus Vogel (Sucupira) tem potencial de modular a fermentação ruminal devido ao seu efeito nos microrganismos ruminais, sendo seu efeito dose dependente, podendo ser usado como aditivo para manipular a fermentação no rúmen e ser um provável substituto da monensina sódica, porém mais estudos devem ser realizados para elucidar e responder as dúvidas referentes a utilização destas substancias, como esclarecer os efeitos desses produtos sobre os microrganismos ruminais, seus efeitos no metabolismo animal, nos parâmetros de fermentação ruminal in vivo, quantificar as doses a serem utilizadas e quantificar os ganhos em desempenho animal. O Sistema de Cultura em Fluxo Contínuo mostrou ser uma importante metodologia alternativa e de precisão para o estudo de digestibilidade ruminal.

Tot i que es coneix l’involucrament de les cèl·lules pancreàtiques acinars en patologies exocrines (pancreatitis i càncer de pàncrees), la manca de models normals basats en cèl·lules ha limitat l’estudi de les alteracions que succeeixen en el programa de diferenciació pancreàtica. Hem demostrat prèviament que les cèl·lules mare embrionàries murines, que són pluripotents, poden adquirir un fenotip acinar in vitro. Això es va aconseguir, en part, amb una combinació de senyals que provenien del cultiu de pàncrees fetals que no era, però, específic del llinatge pancreàtic. L’objectiu d’aquest treball ha estat el de desenvolupar un protocol selectiu pel llinatge acinar basat en l’activació seqüencial de vies de senyalització que recapitulin el desenvolupament pancreàtic in vivo, a través de la formació definitiva de l’endoderm, l’especificació pancreàtica i acinar i l’expansió/diferenciació de progenitors acinars. El tractament de cossos embrionaris amb Activina A va promoure l’expressió de gens d’endoderm com està prèviament descrit. El tractament subsegüent amb àcid Retinoic, FGF10 i Ciclopamina, un inhibidor de la via de Hedgehog, va resutar en la inducció dels marcadors de progenitors pancreàtics Pdx1, Ptf1a i Cpa1 però també d’aquells expressats en el llinatge pancreàtic, que van ser reduïts amb la inhibició de BMPs. Les cèl·lules van ser a continuació cultivades en Matrigel utilitzant un sistema de cultiu en 3D en presència de fol·listatina, dexametasona i KGF comportant una inducció significativa dels nivells de mRNA i proteïna de marcadors acinars i una disminució de l’expressió dels de marcadors acinars. A més, es va veure que Amyl es secretava en el medi. Aquestes dades indiquen que l’activació selectiva del programa de diferenciació acinar en cèl·lules mare embrionàries es pot dur a terme mitjançant una inducció esgraonada de vies de senyalització involucrades en el desenvolupament pancreàtic exocrí proporcionant una eina potencial per estudiar la diferenciació pancreàtica i malalties relacionades amb el pàncrees. / Despite known involvement of pancreatic acinar cells in exocrine pathologies (pancreatitis and pancreatic cancer), the lack of normal cell-based models has limited the study of the alterations that occur in the acinar differentiation program. We have previously shown that mESC (murine embryonic stem cells), which are pluripotent, can acquire an acinar phenotype in vitro. This was achieved, in part, by a combination of signals provided by the culture of foetal pancreases which was, however, no specific for the acinar lineage. The aim of this work was to develop a protocol selective for the acinar lineage based on the sequential activation of signaling pathways that recapitulate pancreatic development in vivo, through the definitive endoderm formation, the pancreatic and acinar specification and the expansion/differentiation of acinar progenitors. Treatment of embryoid bodies with Activin A enhanced the expression of endodermal genes as previously described. Subsequent treatment with Retinoic acid, FGF10 and Cyclopamine, an inhibitor of the Hedgehog pathway, resulted in the enhancement of pancreatic progenitor markers Pdx1, Ptf1a and Cpa1 but also of those expressed in the hepatic lineage, which were reduced by BMPs inhibition. Cells were further cultured in Matrigel using a 3D culture system in the presence of follistatin, dexamethasone, and KGF leading to a significant enhancement of the mRNA and protein levels of acinar markers while decreasing the expression of endocrine ones. Moreover, active Amyl was released into the medium. These data indicate that the selective activation of the acinar differentiation program in ES cells can be achieved by stepwise induction of signaling pathways involved in pancreatic exocrine development providing a potential tool for studying pancreatic differentiation and pancreas-related diseases.

The absolute clarity of the lens of the eye is vital in the visual system. The unique structural and physiological properties of the lens are tightly integrated with highly ordered protein content to allow the lens to remain transparent. Consequently, any alteration or disturbance of these highly ordered proteins can affect the optical properties of the lens. In humans, cataracts are the major cause of blindness, yet the exact aetiology of cataract formation (cataractogenesis) is not fully understood. The purpose of the current research was to investigate whether deregulation of the Ca²⁺-dependent enzyme, calpains, following changes in lens Ca²⁺ homeostasis, is a key mechanism leading to undesired cleavage of a number of proteins that are linked with maintaining lens transparency and contributing to cataractogenesis. An ovine lens culture (in vitro) system and the heritable ovine cataract (in vivo) model were used to test the research hypothesis. The Ca²⁺ ionophore, ionomycin, was used to induce a Ca²⁺ overload and in vitro opacification during lens culture. Opacity in the lens was graded by a computer image analysis program. Protein profile (SDS-PAGE, 2-DE and Western detection), calpain activity (casein zymography), lens structure (microscopic view) and cytotoxicity level (LDH leakage assay) were analysed in Ca²⁺-induced opaque lenses. The involvement of calpain during opacification was further examined by applying synthetic exogenous calpain inhibitors to the in vitro system. Two novel exogenous calpain inhibitors were also assessed for their therapeutic potential in preventing the progression of cataracts in the in vivo cataract model by topical administration of the inhibitor direct to the sheep's eye over a 11 week period. HPLC was used to detect the penetration of these compounds into ocular tissues. Sustained Ca²⁺ influx into cultured lenses caused dense opacification. The opacity was characterised by formation of a turbid fraction and cell death in the outer cortex of the ovine lens. There was increased calpain autolysis associated with the progress of opacification, indicating increased calpain activity. Major degradation of the cytoskeletal proteins, spectrin and vimentin, was observed whilst there was limited degradation of the lens structural soluble proteins, crystallins, in response to a Ca²⁺ flux. Lens proteins were protected from degradation by adding synthetic calpain inhibitors to the culture medium. Topical administration of novel anti-calpain molecules failed to retard the progression of cataractogenesis in the ovine inherited cataract model. Further investigation of drug penetration showed that efficacy of inhibitory compounds was limited by permeability of these molecules across the cornea and the ability of the molecules to reach and penetrate into the lens. The ovine lens Ca²⁺-induced opacification (OLCO) model in this thesis has provided a model to understand the role of Ca²⁺ homeostasis in lens transparency. With sustained intracellular Ca²⁺ level, the degradation of cytoskeletal elements is highly correlated with calpain activity. Cataractogenesis is the pathological response to the loss of lens Ca²⁺ homeostasis in this model. The current results support the hypothesis that the deregulation of calpain activity is a trigger for a series of cascading events, leading to death of the cells in the lens.