The global population is predicted to increase to around 11 billion by 2100. By 2050, the average age in the most populous age group will be over sixty. The ageing population (over sixty-five) is projected to exceed the number of children by 2047. These demographics imply that as the ageing population section increases, there will be a greater need for long-term care services. In order to adequately prepare against this trend, medical experts and evidence-driven policymakers are realising that personalised healthcare can help alleviate the burden related to the planning and commissioning of services allied to long-term care. Central to this picture is conditions that affect the brain - the most important organ of the human body. Dementia, stroke, and other conditions have a tremendous impact on loss of life, quality of life and healthcare cost. The challenge regarding brain disease is exacerbated further due to the difficulty regarding accessibility of this organ, but also due to the immense complexity regarding its morphology and functionality. In this context, advanced biophysical modelling is considered a promising option for studying brain pathophysiology and becomes a priority investment regarding routes for brain research. Simulations offer the promise of improved, clinically relevant, predictive information, acceleration for the pipeline of drug discovery/design and better planning of long-term care for patients. Within this paradigm, a particular model of water transport in the cerebral environment is essential. Numerous brain disorders arise from water imbalance in the cerebral environment, such as hydrocephalus (HCP), oedema and Chiari malformations to name a few. In this research, a novel multiscale model of fluid regulation and tissue displacement in the cerebral environment is developed, arising from the use of Multiple-network Poroelastic Theory (MPET). Characteristics of a four-network poroelastic model (4MPET) are first explored. Then, this model is extended to a fully dynamic (transient) six-network model (6MPET) via the addition of two new compartments, namely the glial cells compartment and the glymphatic system compartment. The introduction of these two compartments in the MPET paradigm reflects recent seminal findings in cerebral physiology, namely the extent and importance regarding transport/clearance of the perivascular spaces of the brain vasculature. We develop and present a numerical implementation of the 6MPET model, and we utilise this framework to analyse acute HCP and cerebral oedema in a variety of settings, in order to show the enhanced capability of the proposed 6MPET model compared to the classical 4MPET. Investigations of acute hydrocephalus through the fully dynamic 6MPET reveal compensatory trans-ependymal pressure behaviour in the glymphatic compartment. It was also shown that aquaporin-4 (AQP4) deficient expression exaggerates ventriculomegaly, and this too is demonstrated in acute hydrocephalus. Additionally, using the 6MPET model, one is able to witness three mitigating factors for cytotoxic oedema. Specifically, these are: reducing water mobility in the glial cells compartment, increasing the compliance of the glial cells compartment and finally AQP4-deficient expression.

Introduction: Nitric oxide (NO) is an integral molecule implicated in the control of vascular function. It has been suggested that vascular dysfunction may lead to the development of acute mountain sickness (AMS), high-altitude cerebral oedema (HACE) and high-altitude pulmonary oedema (HAPE), though data to date remains scarce. Therefore, there is a clear need for further work to address the role of NO in the pathogenesis of high-altitude illness. Aims: There were two primary aims of the current work: (1) To examine whether hypoxia mediated changes in systemic NO metabolism are related to the development of AMS and sub-clinical pulmonary oedema and (2) to examine whether hypoxia mediated changes in the trans-cerebral exchange kinetics of NO metabolites are related to the development of AMS and headache. Hypothesis: We hypothesise that hypoxia will be associated with an increase in reactive oxygen species (ROS) formation, resulting in a decrease in vascular NO bioavailability (O2•- + NO → ONOO•-, k = 109 M.s-1). The reduction in NO will lead to vascular dysfunction and impaired oxygen (O2) delivery. Subsequent hypoxaemia will result in pulmonary vascular vasoconstriction and the development of sub-clinical pulmonary oedema within and mild brain swelling. Symptoms and reductions in NO bioavailability will be more pronounced in those who develop AMS since they are typically more hypoxaemic. Alternatively, a hypoxia mediated increase in NO, during vasodilatation, specifically across the cerebral circulation, may activate the trigminovascular system resulting in headache and by consequence, AMS. Methods: Study 1 – AMS symptoms, systemic venous NO concentration and nasal potential difference (NPD), used as a surrogate biomarker of extravascular lung oedema, were quantified in normoxia, after a 6hr passive exposure to 12% oxygen (O2) and immediately following a hypoxic maximal exercise challenge (≈6.5 hrs). Final measurements were 2 obtained two hours into (hypoxic) recovery. Study 2 – AMS, radial arterial and internal jugular venous NO metabolite concentrations and global cerebral blood flow (CBF), using the Kety-Schmidt technique, were assessed in normoxia and after a 9hr passive exposure to 12.9% O2. AMS was diagnosed if subjects presented with a combined Lake Louise score of ≥5 points and an Environmental Symptoms Questionnaire – Cerebral score of ≥0.7 points. Results: Hypoxia was associated with a reduction in total plasma NO, primarily due to a reduction in nitrate (NO3•) and a compensatory increase in red blood cell (RBC)-bound NO(P < 0.05 vs. normoxia) in both studies. Study 1 – Exercise reduced plasma nitrite (NO2•) (P< 0.05 vs. normoxia) whereas RBC-bound NO did not change. NO was not different in those who developed AMS (AMS+) compared to those who remained comparatively more healthy (AMS-) (P < 0.05). NPD was not affected by hypoxia or exercise and was not different between AMS+ and AMS- (P > 0.05). Study 2 – Hypoxia decreased arterial concentration of total plasma NO due primarily to a reduction in NO2•- and nitrate (NO3•-). Hypoxia did not alter the cerebral metabolism of RSNO, whereas the formation of RBC-bound NO increased. Discussion: These findings suggest that alterations in systemic or trans-cerebral NO metabolism are not implicated in the pathophysiology of AMS or sub-clinical pulmonary oedema. However, hypoxia was associated with an overall reduction in the total NO pool (NOx), whereas, selected alterations in more vasoactive NO metabolites were observed. Reductions in the partial pressure of O2 (pO2) were thought to be a key regulator in these changes. Overall net increases in RBC NO and corresponding reductions in plasma NO2• in the face of no alterations in NOx indicates that rather than being simply consumed, NO is reapportioned to other NO metabolites and this may be implicated in the pathophysiology of AMS.

Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T18:28:56Z (GMT). No. of bitstreams: 1 Vega_SalomonHuancahuire_M.pdf: 1822310 bytes, checksum: 4712d36f0c1a5963a20bb4649d8aa879 (MD5) Previous issue date: 2009 / Resumo: As enzimas PLA2 provenientes de veneno de serpentes são intensamente estudadas devido a que os envenenamentos constituem um dos principais problemas de saúde em muitos paises. Por outro lado, estas toxinas ajudam a revelar aspectos desconhecidos da fisiologia celular e tisular. Neste trabalho, apresentamos a purificação e a caracterização bioquímica e farmacológica de duas miotoxinas fosfolipases A2: BbTX-II e BbTX-III, a partir de veneno de Bothrops brazili. As duas proteínas foram isoladas e purificadas usando um procedimento simples e rápido envolvendo duas etapas cromatográficas, exclusão molecular em Sephadex G-75 e HPLC de fase reversa (C18). A eletroforese de ambas miotoxinas mostrou massas relativas em torno de 13 e 27 kDa (para monômeros e dímeros respectivamente). Espectrometria de massa por MALDI-TOf confirmou a pureza das proteínas e mostrou que possuem massas moleculares em torno de 13,8 kDa. A análise de aminoácidos mostrou alto conteúdo de aminoácidos básicos e hidrofóbicos, assim como 14 resíduos de Cys. BbTX-III apresentou atividade PLA2 na presença de um substrato cromogênico, mostrando comportamento sigmoidal, principalmente à baixas concentrações. Atividade máxima foi alcançada em pH 8 e entre 35-45 oC. BbTX-III mostrou-se completamente dependente de Ca2+ e, na presença dos íons Mg2+, Mn2+, Cd2+ e Zn2+, a atividade enzimática foi reduzida a níveis similares aos observados na ausência de Ca2+. A análise de composição de aminoácidos mostrou alta presença de Lys, His e Arg (pI 8,46). A presença de 14 resíduos de cisteína sugere a formação de 7 pontes dissulfeto. O estudo de homologia da seqüência da PLA2 BbTX-III mostrou que existem posições extremamente conservadas nas PLA2. S(1), L(2), E(4) 7 a 10 (QMIL), Y(21). Os resíduos conservados Y(28), G(30), G(32), D(49), H(48) e Y(52) estão direta ou indiretamente ligados com a catálise. Além disso, BbTX-III apresentou algumas mutações: K(35) -> G(35), R(51) -> Y(51) e D(118) -> A(118) que estão estrategicamente posicionadas para a expressão da atividade catalítica. Apesar destas substituições, as atividades farmacológicas e a atividade catalítica são mantidas. O efeito neurotóxico de BbTX-III foi analisado in vitro na preparação neuromuscular biventer cervicis de pintainhos. O resultado mostrou que a toxina é menos potente quando comparada com venenos crotálicos. BbTX-III demonstrou efeito miotóxico local in vivo através da liberação de creatina quinase (CK) e, efeito inflamatório, através de edema de pata. Como a BbTX-III produziu forte efeito inflamatório, a hidrólise de fosfolipídios poderia ser relevante neste fenômeno. BbTX-II foi caracterizada como uma PLA2 K49 (cataliticamente inativa) em função das características físico-químicas evidenciadas: massa de 13,68 kDa, 121 resíduos de aminoácidos, caráter básico (pI 8.73) e alto grau de homologia seqüencial na sua estrutura primária, quando comparada com outras PLA2B K49 procedentes de veneno de serpentes botrópicas. O alinhamento com outras seqüências completas de PLA2 BK49 mostrou a presença de algumas mutações importantes. Assim, as substituições Y?N(27), N?P(58) e L?F(114) não modificaram os efeitos biológicos aqui estudados, revelando que poderiam estar relacionados com outras atividades. Os resíduos N(28), K(111), L(32) poderiam contribuir com a interrupção da catálise. Esta nova PLA2 K49 BbTX-II mostrou miotoxicidade local in vivo, atividade inflamatória e letalidade, corroborando que se enquadra dentro da família de proteínas PLAB2B K49. Os estudos de neurotoxicidade revelaram um efeito neurotóxico in vitro na preparação biventer cervicis de pintainho (20 µg/ml). BbTX-II e BbTX-III mostraram ser miotoxinas com atividade edematogênica e neurotóxica, independentemente de apresentarem atividade catalítica (BbTX-III) ou não (BbTX-II) Apoiando a existência de regiões moleculares distintas à catalítica responsáveis pelos efeitos farmacológicos. Os efeitos farmacológicos da toxina BbTX-III (PLA2B D49) provavelmente tenham uma estrita relação entre a atividade enzimática e a ligação da toxina com micro-domínios na membrana plasmática onde sua atividade seja maximizada e cause danos relevantes na organização da membrana. No caso da BbTX-II (PLAB2 K49) possivelmente a combinação de aminoácidos aromáticos/hidrofóbicos e positivamente carregados da região C-terminal seja a responsável de alterar a integridade da membrana plasmática. / Abstract: The enzymes PLA2 coming of venom snake are studied intensely due to that the poisonings constitute one of the main problems of health in many countries. On the other hand, these toxins help to reveal unknown aspects of the cellular and tissue physiology. In this work, we presented the purification and biochemical and pharmacological characterization of two myotoxic phospholipases A2: BbTX-II and BbTX-III from Bothrops brazili venom. The two proteins were isolated and purified using a simple and fast procedure involving two chromatographic steps, molecular exclusion in Sephadex G-75 and reverse-phase HPLC (C-18 column). Both myotoxins showed around 13 and 27 kDa (for monomers and dimers, respectively) relative mass. MALDI-TOf mass spectrometry confirmed the purity of the proteins showing molecular masses around 13,8 kDa. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 cysteine residues. BbTX-III presented PLA2 activity in the presence of a chromogenic substrate, showing sigmoidal behavior, mainly at low concentrations. Maximum PLA2 activity was reached at pH 8 and 35-45 oC. Maximum activity required Ca2+ and, in the presence of Mg2+, Mn2+, Cd2+ and Zn2+, was reduced at similar levels as observed in the absence of Ca2+. Amino acids analysis of composition showed high presence of Lys, His and Arg (pI 8,46). The presence of 14 cystein residues suggested the formation of 7 disulfide bridges. Sequence homology of the PLA2 BbTX-III revealed positions extremely conserved in PLA2 S(1), L(2), E(4) 7 to 10 (QMIL), Y(21). The conserved residues Y(28), G(30), G(32), D(49), H(48) and Y(52) are direct or indirectly linked to the catalysis. Besides, BbTX-III presented some mutations: K(35) -> G(35), R(51) -> Y(51) and D(118) -> A(118) that are strategically positioned for the expression of catalytic activity. Despite these substitutions, the pharmacological and catalytic activities are maintained. The neurotoxic effect of BbTX-III was analyzed in vitro at chick biventer cervicis muscle preparation. Our results showed that the blockage of the muscle contraction was lower when compared with crotalic venoms. BbTX-III demonstrated in vivo myotoxic local effect through the liberation of creatine kinase (CK) and inflammatory effect through paw edema. As BbTX-III produced strong inflammatory effect, the phospholipids hydrolysis could be relevant in this phenomenon. BbTX-II was characterized as PLA2 homologous K49 (catalytically inactive), because its chemical and physical evidenced characteristics: mass of 13,68 kDa, 121 amino acids residues, basic character (pI 8.73) and high sequential homology in its primary structure, when compared with other PLA2 K49 from venom of Botrhops serpents. The alignment with other complete sequences of PLA2 homologous K49 showed the presence of some important mutations. Substitutions Y->N(27), N->P(58), and L->F(114) did not modify the biological activities here studied, revealing that it could be related to other activities. The residues N(28), K(111), L(32) could contribute with the interruption of the catalysis. This new PLA2 K49 BbTX-II showed in vivo myotoxic local effect, inflammatory and lethality activities, evidencing it was fitted to the family of proteins PLA2 K49 homologous. Beside BbTX-II revealed in vitro neurotoxyc effect at chick biventer cervicis muscle preparation (20 µg/mL). BbTX-II and BbTX-III showed to be myotoxins to activity edematogenic and neurotoxyc independently of present catalytic activity (BbTX-III) or no (BbTX-II) Supporting the existence of molecular areas different to the catalytic responsible for the pharmacological effects. The pharmacological effects of the toxin BbTX-III (PLA2 D49) they probably have a strict relationship between the enzymatic activity and binding of the toxin with micro-domains in the plasmatic membrane where the activity is maximized and cause relevant damages in the organization of the membrane. In the case of the BbTX-II (PLA2 K49) possibly the combination of amino acids aromatics/hidrophobics and positively loaded of the area C-terminal it is the responsible of altering the integrity of the plasmatic membrane. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Marcos Hikari Toyama / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T11:46:22Z (GMT). No. of bitstreams: 1 Oliveira_SimoneCristinaBuzzo_M.pdf: 2132823 bytes, checksum: b98ccca4e06a6ca0155f84135393b9b5 (MD5) Previous issue date: 2007 / Resumo: Lectinas são proteínas que se ligam de forma específica e reversível a carboidratos. Estão distribuídas pelos mais diversos organismos e apresentam atividades de interesse em pesquisas biológicas e médicas. Neste trabalho, duas isoformas de lectina da alga marinha vermelha Bryothamnion triquetrum foram purificadas por cromatografia de troca iônica seguida por uma de fase reversa. As frações foram nomeadas de BTLD1 e BTLD2 e ambas apresentaram massa molecular aparente em SDSPAGE de aproximadamente 9,0 kDa. A análise dos aminoácidos de ambas as lectinas revelaram moléculas de caráter básico e as regiões Nterminal das lectinas foram seqüenciadas e comparadas entre si e com lectinas de outras algas, apresentando grande similaridade com hypninA1, hypninA2 e BTL. Além disso, o dicroísmo circular revelou que a estrutura secundária das proteínas é predominantemente de arranjo aleatório. A lectina BTLD2, a isoforma mais abundante do extrato da alga, apresentou ação antibacteriana contra a bactéria grampositiva Clavibacter michiganensis subsp. michiganensis (Cmm), mas não foi eficiente contra a bactéria gramnegativa Xanthomonas axonopodis pv. passiflorae (Xap). Também foram utilizados dois modelos de fosfolipases A2, uma isolada do veneno total de Crotalus durissus cascavella que é cataliticamente ativa do tipo Asp 49 (D49), e uma outra PLA2 cataliticamente não ativa do tipo Lys49 (K49), isolada do veneno de Bothrops jararacussu, para ensaios biológicos. A atividade farmacológica e biológica de ambas as PLA2s foi significativamente afetada pela coincubação das mesmas com BTLD2. A formação de heterodímeros de BTLD2 com a PLA2 de Crotalus durissus cascavella ou com a PLA2 de Bothrops jararacussu sugere a formação de um complexo estável em solução com uma massa molecular de aproximadamente 23,0 kDa. A BTLD2 também foi capaz de aumentar significativamente a atividade enzimática da PLA2 de C. d. ascavella em comparação com a PLA2 cataliticamente ativa isolada. As PLA2s de C. d. cascavella e B. jararacussu mostraram forte atividade antibacteriana contra bactérias Cmm e tiveram pouco efeito contra a bactéria Xap. Entretanto, o complexo BTLD2: PLA2 D49 ou K49 mostram um aumento da atividade antibacteriana, principalmente contra a bactéria Xap. As PLA2s induziram atividade edematogênica em pata de ratos e também foram capazes de induzir uma forte agregação plaquetária usando o sistema de plaquetas lavadas. Em ambos os sistemas, o complexo BTLD2: PLA2 mostrou uma atividade menor do que os respectivos controles. Concluindo, os resultados apresentados mostram que esta nova lectina isolada de alga vermelha possui uma evidente capacidade de interação com moléculas de PLA2, tanto cataliticamente ativas quanto inativas, com a formação de heterodímeros. Portanto, esta interação é capaz de modificar significativamente a estrutura terciária da PLA2, uma vez que o complexo mostrou atividade enzimática aumentada além de possivelmente alterar a estrutura de outras regiões moleculares da enzima. É importante verificar que a atividade farmacológica das PLA2s não tem uma correlação direta com a atividade catalítica das mesmas, envolvendo outras regiões que permitem a sua interação com receptores cuja região está distante do sítio catalítico da PLA2 / Abstract: Lectins are proteins that bind specifically and reversibly to carbohydrates. They are widely distributed among living organisms and show many activities of biological and medical interest. In this work, two isoforms of lectins isolated from the red marine alga Bryothamnion triquetrum were purified by ion exchange followed by reverse phase chromatographies. The fractions named BTLD1 and BTLD2 showed apparent molecular mass of approximately 9.0 kDa in SDSPAGE. Both lectins probably have a basic character, as indicated the amino acid analyses using the Compute pI/Mw tool at the ExPASy server. The Nterminal sequences were compared between both lectins and also to other lectins, showing similarity with hypninA1, hypninA2 and BTL. The circular dichroism indicated that the secondary structure of the proteins are predominantly random coiled. Additionaly, BTLD2 (the more abundant lectin isoform in the alga extract) showed antibacterial activity against the grampositive bacteria Clavibacter michiganensis subsp. michiganensis (Cmm) but was not effective against the gramnegative bacteria Xanthomonas axonopodis pv. passiflorae (Xap). A possible modulation of phospholipase activity by BTLD2 was also studied using two phospholipases A2, one isolated from Crotalus durissus cascavella venom that is Asp49 (D49) catalytically active, and other PLA2 Lys49 (K49) catalytically non active from Bothrops jararacussu venom. The pharmacological and biological activities of PLA2s were significantly change by incubation with BTLD2. The heterodimer formation of BTLD2 with Crotalus durissus cascavella or Bothrops jararacussu PLA2s appears to be a stable complex in solution with a molecular mass of approximately 23 kDa. BTLD2 significantly increased the enzymatic activity of the PLA2 from C.d. cascavella compared to the PLA2 alone. Both PLA2s (catalytically active and nonactive) showed strong antibacterial activity against Cmm with little effect against Xap. However, the BTLD2: PLA2 D49 or K49 complexes showed increase in antibacterial activity, particularly against Xap. Moreover, both PLA2s induced edematogenic activity in rat paw and strong platelet aggregation in washed platelets system. Interestingly, addition of BTLD2 with consequent formation of the BTLD2: PLA2 complex decreased both PLA2induced edema and platelet aggregation. Taken toghether, these results show that this new lectin isolated from a red alga and named BTLD2 has the capacity to interact with catalytic or noncatalytic PLA2, forming heterodimers. Therefore, this interaction possibly modify the PLA2 tertiary structure; as the complex BTLD2: PLA2 increase the enzymatic activity of PLA2 (i.e., the phospholipase activity) but at the same time decrease pharmacological and biological PLA2 activities not related to catalysis (i.e., platelet aggregation and edema). This suggests that the PLA2 structure is possibly modified in other sites of the enzyme rather than in the catalytic site. It can be concluded that the PLA2s has several binding sites for different molecules comprising the catalytic site responsible for the phospholipase activity and at least one more pharmacological site responsible for the effects observed after interaction with BTLD2. Therefore, the pharmacological activity of PLA2s has no direct correlation with the catalytic activity, involving other binding sites that allow receptor interaction whose position might be far from the catalytic site of PLA2 / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Atherosclerotic renovascular disease (ARVD) is a significant cause of chronic kidney disease (CKD) and is associated with an increased risk for cardiovascular morbidity and mortality. Randomised controlled trials, representing over 2100 patients, have failed to demonstrate any prognostic benefit of percutaneous renal revascularisation when utilised in addition to standard medical therapy. This negative finding has been interpreted in three ways. Firstly, that ARVD may be an association of CKD and not a specific disease process. Secondly, that published studies have recruited low-risk patients who are least likely to benefit from revascularisation. Thirdly, that the focus of treatment for patients with ARVD should be optimal medical therapy, not renal revascularisation. This research project had a series of linked aims. These were investigated in two large patient cohorts that had been accumulated at this centre over the last decade. These cohorts comprised > 900 patients with ARVD, the Salford Renovascular Database (SRVD), and > 2500 patients with all-cause CKD, the Chronic Renal Insufficiency Standards Implementation Study (CRISIS). The first aim was to consider whether ARVD should be considered as a specific cause of CKD. Here risks for death and progression to renal replacement therapy were compared between patients having ARVD as their primary cause of renal failure and patients with other coded causes of CKD. In this analysis, patients with ARVD had a greater risk for death and a lesser risk for RRT than patients with other forms of CKD.The second aim of this thesis was to consider if specific patient sub-groups of ARVD could be identified. Patients in the SRVD with currently accepted high- risk clinical presentations were selected and outcomes compared to patients without a high-risk presentation. In this analysis, presentation with flash pulmonary oedema (but with not refractory hypertension or rapidly declining renal function) was associated with an increased risk for death and cardiovascular event. When the effects of revascularisation were considered in patients with high-risk presentations, a mortality benefit was observed in patients with flash pulmonary oedema and in patients presenting with rapidly declining renal function and refractory hypertension in combination. A separate analysis was performed in the SRVD to consider if a high-risk sub-group of ARVD patients could be identified using laboratory measurements. Here, a classification tree methodology was employed to identify ARVD patients with the greatest risk for progression to end stage kidney disease. The results of this analysis were converted into a practically applicable clinical scoring system incorporating renal function, proteinuria, medications, smoking history and renal artery occlusion. The final aim of this thesis was to describe how the majority of ARVD patients should be treated. In this analysis of the SRVD effects of treatment with anti- platelet and beta-blocker therapy were considered, and shown to be associated with reduced risks for cardiovascular events and death.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21954号 / 医博第4496号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 伊佐 正, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Master of Science / Department of Horticulture, Forestry, and Recreation Resources / Chad T. Miller / Kimberly A. Williams / Intumescences are a physiological disorder characterized by hypertrophy and possibly hyperplasia of plant cells. Many plant species are susceptible to intumescence development, but the specific causative factors remain uncertain. Ultimately, this disorder results in the death of the affected cells. Previous observations and research suggest that the quality and quantity of light to which plants are exposed may be a factor in development of the disorder. The purpose of the first study was to assess the preventive effect of UVB radiation on intumescence development in ornamental sweet potato (Ipomoea batatas). Two sweet potato cultivars, ‘Sidekick Black’ and ‘Ace of Spades,’ were grown under four light treatments of 1) Normal; 2) UVB; 3) UVB-Blocked; 4) Full-Spectrum. The ‘Ace of Spades’ cultivar was highly susceptible to intumescence development, while ‘Sidekick Black’ was much less susceptible to the disorder. For ‘Ace of Spades,’ the addition of UVB radiation significantly reduced the number of leaves affected with intumescences when compared to plants grown under the other light treatments. This study indicates a cultivar-specific effect of UVB light in minimizing intumescence development on ornamental sweet potato, therefore suggesting a potential genetic component in intumescence susceptibility. Many plant species are prone to similar physiological disorders in which lesions develop on the leaf tissue. Nomenclature for such lesions has varied significantly in the literature. Interchangeably using these terms causes confusion as to whether these names refer to the same or different disorders. The objective of the second study was to characterize the development of lesions on ornamental sweet potato (Ipomoea batatas ‘Blackie’), tomato (Solanum lycopersicum ‘Maxifort’) and interspecific geranium (Pelargonium בCaliente Coral’). Light microscopy, field emission scanning electron microscopy (FESEM), and digital photography were used to observe lesion development on each species. Lesions on ornamental sweet potato predominately involved the hypertrophy of the palisade parenchyma through the upper epidermis, while geranium lesions involved the hypertrophy of spongy parenchyma cells restricted by the lower epidermis. Tomato lesions involved both the hypertrophy and hyperplasia of the lower epidermis and spongy parenchyma. Thus, different species possess varied cellular responses when developing lesions due to physiological causes.

La rétinopathie diabétique (RD) se compose d’une part d’une ischémie rétinienne périphérique et d’autre part d’une exsudation rétinienne responsable d’un œdème maculaire diabétique, première cause de cécité chez les moins 55 ans. Les traitements utilisés actuellement sont non spécifiques et traitent les complications tardives de la RD. Les phases précoces de la RD ne sont donc pas ciblées. L’hyperglycémie chronique entraine un stress oxydant et une activation des PKC qui participent à l’altération des BHR. L’objectif de ce travail a été 1°) d’étudier l’implication de la PKCζ et de la voie Rho/ROCK/Myosine II sur la physiopathogénie de la RD et 2°) de montrer l’effet bénéfique de leurs inhibiteur sur les BHR et sur la reperfusion des capillaires rétiniens. Nous avons confirmé l’hyperactivation de la PKCζ et de la voie Rho/ROCK/Myosine II chez les rats diabétiques et leur participation à la rupture de la BHR externe. Le traitement par leurs inhibiteurs respectifs normalise l’activation des deux enzymes et restaure l’intégrité anatomique et fonctionnelle de la BHR externe. De plus l'hyperactivation de ROCK altère la perfusion rétinienne par 1) constriction focale artériolaire, 2) protrusions membranaires endoluminales des cellules endothéliales (blebbing) et 3) vasoconstriction capillaire diffuse. Nous avons montré que l'ensemble de ces phénomènes étaient réversibles par traitement intravitréen de son inhibiteur le Fasudil. De manière importante le traitement par Fasudil induit également une diminution du VEGF rétinien responsable de la perméabilité des barrières et témoin indirect de l’ischémie rétinienne. Ces travaux éclairent la physiopathogénie de la RD et ouvre des perspectives thérapeutiques permettant de cibler les événements précoces de la RD. / Diabetic retinopathy (DR) mainly results from peripheral retinal ischemia and exudation leading to sight threatening complications such as retinal neovascularization or macular edema. This latter represents the main cause of visual loss among working age individuals. Current treatments address late complications of DR and are non-specific. Therefore, early events are currently not addressed. Chronic hyperglycemia increases oxidative stress and activates PKC leading to blood retinal barrier (BRB) breakdown. The aims of the present work were two fold. First, to assess the implication of PKCζ and the Rho/ROCK/Myosin II pathway on the pathogenesis of DR and second, to assess whether their specific inhibitors have the potential to restore the phenotype. Herein we have demonstrated the pathogenic role of PCKζ and ROCK hyperactivation on the development of diabetes induced external BRB breakdown. Furthermore their inhibitors restored the morphologic and functional aspect of the external BRB. We also found that ROCK hyperactivation was responsible for altered retinal perfusion through several mechanism namely 1) focal constriction of retinal arterioles; 2) endoluminal protrusions of the endothelial cell membrane (blebs) and 3) capillary diffuse vasoconstriction. We were able to demonstrate that all this aspects were reversible by Fasudil, a ROCK inhibitor, administrated into the vitreous. Of importance this treatment decreased also retinal VEGF that is a well-known factor responsible for barrier breakdown and a surrogate marker for retinal ischemia. To conclude the present findings not only shed light on the mechanisms of DR but also open new therapeutic avenues addressing the early events of DR a current unmet medical need.

Patients admitted in similar clinical condition after spontaneous SAH can develop very different clinical courses. This could depend on the severity of the initial global ischemic brain injury at ictus. In the present study, we explored relations between clinical and radiological parameters at admission that indicate a more severe initial impact, and the following days hormone levels and brain metabolism. Early global cerebral oedema (GCE) on computed tomography occurred in 57 % of SAH patients and was associated with a more severe clinical condition. The brain’s glucose metabolism, measured with intracerebral microdialysis (MD), changed the first days. MD-glucose was initially high and MD-pyruvate low. MD-glucose gradually decreased and MD-pyruvate and MD-lactate increased, suggesting a transition to a hyperglycolytic state. This was more pronounced in patients with GCE. Similar patterns were seen for interstitial non-transmitter amino acids. From initial low concentrations, they gradually increased in parallel with MD-pyruvate. The amino acid concentrations were higher for patients admitted in better clinical condition. Insulin lowered MD-glucose and MD-pyruvate even when plasma glucose values remained high. P-ACTH and S-cortisol were elevated early after SAH. GCE was associated with higher S-cortisol acutely. Urine cortisol excretion, indicating levels of free cortisol, were higher in patients in a better clinical condition. Suppressed P-ACTH occurred in periods of brain ischemia. We suggest that GCE on the first CT scan is a warning sign indicating increased vulnerability if the patient is exposed to compromised energy supply or increased energy demand. Reduction of blood glucose after SAH should be done with caution. The temporal change of the glucose metabolism and the amino acid concentrations probably reflect activated repair mechanisms. This should be considered in the intensive care treatment of SAH patients. Finally, our results support earlier observations that the response of the hypothalamic-pituitary-adrenal system is important in critical care.

Das Auftreten pulmonaler Belüftungsstörungen nach Injektion von Xylazin beim Schaf ist in der wissenschaftlichen Literatur an Einzeltieren beschrieben. Der dabei noch ausstehende Nachweis eines postulierten Lungenödems anhand objektiver Parameter in statistisch relevanter Anzahl wurde in der hier vorliegenden Arbeit angestrebt. Weiterhin wurden ein Einfluss der wiederholten Exposition und eine Dosisabhängigkeit überprüft. Zur Bearbeitung dieser Fragen wurden 16 weibliche Merinolandschafe dreimalig in einem Abstand von 8 Wochen untersucht. Nach Prämedikation mit Midazolam (0,25 mg/kg) und Sufentanil (0,6 µg/kg) erfolgte die Allgemeinanästhesie mit Propofol (5-10 mg/kg/h). Zu den ersten beiden Versuchsabschnitten wurde Xylazin in einer Dosis von 0,15 mg/kg, im dritten Versuchsdurchgang in Höhe von 0,3 mg/kg intravenös verabreicht. Jeweils 10 Minuten vor und 5, 15, 30 Minuten nach Applikation von Xylazin wurden computertomographische Untersuchungen durchgeführt. Mit Hilfe der quantitativen computertomographischen Analyse konnte das totale Lungengewicht, der Anteil nicht belüftetes Lungengewicht und das totale Lungenvolumen ermittelt werden. Zusätzlich wurden mittels arterieller Blutgasanalysen der arterielle Sauerstoff- und Kohlenstoffdioxidpartialdruck bestimmt. In der dieser Arbeit zu Grunde liegenden Annahme nimmt im Falle eines Lungenödems das totale Lungengewicht bei konstantem Lungenvolumen zu. Eine Zunahme des totalen Lungengewichts war in allen drei Versuchsdurchgängen statitisch signifikant nachweisbar. Im Vergleich zu den Angaben in der Literatur wurden dabei jedoch keine Zunahmen in Höhe eines klinisch relevanten Lungenödems erreicht. Unerwartet konnte zusätzlich ein signifikanter Rückgang des totalen Lungenvolumens detektiert werden. Weiterhin waren bereits 5 Minuten nach Xylazininjektion bis zu einem Drittel des totalen Lungengewichts nicht belüftet. Diese pulmonalen Belüftungsstörungen nach Applikation von Xylazin beim Schaf wurden aufgrund der vorliegenden Ergebnisse nicht ausschließlich der Entstehung eines Lungenödems zugeordnet. Die detektierte Reduktion des totalen Lungenvolumens bei konstanter Beatmung kann nur durch Atelektasen begründet werden. Entsprechend dem Ausmaß der detektierten pulmonalen Reaktionen nach Xylazingabe wurden eine schwere Hypoxämie sowie eine Hyperkapnie festgestellt. Durch die mehrfache Exposition von Xylazin erfolgte der Nachweis der Wiederholbarkeit dieser Ergebnisse. Eine Dosisabhängigkeit des Ausprägungsgrades der pulmonalen Befunde hingegen konnte nicht statistisch signifikant bestätigt werden. Anhand der hier vorliegenden Ergebnisse muss die Ätiologie der pulmonalen Veränderungen nach Injektion von Xylazin beim Schaf neu durchdacht und in weiteren Studien verfolgt werden. Einflussfaktoren wie die Form der Applikation oder eine genetische Prädisposition gilt es in Zukunft zu analysieren. Neben der klinischen Anwendung von Xylazin sind die erarbeiteten Resultate relevant für humanmedizinische Fragestellungen in der Pulmologie. Dort sollte in der häufigen Verwendung des Schafes als Tiermodell in Hinblick auf mögliche Interaktionen mit den experimentellen Ergebnissen auf die Applikation von Xylazin verzichtet werden.