Neste trabalho foi avaliada a permeação/penetração cutânea in vitro (pele suína) de propionato de clobetasol nanoencapsulado incorporado em um semissólido, empregando células de difusão de Franz. A nanoencapsulação foi capaz de reduzir a quantidade de fármaco que penetra nas camadas da pele (estrato córneo, epiderme e derme) sem alterar a forma (distribuição percentual) como o propionato de clobetasol se distribui. A adequabilidade de diferentes membranas sintéticas (acetato de celulose, policarbonato e membrana de diálise) para a avaliação da liberação in vitro, empregando células de difusão de Franz, a partir desta formulação foi também estudada. A partir da combinação de diferentes técnicas analíticas (espalhamento de luz dinâmica, microscopias eletrônicas de transmissão e varredura) foi observado que a membrana de menor tamanho de poro (membrana de diálise, 12 kDa de cut off) é a mais adequada para a condução deste tipo de avaliação, pois é a única capaz de evitar a passagem de nanocápsulas íntegras da formulação para o meio receptor das células de difusão, em detrimento das membranas de policarbonato e acetato de celulose (0,05 μm e 0,45 μm de tamanho de poro, respectivamente). Além disso, uma nova estratégia para a avaliação da liberação in vitro de fármacos associados a nanocápsulas de núcleo lipídico, combinando fluxo contínuo de meio de liberação e sacos de diálise foi proposta neste trabalho. A técnica mostrou-se adequada para a obtenção do perfil de liberação in vitro a partir de suspensões de nanocápsulas contendo diferentes fármacos modelo (prednisolona e propionato de clobetasol), possibilitando a diferenciação destes sistemas de soluções contendo os fármacos livres, graficamente e pelos valores de fluxo calculados. Adicionalmente, esta estratégia mostrou-se apropriada para a manutenção da concentração de fármaco no meio de liberação afastada da saturação, contribuindo para o atendimento da condição sink. Ainda, classificamos o sistema como um protótipo semi-automatizado para a avaliação da liberação in vitro de fármacos, capaz de gerar resultados com maior precisão em relação à diálise convencional. / The in vitro cutaneous permeation/penetration (porcine skin) of clobetasol propionate-loaded lipid-core nanocapsules incorporated into a semisolid dosage form was evaluated, using the Franz diffusion cells technique. It was shown that the nanoencapsulation was able to reduce the drug amount penetration into skin layers (stratum corneum, epidermis and dermis) without changing the way (percentual distribution) that it was distributed. The suitability of different synthetic membranes (cellulose acetate, polycarbonate, and dialysis membrane) to assess the in vitro drug release using Franz diffusion cells from this formulation was also studied. It was ascertained by combining different analytical techniques (dynamic light scattering, scanning and transmition electron microscopy) that the membrane with smaller pore size (dialysis membrane, 12 kDa cut off) is the most appropriate for conducting this kind of study, because it is the only one able of preventing the passage of intact nanocapsules from formulation to Franz diffusion cells receptor media, instead of polycarbonate and cellulose acetate membranes (0.05 and 0.45 pore size, respectively). In addition, a new strategy to assess in vitro drug release drug-loaded lipid-core nanocapsules was proposed, associating continuous flow of release media and dialysis sac. The proposed system was adequate to assess the in vitro drug release profiles from nanocapsule suspensions containing different model drugs (prednisolone and clobetasol propionate), enabling the differentiation of these systems from drug solutions, graphically and by the calculated flux values. Furthermore, this strategy was suitable to maintain the drug concentration into release media far away from saturation, contributing to the sink condition. Also, the proposed system was described as a semi-automated prototype for in vitro drug release evaluation, able to produce results with greater accuracy than conventional dialysis technique.

Terpenes are naturally occurring compounds obtained from the plants secondary metabolism. Despite presenting pharmacological effects, structural changes within their skeleton may increasing their pharmacological activity and attenuate the toxicological effects. Carvacrol is a phenolic monoterpene present in essential oils from plants belonging to the Labiatae family. Studies have demonstrated that carvacrol has anti-inflammatory activity. However, sctructural changes may reduce the effective dose of this monoterpene. Thus, in this study, we conducted an extensive systematic review evaluating the antiinflammatory activity of terpenes that suffered an alteration in its structure through synthesis and semi-synthesis, synthesize the carvacrol propionate (CP) from the carvacrol and evaluate its potential antinociceptive, anti-hyperalgesic and anti-inflammatory effects. To build the revision, it was made the search in Scopus, Embase and PubMed databases, using the descriptors anti-inflammatory agents, terpenes and structure activity relationship. In the experimental part, it was used Male Swiss mice (25-35 g) with 2 to 3 months age. The animals were divided in groups and were treated with CP (25, 50 and 100 mg/kg), vehicle (saline solution 0.9% + Tween 80 0.2%) or standard drug, intraperitoneally (i.p.). The antinociceptive effect was evaluated through the formalin (1%) protocol and the hot plate test. The mechanical hyperalgesia was evaluated through the algic agents injection: carragee nan (CG; 300 µg/paw), tumor necrosis factor-a (TNF-a; 100 pg/paw), prostaglandin E2 (PGE2; 100 ng/paw) or dopamine (DA; 30 µg/paw) using a digital analgesimeter (von Frey). To assess the anti-inflammatory effect, it was used the pleurisy and paw edema induced by GC (1 %) in digital plethysmometer. The cytotoxicity of CP was evaluated by the MTT colorimetric method. The experimental protocols were approved by the UFS ethics committee (CEPA/UFS: 35/12). The results are expressed as mean ± SEM and differences between groups were analyzed by one-way or two-way ANOVA test followed by Tukey or Bonferroni tests. Values of p < 0.05 were considered statistically significant. In systematic review, 27 papers were found concerning about terpenes structural modification and the evaluation of their anti-inflammatory activity. In the experimental part, the administration of CP produced a significant decrease (p < 0.01 and 0.001) in the test formalin-induced nociceptive in both phases of the test. In the hot plate test, the reaction time increased significantly at doses 50 and 100 mg/kg (p < 0.05, 0.01 and 0.001). CP inhibited the development of mechanical hyperalgesic induced by all agents tested (p < 0.05, 0.01 and 0.001). In the evaluation of anti-inflammatory activity, the treatment with CP was able to decrease significantly the leukocyte recruitment (p < 0.001), the TNF-a (p < 0.001), the IL-1ß (p < 0.05) and protein leakage (p < 0.01). In addition, the paw edema induced by CG in mice was inhibited significantly by CP (p < 0.05, 0.01 and 0.001). Thus, it is concluded that the CP attenuates nociception, mechanical hyperalgesia and inflammation, through an inhibition of cytokines. Therefore, structural modification terpene can be an interesting alternative for obtaining molecules with pharmacological properties. / Os terpenos sao compostos naturais obtidos do metabolismo secundario das plantas. Apesar de apresentar efeitos farmacologicos, modificacoes estruturais realizadas no seu esqueleto podem levar o aumentando de suas atividades farmacologicas e atenuar os efeitos toxicologicos. Neste contexto, insere-se o carvacrol, um monoterpeno fenolico, presente em oleos essenciais de plantas pertencentes a familia Labiatae. Estudos comprovam a atividade farmacologica deste monoterpeno. No entanto, modificacoes estruturais podem diminuir a dose efetiva deste composto. Desta forma, no presente estudo realizamos uma extensa revisao sistematica que avaliou a atividade anti-inflamatoria de terpenos que sofreram modificacoes em sua estrutura, atraves de sintese. Adicionalmente, sintetizar o propionato de carvacrol (CP), a partir do carvacrol, e avaliar seus possiveis efeitos antinocicepivo, anti-hiperalgesico e anti-inflamatorio. Para construir a revisao, foi realizada a busca nas bases de dados Scopus, PubMed e Embase, utilizando os descritores agentes anti-inflamatorios, terpenos e relacao estrutura atividade. Ja para a parte experimental, foram utilizados camundongos Swiss machos (25-35 g) com 2 a 3 meses de idade. Os animais foram divididos em grupos e foram tratados com CP (25, 50 e 100 mg/kg), veiculo (solucao salina 0,9% + Tween 80 0,2%) ou droga padrao, por via intraperitoneal (i.p.). O efeito antinociceptivo foi avaliado utilizando o protocolo de formalina (1%) e o teste da placa quente. A hiperalgesia mecanica foi avaliada apos a administracao dos agentes algicos carragenina (CG; 300 Êg/pata), fator de necrose tumoral- ¿ (TNF- ¿; 100 pg/pata), prostaglandina E2 (PGE2; 100 .g/pata) ou dopamina (DA; 30 Êg/pata) utilizando o analgesimetro digital Von Frey. Na avaliacao do efeito antiinflamatorio utilizou-se o teste de pleurisia e edema de pata induzido por CG (1%) em pletismometro digital. A citotoxicidade foi avaliada atraves do metodo colorimetrico MTT. Os protocolos experimentais foram aprovados pelo comite de etica da UFS (CEPA/UFS: 35/12). Os resultados foram expressos como media } erro padrao da media e as diferencas entre os grupos foram analisadas por meio do teste de variancia ANOVA, uma via ou duas vias, seguido pelo teste de Tukey ou Bonferroni. Valores de p < 0,05 foram considerados estatisticamente significantes. Na revisao sistematica foram encontrados 27 artigos sobre modificacao estrutural de terpenos e atividade anti-inflamatoria. Na parte experimental, a administracao do CP produziu uma reducao significativa (p < 0,01 ou 0,001) no teste da nocicepcao induzida por formalina, em ambas as fases do teste. No teste da placa quente, o tempo de reacao aumentou significativamente nas doses de 50 e 100 mg/kg (p < 0,05; 0,01 ou 0,001). O CP tambem foi capaz de inibir o desenvolvimento da hiperalgesia mecanica induzida por todos os agentes testados (p < 0,05; 0,01 ou 0,001). Na avaliacao da atividade anti-inflamatoria, o tratamento com CP causou uma diminuicao significativa (p < 0,001) no numero total de leucocitos, diminuindo os niveis de TNF- ¿ (p < 0,001), IL-1 À (p < 0,05) e extravasamento de proteinas (p < 0,01). Alem disso, o edema de pata induzido por CG tambem foi inibido pelo CP (p < 0,05; 0,01 ou 0,001). Desta forma, conclui-se que o CP possui atividade antinociceptiva, anti-hiperalgesica e anti-inflamatoria, provavelmente por inibicao de citocinas. Dessa maneira, a modificacao estrutural em terpeno pode ser uma alternativa interessante para obtencao de moleculas com propriedades farmacologicas.

It has always been a challenge to imitate the lung environment, therefore there is a constant development of standardized in vitro dissolution methods for inhaled products. Dissolution in vitro has been considered as an important parameter, because low solubility determines the bioavailability of inhaled drugs. The in vitro dissolution data generated by the dissolution test experiment can be correlated with in vivo pharmacokinetic data through in vitro-in vivo correlation (IVIVC), because a completed predictive IVIVC model is very useful for drug formulation design and manufacturing changes after approval. The aim of this study was to investigate the effect of the membrane on the dissolution profile of orally inhaled drugs with different solubility, Budesonide (BUD) and Fluticasone propionate (FP) in the different pore sizes of the membrane 8.0 μm, 3.0 μm and 0.4 μm. The method in this study builds on previous dissolution methods, a Transwell® setup to dissolve the drugs with a small amount of dissolution medium, which mimics more the limited lung fluid capacity in vivo. In order to collect the dose from the drugs, Andersen Cascade Impact was used. The dissolution rate of BUD was first in the ranking in all of the pore sizes in the membrane.

Einleitung: Bei Milchkühen häufen sich Erkrankungen in der Frühlaktation. Sie gehören zu den wichtigsten Ursachen frühzeitiger Merzung und damit der aktuell unbefriedigenden Nutzungsdauer. Ziele der Untersuchungen: Ziel dieser Arbeit war es, den Stoffwechsel von Milchkühen in der kritischen Übergangszeit vom Trockenstehen zur Laktation (Transitphase) durch drei verschiedene prophylaktische Maßnahmen zu stabilisieren: mittels Huminsäuren Belastungen aus dem Darm einschließlich Endotoxinen zu mindern, mit einem Ammoniumpropionat-Propylenglykol- Gemisch die Energieversorgung zu verbessern sowie mit Dexamethason-21-isonicotinat die Stoffwechselfunktion der Leber zu fördern sowie gleichzeitig Entzündungsprozesse infolge der Kalbung zu hemmen. Materialien und Methoden: Die Untersuchungen wurden in einem sächsischen Bestand an 312 Kühen der Rasse „Holstein Friesian“ randomisiert innerhalb eines Jahres durchgeführt. An jeweils 78 Kühe wurden 300 ml Ammoniumpropionat-Propylenglykol-Gemisch(C3) täglich vom 14. Tag ante partum (a.p.) bis zum 14. Tag post partum (p.p.) oral verabreicht; ebenfalls oral wurden 100 g Huminsäure-Fertigpräparat (HS-FP) bzw. 50 g Huminsäuren-Rohstoff (HS-RS) im selben Zeitraum appliziert, und Dexamethason-21-isonicotinat (DEXA21) wurde einmalig am 1. Tag p.p. intramuskulär in der Dosierung 0,02 mg/kg Körpermasse verabreicht. 78 unbehandelte Kühe dienten als Kontrollgruppe. Die Auswirkungen dieser Maßnahmen auf Gesundheit, Leistung und Stoffwechsel wurden durch klinische Untersuchungen, durch Blutkontrollen am 14. Tag a.p., am 3. und 28. Tag p.p. (Leukozyten, freie Fettsäuren [FFS], Bilirubin, ß-0H-Butyrat[BHB], Glucose, Cholesterol, Creatinkinase [CK], Aspartat-Amino-Transferase [ASAT], Glutamat-Dehydrogenase [GLDH], gamma-Glutaryl-Transferase [GGT], Protein, Albumin, Mg, Fe, Ca, anorganisches Phosphat [Pi], Na, K) sowie durch die Erfassung von Gesundheitsstatus, Milchleistung und Fruchtbarkeit zu bestimmten Zeitpunkten geprüft. Ergebnisse: Die verschiedenen prophylaktischen Maßnahmen hatten keinen signifikanten Einfluss auf Fruchtbarkeits- und Gesundheitsparameter. Bei den absoluten und fettkorrigierten Milchmengen konnten ebenfalls keine statistisch gesicherten Unterschiede zwischen den Versuchsgruppen und der Kontrollgruppe festgestellt werden. Der Milcheiweißgehalt von C3 28 d p.p. sowie der Milchfettgehalt von DEXA21 und C3 100 d p.p. waren signifikant erhöht. Die Ergebnisse der Blutuntersuchungen ergaben hauptsächlich am 3., aber auch am 28. Tag p.p. gesicherte Unterschiede bei wichtigen Stoffwechselparametern wie Glucose, Cholesterol, Bilirubin, Protein, Albumin, Ca, Fe und CK. Die einmalige Gabe von Dexamethason-21-isonicotinat am 1. Tag p.p. hatte den besten Einfluss auf den Leber- und Energiestoffwechsel. In dieser Gruppe waren am 3. Tag p.p. die Glucose-, Bilirubin-, Cholesterol-, Protein, Ca- und Fe-Konzentrationen sowohl gegenüber der KG wie auch gegenüber allen anderen Versuchsgruppen signifikant günstiger. Für die Albumin- und Na-Konzentrationen sowie die CK-Aktivität traf das gegenüber der Kontroll- sowie der C3-Gruppe zu. Der Einsatz der Wirkstoffe mit HS-RS, HS-FP sowie C3 führte ebenfalls zu positiven Effekten auf die Leistung und den Stoffwechsel gegenüber der Kontrollgruppe, jedoch ließen sich diese nur in wenigen Fällen statistisch sichern. Schlussfolgerungen: Die Applikation von Dexamethason-21-isonicotinat einen Tag p.p. stabilisiert signifikant den Stoffwechsel von Kühen nach dem Partus. Gleichartige Effekte auf Milch- und Fruchtbarkeitsleitung sowie die Morbidität konnten nicht gesichert nachgewiesen werden. Für Huminsäure-Rohstoff, Huminsäure-Fertigpräparat sowie Ammoniumpropionat-Propylenglykol-Gemisch waren solche Effekte tendenziell erkennbar, statistisch aber nicht zu sichern. Auch wenn besonders mit Dexamethason-21-isonicotinat der Stoffwechsel in Belastungssituationen kurzfristig stabilisiert werden kann, müssen generell Haltung und Fütterung analysiert sowie Mängel beseitigt werden.:Inhaltsverzeichnis Inhaltsverzeichnis .I Abkürzungsverzeichnis IV 1 Einleitung .......................................................................................... 1 2 Literaturübersicht ............................................................................. 3 2.1 Stoffwechsel der Milchkuh im geburtsnahen Zeitraum ....................... 3 2.2 Bovine Ketose .................................................................................... 5 2.3 Fettmobilisationssyndrom ................................................................... 7 2.4 Möglichkeiten der Stabilisierung des Stoffwechsels der Milchkuh im geburtsnahen Zeitraum ...................................................................... 9 2.4.1 Allgemeines zur Stoffwechselstabilisierung ........................................ 9 2.4.2 Energiereiche C3-Verbindungen ...................................................... 11 2.4.2.1 Propionat .......................................................................................... 12 2.4.2.2 Propylenglykol .................................................................................. 14 2.4.2.3 Ammoniumpropionat-Propylenglykol-Gemisch ................................ 15 2.4.3 Huminsäuren .................................................................................... 16 2.4.3.1 Einsatz, Vorkommen, Aufbau ........................................................... 16 2.4.3.2 Effekte .............................................................................................. 16 2.4.3.3 Wirkungsweise im Organismus ........................................................ 17 2.4.3.4 Anwendungen in der Veterinärmedizin ............................................. 18 2.4.3.5 Huminsäurenpräparate ..................................................................... 20 2.4.4 Glukokortikoide................................................................................. 21 2.4.4.1 Aufbau .............................................................................................. 21 2.4.4.2 Wirkungsweise ................................................................................. 21 2.4.4.3 Effekte .............................................................................................. 22 2.4.4.4 Dexamethason-21-isonicotinat ......................................................... 25 3 Tiere, Material und Methoden ........................................................ 27 3.1 Untersuchte Tiere, Betrieb, Fütterung .............................................. 27 3.2 Versuchsanordnung, Gruppeneinteilung .......................................... 28 3.3 Entnahme, Aufbereitung und Aufbewahrung der Blutproben ........... 30 3.4 Bestimmung der Blutparameter, Referenzbereiche ......................... 31 3.4.1 Bestimmung der Leistungs-, Gesundheits- und Fruchtbarkeitsparameter .................................................................. 33 3.5 Statistische Prüfung der ermittelten Daten ....................................... 35 4 Ergebnisse ...................................................................................... 36 4.1 Methodische Aspekte ....................................................................... 36 4.1.1 Wertung der Untersuchungsergebnisse kranker und selektierter Kühe ................................................................................................ 36 4.1.2 Akzeptanz der verabreichten Futterzusatzstoffe .............................. 37 4.2 Klinische Befunde ............................................................................. 38 4.3 Leistungsparameter .......................................................................... 41 4.3.1 Milchleistung .................................................................................... 41 4.3.2 Fruchtbarkeit .................................................................................... 44 4.4 Labordiagnostische Parameter......................................................... 45 4.4.1 Energie-Fett-Leberstoffwechsel ....................................................... 45 4.4.1.1 Glucose ............................................................................................ 45 4.4.1.2 Cholesterol ....................................................................................... 47 4.4.1.3 Bilirubin ............................................................................................ 48 4.4.1.4 Beta-Hydroxy-Butyrat ....................................................................... 49 4.4.1.5 Freie Fettsäuren ............................................................................... 50 4.4.1.6 Aspartat-Amino-Transferase ............................................................ 51 4.4.1.7 Gamma-Glutamyl-Transferase ......................................................... 52 4.4.1.8 Glutamat-Dehydrogenase ................................................................ 53 4.4.2 Eiweißstoffwechsel ........................................................................... 54 4.4.2.1 Gesamtprotein .................................................................................. 54 4.4.2.2 Albumin ............................................................................................ 55 4.4.3 Mineralstoff- und Spurenelementstoffwechsel .................................. 56 4.4.3.1 Natrium ............................................................................................. 56 4.4.3.2 Kalium .............................................................................................. 57 4.4.3.3 Calcium ............................................................................................ 58 4.4.3.4 anorganisches Phosphat .................................................................. 59 4.4.3.5 Magnesium ....................................................................................... 60 4.4.3.6 Eisen ................................................................................................ 61 4.4.4 Muskelstoffwechsel .......................................................................... 62 4.4.4.1 Kreatinkinase ................................................................................... 62 4.4.5 Leukozyten ....................................................................................... 63 5 Diskussion ...................................................................................... 64 5.1 Klinische Parameter ......................................................................... 64 5.1.1 Morbidität ......................................................................................... 64 5.1.2 Milchleistung .................................................................................... 67 5.1.3 Fruchtbarkeit .................................................................................... 70 5.2 Klinisch-chemische Parameter, Stoffwechsel ................................... 71 5.2.1 Wirkung von Huminsäuren auf den Stoffwechsel ............................. 71 5.2.2 Wirkung einer energiereichen C3-Verbindung auf den Stoffwechsel 71 5.2.3 Wirkung von Dexamethason-21-isonicotinat auf den Stoffwechsel .. 74 6 Zusammenfassung ......................................................................... 83 7 Summary ......................................................................................... 85 8 Literaturverzeichnis ....................................................................... 87 / Problem: In dairy cattle diseases are common in early lactation. They are among the main causes of early culling and the current unsatisfactory productive life. Objective: The aim of this work was to stabilize metabolism of dairy cows in the critical transition period from standing dry to lactation by three different prophylactic applications: using humic acids to minimize strain from the gut including endotoxins, using ammonium propionate mixed with propylene glycol to improve energy supply and dexamethasone-21-isonicotinate to promote metabolic function of the liver and at the same time to inhibit inflammatory processes following parturition. Experimental design: The studies were performed in a Saxon dairy farm on 312 cows of the „Holstein Friesian\" breed, randomly performed within one year. 78 cows were administered orally 300 ml ammonium propionate mixed with propylene glycol (C3) daily from 14 days before parturition (a.p.) to 14 days after parturition (p.p.), another 78 cows 100 g of a humic acid drug (HS-FP) or 50 g of humic acid raw material (HS-RS) were administered orally in the same period and dexamethasone-21-isonicotinate (DEXA21) was applied intramuscularly to another 78 cows on the first day p.p. in a dose of 0.02 mg/kg body weight. 78 untreated cows were used as control group. The impact of these administrations on health, performance and metabolism has been measured by clinical examinations and blood tests on 14. day a.p., on 3. and 28. day p.p. (Leukocytes, free fatty acids [ FFS ], bilirubin, beta-0H-butyrate [BHB] , glucose, cholesterol, creatine kinase [CK], aspartate aminotransferase [AST], glutamate dehydrogenase [GLDH], gamma glutaryl transferase [GGT], protein, albumin, Mg, Fe, Ca, inorganic phosphate [Pi] , Na, K) and was verified by detection of health status, milk yield and fertility. Results: The different prophylactic administrations had no significant effect on fertility and health parameters. The absolute and fat- corrected milk yields also showed no statistically reliable differences between experimental groups and control group. Milk protein content in C3 28 days p.p. and milk fat content in DEXA21 and C3 100 days p.p. were significantly increased. Blood control results showed mainly on 3. and 28. day p.p. important differences in metabolic parameters, such as glucose, cholesterol, bilirubin, protein, albumin, Ca, Fe and CK, which are statistically secured. A single dose of dexamethasone-21- isonicotinate on first day p.p. had the best effect on liver and energy metabolism. Three days p.p. glucose, bilirubin, cholesterol, protein, Ca and Fe concentrations performed significantly better in DEXA21 group compared both to control group and all other treatment groups. For albumin and Na concentrations and CK activity that was true with respect to control and C3 group. The use of a humic acid drug, humic acid raw material and ammonium propionate mixed with propylene glycol had positive impact on performance and metabolism compared with control group too, but could be statistically secured in only a few cases. Conclusions: The application of dexamethasone-21-isonicotinate at the first day p.p. significantly stabilizes metabolism in cows after parturition. Similar effects on milk yield and fertility as well as morbidity could not be observed. For humic acid drug, humic acid raw material and ammonium propionate mixed with propylene glycol such effects tended to be recognizable, but cannot be statistically secured. Metabolism can be stabilized in short term stress situations with dexamethasone-21-isonicotinate, general care and feeding must be analyzed and deficiencies have to be eliminated.:Inhaltsverzeichnis Inhaltsverzeichnis .I Abkürzungsverzeichnis IV 1 Einleitung .......................................................................................... 1 2 Literaturübersicht ............................................................................. 3 2.1 Stoffwechsel der Milchkuh im geburtsnahen Zeitraum ....................... 3 2.2 Bovine Ketose .................................................................................... 5 2.3 Fettmobilisationssyndrom ................................................................... 7 2.4 Möglichkeiten der Stabilisierung des Stoffwechsels der Milchkuh im geburtsnahen Zeitraum ...................................................................... 9 2.4.1 Allgemeines zur Stoffwechselstabilisierung ........................................ 9 2.4.2 Energiereiche C3-Verbindungen ...................................................... 11 2.4.2.1 Propionat .......................................................................................... 12 2.4.2.2 Propylenglykol .................................................................................. 14 2.4.2.3 Ammoniumpropionat-Propylenglykol-Gemisch ................................ 15 2.4.3 Huminsäuren .................................................................................... 16 2.4.3.1 Einsatz, Vorkommen, Aufbau ........................................................... 16 2.4.3.2 Effekte .............................................................................................. 16 2.4.3.3 Wirkungsweise im Organismus ........................................................ 17 2.4.3.4 Anwendungen in der Veterinärmedizin ............................................. 18 2.4.3.5 Huminsäurenpräparate ..................................................................... 20 2.4.4 Glukokortikoide................................................................................. 21 2.4.4.1 Aufbau .............................................................................................. 21 2.4.4.2 Wirkungsweise ................................................................................. 21 2.4.4.3 Effekte .............................................................................................. 22 2.4.4.4 Dexamethason-21-isonicotinat ......................................................... 25 3 Tiere, Material und Methoden ........................................................ 27 3.1 Untersuchte Tiere, Betrieb, Fütterung .............................................. 27 3.2 Versuchsanordnung, Gruppeneinteilung .......................................... 28 3.3 Entnahme, Aufbereitung und Aufbewahrung der Blutproben ........... 30 3.4 Bestimmung der Blutparameter, Referenzbereiche ......................... 31 3.4.1 Bestimmung der Leistungs-, Gesundheits- und Fruchtbarkeitsparameter .................................................................. 33 3.5 Statistische Prüfung der ermittelten Daten ....................................... 35 4 Ergebnisse ...................................................................................... 36 4.1 Methodische Aspekte ....................................................................... 36 4.1.1 Wertung der Untersuchungsergebnisse kranker und selektierter Kühe ................................................................................................ 36 4.1.2 Akzeptanz der verabreichten Futterzusatzstoffe .............................. 37 4.2 Klinische Befunde ............................................................................. 38 4.3 Leistungsparameter .......................................................................... 41 4.3.1 Milchleistung .................................................................................... 41 4.3.2 Fruchtbarkeit .................................................................................... 44 4.4 Labordiagnostische Parameter......................................................... 45 4.4.1 Energie-Fett-Leberstoffwechsel ....................................................... 45 4.4.1.1 Glucose ............................................................................................ 45 4.4.1.2 Cholesterol ....................................................................................... 47 4.4.1.3 Bilirubin ............................................................................................ 48 4.4.1.4 Beta-Hydroxy-Butyrat ....................................................................... 49 4.4.1.5 Freie Fettsäuren ............................................................................... 50 4.4.1.6 Aspartat-Amino-Transferase ............................................................ 51 4.4.1.7 Gamma-Glutamyl-Transferase ......................................................... 52 4.4.1.8 Glutamat-Dehydrogenase ................................................................ 53 4.4.2 Eiweißstoffwechsel ........................................................................... 54 4.4.2.1 Gesamtprotein .................................................................................. 54 4.4.2.2 Albumin ............................................................................................ 55 4.4.3 Mineralstoff- und Spurenelementstoffwechsel .................................. 56 4.4.3.1 Natrium ............................................................................................. 56 4.4.3.2 Kalium .............................................................................................. 57 4.4.3.3 Calcium ............................................................................................ 58 4.4.3.4 anorganisches Phosphat .................................................................. 59 4.4.3.5 Magnesium ....................................................................................... 60 4.4.3.6 Eisen ................................................................................................ 61 4.4.4 Muskelstoffwechsel .......................................................................... 62 4.4.4.1 Kreatinkinase ................................................................................... 62 4.4.5 Leukozyten ....................................................................................... 63 5 Diskussion ...................................................................................... 64 5.1 Klinische Parameter ......................................................................... 64 5.1.1 Morbidität ......................................................................................... 64 5.1.2 Milchleistung .................................................................................... 67 5.1.3 Fruchtbarkeit .................................................................................... 70 5.2 Klinisch-chemische Parameter, Stoffwechsel ................................... 71 5.2.1 Wirkung von Huminsäuren auf den Stoffwechsel ............................. 71 5.2.2 Wirkung einer energiereichen C3-Verbindung auf den Stoffwechsel 71 5.2.3 Wirkung von Dexamethason-21-isonicotinat auf den Stoffwechsel .. 74 6 Zusammenfassung ......................................................................... 83 7 Summary ......................................................................................... 85 8 Literaturverzeichnis ....................................................................... 87

Today, orally inhaled drugs found on the market suffer from variable and discontinuous pulmonary drug release which lowers efficacy and patience compliance. This is usually a consequence of the poor understanding of the interaction and dissolution behavior of drug particles in the lung environment. Thus, the aim of this project was to investigate the effect of the dissolution medium on dissolution profiles for the well-known orally inhaled drug budesonide (BD) and fluticasone propionate (FP), in order to assess the importance of a proper selection of dissolution media for in vitro dissolution methods. In order to achieve this a modified Andersen Cascade Impactor was used to simulate deposition of particles onto filters. The dissolution was measured using a Transwell set up with polycarbonate membranes that can hold the filters with the deposited drug on it. Different media were prepared, from simple to more biorelevant. The samples taken during the dissolution experiments were analyzed quantitatively using UPLC-UV and the experimental data was processed by fitting to the Weibull function. The aim of this project was successfully achieved and the dissolution media that worked best for both BD and FP was PBS with the addition of 0.5% SDS. On the other hand, the dissolution media that performed the least for both BD and FP was the simulated lung fluid (SLF) with presence of 0.02% (w/v) DPPC. This may be due to the fact that DPPC forms liposomal aggregates which probably results in the media becoming more viscous and hence the dissolution time becomes slower.

With the advent of industrialization and food processing techniques the sizes of the cereal bran have been drastically reduced. In my thesis, I have tested the effect, if any, of wheat bran and maize bran particle size, in vitro, on the gut microbiota community structure by 16S rRNA sequencing and their function, by Short chain fatty acids (acetate, propionate, butyrate) production. In turn, we also linked the microbiota and SCFA differences to different chemical composition amongst variously sized fractions of wheat and maize bran.

Three-dimensional structures of proteins provide insights into the mechanisms of macromolecular assembly, enzyme catalysis and mode of activation, substrate-specificity, ligand-binding properties, stability and dynamical features. X-ray crystallography has become the method of choice in structural biology due to the remarkable methodological advances made in the generation of intense X-ray beams with very low divergence, cryocooling methods to prolong useful life of irradiated crystals, sensitive methods of Xray diffraction data collection, automated and fast methods for data processing, advances and automation in methods of computational crystallography, comparative analysis of macromolecular structures along with parallel advances in biochemical and molecular biology methods that allow production of the desired biomolecule in quantities sufficient for X-ray diffraction studies. Advances in molecular biology techniques and genomic data have helped in identifying metabolic pathways responsible for metabolism of short-chain fatty acids (SCFAs). The primary objective of this thesis is application of crystallographic techniques for understanding the structure and function of enzymes involved in the metabolism of SCFAs in S. typhimurium. Pathways chosen for the present study are (i) propionate degradation to pyruvate and succinate by 2-methylcitrate pathway involving gene products of the prp operon, (ii) acetate activation to acetyl-CoA by AckA-Pta pathway involving gene products of the ack-pta operon, (iii) threonine degradation to propionate involving gene products of the tdc operon, (iv) 1,2-propanediol (1,2-PD) degradation to propionate involving gene products of the pdu operon. These metabolic pathways utilize a large number of enzymes with diverse catalytic mechanisms. The main objectives of the work include structural and functional studies on 2-methycitrate synthase (PrpC), acetate kinase (AckA), propionate kinase isoforms (PduW and TdcD) and propanol dehydrogenase (PduQ) from S. typhimurium. In the present work, these proteins were cloned, expressed, purified and characterized. The purified proteins were crystallized using standard methods. The crystals were placed in an X-ray beam and diffraction data were collected and used for the elucidation of structure of the proteins. The structures were subjected to rigorous comparative analysis and the results were complemented with suitable biochemical and biophysical experiments. The thesis begins with a review of the current literature on SCFAs metabolism in bacteria, emphasizing studies carried out on S. typhimurium and the closely related E. coli as well as organisms for which the structure of a homologue has been determined (Chapter 1). Metabolic pathways involving acetate utilization by activation to acetyl- CoA, propionate degradation to pyruvate and succinate, anaerobic degradation of Lthreonine to propionate and, 1,2-PD degradation to propionate are described in this chapter. Common experimental and computational methods used during the course of investigations are described in Chapter 2, as most of these are applicable to all structure determinations and analyses. Experimental procedures described here include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and structural analysis. In Chapter 3, X-ray crystal structure of S. typhimurium 2-methylcitrate synthase (StPrpC; EC 2.3.3.5) determined at 2.4 Å resolution and its functional characterization is reported. StPrpC catalyzes aldol-condensation of oxaloacetate and propionyl-CoA to 2- methylcitrate and CoA in the second step of 2-methylcitrate pathway. StPrpC forms a dimer in solution and utilizes propionyl-CoA more efficiently than acetyl-CoA or butyryl- CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might be responsible for the specificities for acyl-CoAs of these enzymes. Structural comparison with the ligand free (open) and bound (closed) states of CSs showed that StPrpC represents the first apo structure among xvi CS homologs in a nearly closed conformation. StPrpC molecules were organized as decamers, composed of five identical dimer units, in the P1 crystal cell. Higher order oligomerization of StPrpC is likely to be due to high pH (9.0) of the crystallization condition. In gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural and functional studies on S. typhimurium acetate kinase (StAckA; EC 2.7.2.1) are described in Chapter 4. Acetate kinase, an enzyme widely distributed in the bacteria and archaea domains, catalyzes the reversible phosphoryl transfer from ATP to acetate in the presence of a metal ion during acetate metabolism. StAckA catalyzes Mg2+ dependent phosphate transfer from ATP to acetate 10 times more efficiently when compared to propionate. Butyrate was found to inhibit the activity of the enzyme. Kinetic analysis showed that ATP and Mg2+ could be effectively substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. The X-ray crystal structure of StAckA was determined in two different forms at 2.70 Å (Form-I) and 1.90 Å (Form-II) resolutions, respectively. StAckA contains a fold with the topology βββαβαβα, similar to those of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin. StAckA consists of two domains with an active site cleft at the domain interface. Comparison of StAckA structure with those of ligand complexes of other acetokinase family proteins permitted the identification of residues essential for substrate binding and catalysis. Conservation of most of these residues points to both structural and mechanistic similarities between enzymes of this family. Examination of the active site pocket revealed a plausible structural rationale for the greater specificity of the enzyme towards acetate than propionate. Intriguingly, a major conformational reorganization and partial disorder in a large segment consisting of residues 230-297 of the polypeptide was observed in Form-II. Electron density corresponding to a plausible xvii citrate was observed at a novel binding pocket present at the dimeric interface. Citrate bound at this site might be responsible for the observed disorder in the Form-II structure. A similar ligand binding pocket and residues lining the pocket were also found to be conserved in other structurally known enzymes of acetokinase family. These observations and examination of enzymatic reaction in the presence of citrate and succinate (tricarboxylic acid cycle intermediates) suggested that binding of ligands at this pocket might be important for allosteric regulation in this family of enzymes. Propionate kinase (EC 2.7.2.15) catalyzes reversible conversion of propionylphosphate and ADP to propionate and ATP. S. typhimurium possess two isoforms of propionate kinase, PduW and TdcD, involved in 1,2-propanediol degradation to propionate and in L-threonine degradation to propionate, respectively. In Chapter 5, structural and functional analyses of PduW and TdcD, carried out to gain insights into the substrate-binding pocket and catalytic mechanism of these enzymes, are described. Both isoforms showed broad specificity for utilization of SCFAs (propionate > acetate), nucleotides (ATP ≈ GTP > UTP > CTP) and metal ions (Mg2+ ≈ Mn2+). Molecular modeling of StPduW indicated that the enzyme is likely to adopt a fold similar to other members of acetokinase family. The residues at the active site are well conserved. Differences in the size of hydrophobic pocket where the substrate binds, particularly the replacement of a valine residue in acetate kinases (StAckA: Val93) by an alanine in propionate kinases (StPduW: Ala92; StTdcD: Ala88), could account for the observed greater affinity towards their cognate SCFAs. Crystal structures of TdcD from S. typhimurium in complex with various nucleotides were determined using native StTdcD as the phasing model. Nucleotide complexes of StTdcD provide a structural rationale for the broad specificity of the enzyme for its cofactor. Binding of ethylene glycol close to the γ-phosphate of GTP might suggest a direct in-line transfer mechanism. The thesis concludes with a brief discussion on the future prospects of the work. xviii Projects carried out as part of Master of Science projects and as additional activity during the course of the thesis work are described in three appendices. Analysis of the genomic sequences of E. coli and S. typhimurium has revealed the presence of hpa operon essential for 4-hydroxyphenylacetate (4-HPA) catabolism. S. typhimurium hpaE gene encodes for a 55 kDa polypeptide (StHpaE; EC 1.2.1.60) which catalyzes conversion of 5-carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) to 5-carboxymethyl-2-hydroxymuconic aldehyde (CHMA) in 4-HPA metabolism. Sequence analysis of StHpaE showed that it belongs to aldehyde dehydrogenase (ALDH) superfamily and possesses residues equivalent to the catalytic glutamate and cysteine residues of homologous enzymes (Appendix A). The gene was cloned in pRSET C expression vector and the recombinant protein was purified using Ni-NTA affinity chromatography. The enzyme forms a tetramer in solution and shows catalytic activity toward the substrate analog adipic semialdehyde. Crystal structure of StHpaE revealed that it contains three domains; two dinucleotide-binding domains, a Rossmann-fold type domain, and a small three-stranded β-sheet domain, which is involved in tetrameric interactions. NAD+-bound crystal of StHpaE permitted identification of active site pocket and residues important for ligand anchoring and catalysis. Mutarotases or aldose 1-epimerases (EC 5.1.3.3) play a key role in carbohydrate metabolism by catalyzing the interconversion of α- and β-anomers of sugars. S. typhimurium YeaD (StYeaD), annotated as aldose 1-epimerase, has very low sequence identity with other well characterized mutarotases. In Appendix B, the crystal structure of StYeaD determined in orthorhombic and monoclinic crystal forms at 1.9 Å and 2.5 Å resolutions, respectively are reported. StYeaD possesses a fold similar to those of galactose mutarotases (GalMs). Structural comparison of StYeaD with GalMs has permitted identification of residues involved in catalysis and substrate anchoring. In spite xix of the similar fold and conservation of catalytic residues, minor but significant differences in the substrate binding pocket were observed compared to GalMs. Therefore, the substrate specificity of YeaD like proteins seems to be distinct from those of GalMs. Pepper Vein Banding Virus (PVBV) is a member of the genus potyvirus and infects Solanaceae plants. PVBV is a single-stranded positive-sense RNA virus with a genome-linked viral protein (VPg) covalently attached at the 5'-terminus. In order to establish the role of VPg in the initiation of replication of the virus, recombinant PVBV VPg was over-expressed in E. coli and purified using Ni-NTA affinity chromatography (Appendix C). PVBV NIb was found to uridylylate Tyr66 of VPg in a templateindependent manner. Studies on N- and C-terminal deletion mutants of VPg revealed that N-terminal 21 and C-terminal 92 residues of PVBV VPg are dispensable for in vitro uridylylation by PVBV NIb.

The cell wall provides a complex and rigid structure to the plant for support, protection from environmental factors, and transport. It is mainly composed of polysaccharides, proteins, and lignin. Arabinoxylan (AX), pectin (P), and cellulose (C) are the main components of cereal cell walls and are particularly concentrated in the bran portion of the grain. Cereal arabinoxylans create networks in plant cell walls in which other cell wall polysaccharides are imbedded forming complex matrices. These networks give an insolubility profile to plant cell wall. A previous study in our lab showed that soluble crosslinked arabinoxylan with relatively high residual ferulic acid from corn bran provided advantageous <i>in vitro </i>human fecal fermentation products and promoted butyrogenic gut bacteria. In the present work, arabinoxylan was isolated from corn bran with a mild sodium hydroxide concentration to keep most of its ferulic acid content. Highly ferulated corn bran arabinoxylan was crosslinked to create an insoluble network to mimic the cereal grain cell wall matrices. Firstly, arabinoxylan film (Cax-F), pectin film (P-F), the film produced by embedding pectin into arabinoxylan networks (CaxP-F), and cellulose embedding arabinoxylan matrices (CaxC-F), and embedding the mixture of cellulose and pectin into arabinoxylan networks (CaxCP-F) were fabricated into simulated plant cell wall materials. Water solubility of films in terms of monosaccharide content was examined and revealed that Cax-F was insoluble, and P-F was partially insoluble, and nanosized pectin and cellulose were partially entrapped inside the crosslinked-arabinoxylan matrices. In a further study, these films were used in an <i>in vitro </i>human fecal fermentation assay to understand how gut microbiota access and utilize the different simulated plant cell walls to highlight the role of each plant cell wall component during colonic fermentation. <i>In vitro </i>fecal samples, obtained from three healthy donors were used to ferment the films (Cax-F, P-F, CaxP-F, CaxC-F, and CaxCP-F) and controls (free form of cell wall components -Cax, P and C). The fabricated films that were compositionally similar to cell walls were fermented more slowly than the free polysaccharides (Cax and P). Besides, CaxP-F produced the highest short chain fatty acids (SCFA) amount among the films after 24 hour <i>in vitro </i>fecal fermentation. Regarding specific SCFA, butyrate molar ratio of all films was significantly higher than the free, soluble Cax and P. 16S rRNA gene sequencing explained the differences of the butyrate proportion derived from specific butyrogenic bacteria. Particularly, some bacteria, especially in a butyrogenic genera from Clostridium cluster XIVa, were increased in arabinoxylan films forms compared to the native free arabinoxylan polysaccharide. However, no changes were observed between P and P-F in terms of both end products (SCFA) and microbiota compositions. Moreover, CaxP-F promoted the butyrogenic bacteria in fecal samples compared with pectin alone, arabinoxylan alone, and the arabinoxylan film. Differences in matrix insolubility of the film, which was high for the covalently linked arabinoxylan films, but low for the non-covalent ionic-linked pectin film, appears to play an important role in targeting Clostridial bacterial groups. Overall, the cell wall-like films were useful to understand which bacteria degrade them related to their physical form and location of the fiber polymers. This study showed how fabricated model plant cell wall films influence specificity and competitiveness of some gut bacteria and suggest that fabricated materials using natural fibers might be used for targeted support of certain gut bacteria and bacterial groups.

No description available.

Tuberkulose ist auch heute noch eine der bedrohlichsten Infektionskrankheiten weltweit, verantwortlich für über 1.5 Millionen Todesfälle jährlich. Diese „Erfolgsgeschichte“ ihres Erregers Mycobacterium tuberculosis ist dabei wesentlich durch einen extrem flexiblen Stoffwechsel bestimmt, der dem Bakterium das Wachstum unter den restriktiven Bedingungen der menschlichen Wirtszelle erlaubt. Diese Arbeit erkundet die Flexibilität des zentralen Kohlenstoffmetabolismus in Mykobakterien mit Hilfe mathematischer Modellierungsansätze, ergänzt durch die Integration von qualitativ hochwertigen experimentellen Daten. Ausgehend von einem Überblick über die metabolische Landschaft des zentralen Kohlenstoffmetabolismus, erhöht sich Schritt für Schritt die Detailtiefe bis hin zur genauen Analyse spezieller infektionsrelevanter metabolischer Wege. Die Verknüpfung des zentralen Kohlenstoffmetabolismus zu umgebenden Stoffwechsel- und Biosynthesewegen wird systematisch offen gelegt, als Voraussetzung für eine thermodynamische Charakterisierung des Systems, welche die Glykolyse als limitierenden Stoffwechselweg unter verschiedenen Wachstumsbedingungen charakterisiert. Basierend auf Protein- und Metabolitdaten im Fleißgleichgewicht, erlaubt eine neu vorgestellte Methode die Vorhersage regulatorischer Punkte für den metabolischen Übergang zwischen verschiedenen Kohlenstoffquellen. Abschließend wird mit Hilfe thermodynamisch-kinetischer Modellierung das Zusammenspiel zweier Stoffwechselwege mechanistisch erklärt, welche den robusten Abbau einer intrazellulären Kohlenstoffquelle ermöglichen. Durch die Entwicklung neuer Modellierungstechniken in Kombination mit hochauflösenden experimentellen Daten, trägt diese Arbeit zum besseren Verständnis der kontextabhängigen Flexibilität des mycobakteriellen Stoffwechsels bei, einem vielversprechenden Angriffspunkt für die Entwicklung neuer Medikamente gegen Tuberkulose. / Tuberculosis remains one of the major global health threats responsible for over 1.5 million deaths each year. This ’success story’ of the causative agent Mycobacterium tuberculosis is thereby closely linked to a flexible metabolism, allowing growth despite the restrictive conditions within the human host. In this thesis, the flexibility of the mycobacterial central carbon metabolism is explored by modeling approaches integrating high-quality experimental data. The analyses zoom in from a network based view to the detailed functionalities of individual, virulence relevant pathways. The interconnection of the central carbon metabolism to the remaining metabolic network is charted as a prerequisite to characterize its thermodynamic landscape, debunking glycolysis as bottleneck in different nutritional conditions. Based on steady state metabolomics and proteomics data, regulatory sites for the metabolic transition between different carbon sources are predicted by a novel method. Finally, the flexible interplay between two seemingly redundant pathways for the catabolism of an in vivo-like carbon source is explained mechanistically by means of thermodynamic-kinetic modeling. By employing novel modeling methods in combination with high-resolution experimental data, this work adds to the mechanistic understanding of the context dependent flexibility of mycobacterial metabolism, an important target for the development of novel drugs in the battle against tuberculosis.