Master of Science / Department of Animal Sciences and Industry / J. Ernest Minton / Enteric disease and immune challenge are processes that have detrimental effects on growth performance of young swine. The current study tested the hypothesis salmonellae-induced enteric disease would perturb the endocrine growth axis in a serovar dependent fashion. Specifically, we evaluated effects of Salmonella enterica serovar Typhimurium (Typhimurium) and serovar Choleraesuis (Choleraesuis) on critical regulatory components of growth in young swine. Weaned pigs were housed 2 per pen with ad libitum access to feed and water in a 14 d experiment. Pigs were then repeatedly fed either 10[superscript]8 CFU Choleraesuis or 10[superscript]8 Typhimurium in dough balls, with control pigs receiving dough without bacteria. Bacteria were re-fed twice weekly. Rectal temperatures were monitored daily from d 0 to7 and ADFI was measured through d 14. Pigs were weighed and samples of serum were obtained for circulating IGF-I on days 0, 7, and 14. At the conclusion of the study, samples of semitendinosus muscle and liver were obtained and subsequently assayed for IGF-I, IGFBP-3, and IGFBP-5 mRNA. Rectal temperatures were elevated in pigs given Choleraesuis pigs from d 2 through d 7 (P < 0.05) when compared to control pigs and pigs fed Typhimurium. Pigs receiving Choleraesuis had substantially decreased feed intake on days 2, 3, 4, 7, 8, 9, and 10 (P < 0.01), with a trend for reduction on d 5 (P = .08), and they experienced an approximate 25% reduction in BW compared to control and Typhimurium pigs by the conclusion of the study. Pigs given Choleraesuis also experienced marked reductions in circulating IGF-I on d 7 (P < 0.01 vs. control and Typhimurium) with reductions of lesser magnitude on d 14 (P = 0.07 vs. control and P < 0.05 vs. Typhimurium). Treatment tended to affect liver IGFBP-3 mRNA (P = 0.08), where expression tended to be elevated in Typhimurium and Choleraesuis pigs. In contrast, IGFBP-3 mRNA relative abundance was increased (P < 0.03) in pigs given Typhimurium versus control pigs. Muscle IGF-I mRNA was reduced in Choleraesuis pigs compared to control and Typhimurium (P < 0.05). Treatment tended to affect muscle IGFBP-3 mRNA (P = 0.10), where Choleraesuis had numerically less relative abundance than controls. Oral inoculation of growing pigs with Choleraesuis disrupted feed intake and BW gain, and this was accompanied by decreases in circulating IGF-I and reduced muscle expression of mRNA for IGF-I and IGFBP-3.

During the last decades the number of biologics increased dramatically and several biopharmaceutical drugs such as peptides, therapeutic proteins, hormones, enzymes, vaccines, monoclonal antibodies and antibody-drug conjugates conquered the market. Moreover, administration and local delivery of growth factors has gained substantial importance in the field of tissue engineering. Despite progress that has been made over the last decades formulation and delivery of therapeutic proteins is still a challenge. Thus, we worked on formulation and delivery strategies of therapeutic proteins to improve their biological performance. Phase I of this work deals with protein stability with the main focus on a liquid protein formulation of the dimeric fusion protein PR-15, a lesion specific platelet adhesion inhibitor. In order to develop an adequate formulation ensuring the stability and bioactivity of PR-15 during storage at 4 °C, a pH screening, a forced degradation and a Design of Experiments (DoE) was performed. First the stability and bioactivity of PR-15 in 50 mM histidine buffer in relation to pH was evaluated in a short-term storage stability study at 25 °C and 40 °C for 4 and 8 weeks using different analytical methods. Additionally, potential degradation pathways of PR-15 were investigated under stressed conditions such as heat treatment, acidic or basic pH, freeze-thaw cycles, light exposure, induced oxidation and induced deamidation during the forced degradation study. Moreover, we were able to identify the main degradation product of PR-15 by performing LC/ESI-MS analysis. Further optimization of the injectable PR 15 formulation concerning pH, the choice of buffer and the addition of excipients was studied in the following DoE and finally an optimal PR-15 formulation was found. The growth factors BMP-2, IGF-I and TGF-β3 were selected for the differentiation of stem cells for tissue engineering of cartilage and bone in order to prepare multifunctionalized osteochondral implants for the regeneration of cartilage defects. Silk fibroin (SF) was chosen as biomaterial because of its biocompatibility, mechanical properties and its opportunity for biofunctionalization. Ideal geometry of SF scaffolds with optimal porosity was found in order to generate both tissues on one scaffold. The growth factors BMP-2 and IGF-I were modified to allow spatially restricted covalent immobilization on the generated porous SF scaffolds. In order to perform site-directed covalent coupling by the usage of click chemistry on two opposite sides of the scaffold, we genetically engineered BMP-2 (not shown in this work; performed by Barbara Tabisz) and IGF-I for the introduction of alkyne or azide bearing artificial amino acids. TGF β3 was immobilized to beads through common EDC/NHS chemistry requiring no modification and distributed in the pores of the entire scaffold. For this reason protein modification, protein engineering, protein immobilization and bioconjugation are investigated in phase II. Beside the synthesis the focus was on the characterization of such modified proteins and its conjugates. The field of protein engineering offers a wide range of possibilities to modify existing proteins or to design new proteins with prolonged serum half-life, increased conformational stability or improved release rates according to their clinical use. Site-directed click chemistry and non-site-directed EDC/NHS chemistry were used for bioconjugation and protein immobilization with the aim to underline the preferences of site-directed coupling. We chose three strategies for the incorporation of alkyne or azide functionality for the performance of click reaction into the protein of interest: diazonium coupling reaction, PEGylation and genetic engineering. Azido groups were successfully introduced into SF by implementation of diazonium coupling and alkyne, amino or acid functionality was incorporated into FGF-2 as model protein by means of thiol PEGylation. The proper folding of FGF-2 after PEGylation was assessed by fluorescence spectroscopy, WST-1 proliferation assay ensured moderate bioactivity and the purity of PEGylated FGF-2 samples was monitored with RP-HPLC. Moreover, the modification of native FGF-2 with 10 kDa PEG chains resulted in enhanced thermal stability. Additionally, we genetically engineered one IGF-I mutant by incorporating the unnatural amino acid propargyl-L-lysine (plk) at position 65 into the IGF-I amino acid sequence and were able to express hardly verifiable amounts of plk-IGF-I. Consequently, plk-IGF-I expression has to be further optimized in future studies in order to generate plk-IGF-I with higher yields. Bioconjugation of PEGylated FGF-2 with functionalized silk was performed in solution and was successful for click as well as EDC/NHS chemistry. However, substantial amounts of unreacted PEG-FGF-2 were adsorbed to SF and could not be removed from the reaction mixture making it impossible to expose the advantages of click chemistry in relation to EDC/NHS chemistry. The immobilization of PEG-FGF-2 to microspheres was a trial to increase product yield and to remove unreacted PEG-FGF-2 from reaction mixture. Bound PEG-FGF-2 was visualized by fluorescence imaging or flow cytometry and bioactivity was assessed by analysis of the proliferation of NIH 3T3 cells. However, immobilization on beads raised the same issue as in solution: adsorption caused by electrostatic interactions of positively charged FGF-2 and negatively charged SF or beads. Finally, we were not able to prove superiority of site-directed click chemistry over non-site-directed EDC/NHS. The skills and knowledge in protein immobilization as well as protein characterization acquired during phase II helped us in phase III to engineer cartilage tissue in biofunctionalized SF scaffolds. The approach of covalent immobilization of the required growth factors is relevant because of their short in vivo half-lives and aimed at controlling their bioavailability. So TGF-β3 was covalently coupled by means of EDC/NHS chemistry to biocompatible and biostable PMMA beads. Herein, we directly compared bioactivity of covalently coupled and adsorbed TGF-β3. During the so-called luciferase assay bioactivity of covalent coupled as well as adsorbed TGF-β3 on PMMA beads was ensured. In order to investigate the real influence of EDC/NHS chemistry on TGF-β3’s bioactivity, the amount of immobilized TGF-β3 on PMMA beads was determined. Therefore, an ELISA method was established. The assessment of total amount of TGF-β3 immobilized on the PMMA beads allowed as to calculate coupling efficiency. A significantly higher coupling efficiency was determined for the coupling of TGF-β3 via EDC/NHS chemistry compared to the reaction without coupling reagents indicating a small amount of adsorbed TGF-β3. These results provide opportunity to determine the consequence of coupling by means of EDC/NHS chemistry for TGF β3 bioactivity. At first sight, no statistically significant difference between covalent immobilized and adsorbed TGF-β3 was observed regarding relative luciferase activities. But during comparison of total and active amount of TGF-β3 on PMMA beads detected by ELISA or luciferase assay, respectively, a decrease of TGF-β3’s bioactivity became apparent. Nevertheless, immobilized TGF β3 was further investigated in combination with SF scaffolds in order to drive BMSCs to the chondrogenic lineage. According to the results obtained through histological and immunohistochemical studies, biochemical assays as well as qRT-PCR of gene expression from BMSCs after 21 days in culture immobilized TGF-β3 was able to engineer cartilage tissue. These findings support the thesis that local presentation of TGF β3 is superior towards exogenous TGF β3 for the development of hyaline cartilage. Furthermore, we conclude that covalent immobilized TGF β3 is not only superior towards exogenously supplemented TGF-β3 but also superior towards adsorbed TGF-β3 for articular hyaline cartilage tissue engineering. Diffusion processes were inhibited through covalent immobilization of TGF-β3 to PMMA beads and thereby a stable and consistent TGF-β3 concentration was maintained in the target area. With the knowledge acquired during phase II and III as well as during the studies of Barbara Tabisz concerning the expression and purification of plk-BMP-2 we made considerable progress towards the formation of multifunctionalized osteochondral implants for the regeneration of cartilage defects. However, further studies are required for the translation of these insights into the development of multifunctionalized osteochondral SF scaffolds. / In den letzten Jahrzehnten stieg die Zahl der Biologika dramatisch an und mehrere biopharmazeutische Arzneimittel wie Peptide, therapeutische Proteine, Hormone, Enzyme, Impfstoffe, monoklonale Antikörper und Antikörper-Wirkstoff-Konjugate eroberten den Markt. Darüber hinaus hat die Applikation und lokale Verabreichung von Wachstumsfaktoren im Bereich des Tissue Engineerings eine wesentliche Bedeutung erlangt. Trotz der in den letzten Jahrzehnten erzielten Fortschritte ist die Formulierung und Verabreichung therapeutischer Proteine noch immer eine Herausforderung. Daher haben wir uns in dieser Arbeit mit der Formulierung und Verabreichung therapeutischer Proteine beschäftigt und Strategien entwickelt, um deren biologische Wirkung zu verbessern. In Phase I dieser Arbeit konzentrieren wir uns auf die Stabilität des dimeren Fusionsproteins PR 15, einem Inhibitor der Adhäsion von Plättchen an arterielle Gefäßläsionen. Um eine geeignete flüssige Formulierung zu entwickeln, welche die Stabilität und Bioaktivität von PR-15 während der Lagerung bei 4 °C sicherstellt, wurde ein pH Screening, eine Forced Degradation Studie und ein Design of Experiments (DoE) durchgeführt. Zuerst wurde die Stabilität und Bioaktivität von PR-15 bei verschiedenen pH Werten in 50 mM Histidinpuffer in einer Kurzzeitstabilitätsstudie bei 25 °C und 40 °C nach 4 und 8 Wochen mit Hilfe verschiedener analytischer Methoden beobachtet. Des Weiteren wurden mögliche Abbauwege von PR-15 unter Stressbedingungen wie erhöhter Temperatur, saurem oder basischem pH-Wert, Einfrier-Auftau-Zyklen, Lichteinwirkung, induzierter Oxidation sowie induzierter Deamidierung während der Forced Degradation Studie untersucht. Darüber hinaus konnten wir das Hauptabbauprodukt von PR-15 durch LC/ESI-MS Analysen identifizieren. Im folgenden DoE wurde die injizierbare PR-15 Formulierung weiter optimiert und bezüglich pH, der Wahl des Puffers sowie der Zugabe von Hilfsstoffen analysiert, bis letztendlich eine optimale PR 15-Formulierung gefunden wurde. Die Wachstumsfaktoren BMP-2, IGF-I und TGF-β3 wurden zur Differenzierung von Stammzellen für das Tissue Engineering von Knochen und Knorpel ausgewählt, um multifunktionalisierte osteochondrale Implantate zur Regeneration von Knorpeldefekten herzustellen. Seidenfibroin (SF) wurde aufgrund seiner Biokompatibilität, seiner mechanischen Eigenschaften und seiner Möglichkeiten zur Biofunktionalisierung als Biomaterial gewählt. Zuerst wurden SF-Scaffolds mit idealer Geometrie und optimaler Porosität erzeugt, um sowohl Knochen also auch Knorpel auf einem Scaffold herzustellen. Um eine räumlich begrenzte kovalente Immobilisierung der Wachstumsfaktoren BMP-2 und IGF-I auf den porösen SF-Scaffolds zu ermöglichen, wurden diese mit unnatürlichen Aminosäuren genetisch modifiziert. Das Einführen von Alkin- bzw. Azidresten in die Aminosäuresequenz von BMP-2 (in dieser Arbeit nicht gezeigt; von Barbara Tabisz durchgeführt) und IGF-I erlaubt unter Verwendung der Click-Chemie eine ortsgerichtete kovalente Kopplung der Wachstumsfaktoren an zwei gegenüberliegenden Seiten der Scaffolds. TGF-β3 wurde durch gewöhnliche EDC/NHS-Chemie, welche keine Modifikation erforderte, kovalent an Mikrosphären immobilisiert und in den Poren des gesamten SF-Scaffolds verteilt. Daher beschäftigen wir uns in Phase II mit der Modifikation von Proteinen, dem Protein Engineering, der Immobilisation von Proteinen und mit Biokonjugation. Neben der Synthese lag der Fokus auf der Charakterisierung modifizierter Proteine und deren Konjugaten. Das Gebiet des Protein Engineerings bietet eine Vielzahl von Möglichkeiten, bestehende Proteine zu modifizieren oder neue Proteine mit verlängerter Serumhalbwertszeit, erhöhter konformativer Stabilität oder verbesserten Freisetzungsraten entsprechend der klinischen Anwendung zu entwickeln. Die ortsspezifische Click-Chemie und die nicht-ortsspezifische EDC/NHS-Chemie wurden für die Biokonjugation und die Immobilisierung von Proteinen verwendet mit dem Ziel, die Vorzüge der ortsgerichteten Kopplung hervorzuheben. Für den Einbau der für die Durchführung der Click-Reaktion erforderlichen Alkin- bzw. Azidfunktionalität in das betreffende Protein wurden drei Strategien ausgewählt: die Azokupplung, die PEGylierung und die gentechnische Modifizierung. Azidgruppen wurden mittels Azokupplung erfolgreich in SF eingebaut und die Alkin-, Amino- oder Säurefunktionalität wurde mittels PEGylierung der Cysteine in das Modellprotein FGF-2 integriert. Die korrekte Faltung von FGF-2 nach erfolgreicher PEGylierung wurde durch Fluoreszenzspektroskopie bestätigt, im WST-1 Proliferationsassay wurde eine angemessene Bioaktivität festgestellt und die Reinheit von PEGylierten FGF-2 wurde mittels RP-HPLC analysiert. Darüber hinaus führte die Modifikation von nativem FGF-2 mit 10 kDa PEG-Ketten zu einer erhöhten thermischen Stabilität. Des Weiteren wurde ein IGF-I-Mutant gentechnisch hergestellt, indem die unnatürliche Aminosäure Propargyl-L-Lysin (Plk) an Position 65 in die IGF-I-Sequenz eingebaut wurde. Da letztendlich lediglich kaum nachweisbare Mengen an Plk-IGF-I exprimiert werden konnten, muss die Plk-IGF-I-Expression in anschließenden Studien weiter optimiert werden, um Plk-IGF-I mit höheren Ausbeuten erzeugen zu können. Die Biokonjugation von PEGyliertem FGF-2 und funktionalisierter Seide wurde sowohl mittels Click- als auch mittels EDC/NHS-Chemie erfolgreich durchgeführt. Allerdings wurden erhebliche Mengen PEG-FGF-2 lediglich an SF adsorbiert und nicht kovalent gekoppelt und konnten schlussendlich nicht aus dem Reaktionsgemisch entfernt werden. Die anschließende Immobilisierung von PEG-FGF-2 an Mikrosphären, war ein Versuch die Ausbeute der Reaktion zu erhöhen und adsorbiertes PEG-FGF-2 leichter zu entfernen. Immobilisiertes PEG-FGF-2 wurde mittels Fluoreszenzmikroskopie und/oder Durchflusszytometrie nachgewiesen und die Bioaktivität wurde durch die Analyse der Proliferation von NIH-3T3-Zellen ermittelt. Die Immobilisierung auf Mikrosphären führte jedoch zu demselben Problem wie in Lösung: Adsorption von positiv geladenem FGF-2 an negativ geladenes SF bzw. negativ geladenen Mikrosphären durch elektrostatische Wechselwirkungen. Schließlich waren wir nicht in der Lage, die Überlegenheit der ortsgerichteten Click-Chemie gegenüber der nicht-ortsgerichteten EDC/ NHS-Chemie zu beweisen. Die während Phase II erworbenen Fähigkeiten und Kenntnisse im Bereich der Immobilisierung und Charakterisierung von Proteinen halfen uns in Phase III Knorpelgewebe in biofunktionalisierten SF-Scaffolds zu erzeugen. Der Ansatz der kovalenten Immobilisierung, der für das Tissue Engineering von Knorpel erforderlichen Wachstumsfaktoren, ist aufgrund ihrer kurzen in vivo Halbwertszeiten von Bedeutung und zielt darauf ab, ihre Bioverfügbarkeit zu kontrollieren. So wurde TGF-β3 mittels EDC/NHS-Chemie kovalent an biokompatible und biostabile PMMA-Mikrosphären gekoppelt. Mit Hilfe des sogenannten Luciferase-Assays wurden die Bioaktivitäten von kovalent gekoppeltem sowie von adsorbiertem TGF-β3 auf PMMA-Mikrosphären ermittelt. Um die Kopplungseffizienz zu berechnen und den tatsächlichen Einfluss der EDC/NHS-Chemie auf die Bioaktivität von TGF-β3 zu untersuchen, wurde die Menge an immobilisiertem TGF-β3 auf PMMA-Mikrosphären mittels ELISA bestimmt. Für die Kopplung von TGF-β3 mittels EDC/NHS-Chemie wurde eine signifikant höhere Kopplungseffizienz im Vergleich zu der Reaktion ohne Kopplungsreagenzien, welche eine geringe Menge an adsorbiertem TGF-β3 zeigte, bestimmt. Bei alleiniger Betrachtung der Ergebnisse des Luciferase-Assays, bei welchem kein statistisch signifikanter Unterschied zwischen kovalent immobilisiertem und adsorbiertem TGF-β3 bezüglich der relativen Luciferase-Aktivität beobachtet wurde, scheint es als hätte die EDC/NHS-Kopplung keinen Einfluss auf die Bioaktivität von TGF β3. Beim Vergleich der mittels ELISA bestimmten TGF β3 Gesamtmenge und der mittels Luciferase-Assay bestimmten Menge an aktivem TGF-β3 auf den PMMA-Mikrosphären, wurde jedoch ein Verlust der Bioaktivität von TGF-β3 durch die EDC/NHS-Kopplung deutlich. Ungeachtet dessen, wurde immobilisiertes TGF-β3 genutzt, um Knorpelgewebe in SF-Scaffolds aus BMSCs zu generieren. Nach den Ergebnissen der histologischen und immunhistochemischen Untersuchungen, der biochemischen Assays sowie der qRT-PCR der Genexpression von BMSCs nach 21 Tagen in Kultur, gelang es uns unter Verwendung von immobilisiertem TGF-β3 Knorpelgewebe aufzubauen. Diese Ergebnisse unterstützen die These, dass die lokale Präsentation von TGF-β3 gegenüber exogen zugegebenem TGF-β3 für die Entwicklung von hyalinem Knorpel überlegen ist. Außerdem schließen wir daraus, dass kovalent immobilisiertes TGF-β3 nicht nur gegenüber exogen zugegebenem TGF-β3 für die Entwicklung von hyalinem Knorpelgewebe überlegen ist, sondern auch gegenüber adsorbiertem TGF-β3. Diffusionsprozesse konnten durch kovalente Immobilisierung von TGF-β3 an PMMA-Mikrosphären verhindert werden und damit eine stabile und gleichmäßige TGF β3-Konzentration am Wirkort aufrechterhalten werden. Mit den in Phase II und III gewonnenen Erkenntnissen und den Untersuchungen von Barbara Tabisz zur Expression und Aufreinigung von plk-BMP-2 haben wir erhebliche Fortschritte bei der Entwicklung multifunktionaler osteochondraler Implantate zur Regeneration von Knorpeldefekten gemacht. Für die Umsetzung dieser Erkenntnisse zur Herstellung multifunktionaler osteochondraler SF-Scaffolds sind jedoch weitere Studien erforderlich.

The ability of exogenous growth hormone (GH) to increase milk yield through insulin-like growth factor-1 (IGF-I) in dairy cows is well characterized. However, recent studies utilizing mammary epithelial cell lines indicate a direct effect of GH on mammary epithelial cells (MEC). To test if these observations are relevant in vivo and if this response differs between dairy breeds, three mammary models were utilized. Mammary explants from a lactating Jersey cow were cultured in classical lactogenic media (dexamethasone, insulin, and prolactin) with 0 or 10 ng/mL of recombinant bovine GH for 12h. Primary MEC from lactating Holstein and Jersey cows were cultured in classical lactation media with 0 or 10 ng/mL of GH for 2, 4, and 7 days. And lastly, MEC isolated from pooled Holstein or pooled Jersey milk were cultured in the same conditions as primary MEC. The response to GH was quantified by the relative abundance of mRNA for two milk protein genes (α-lactalbumin and αS1-casein), the GH receptor, IGF-I and insulin-like growth factor binding protein-3 (IGFBP-3) as determined by quantitative RT-PCR. The abundance of α-lactalbumin mRNA in explants was increased in response to GH. After 2 days, Jersey primary MEC showed an increase in GH receptor mRNA, in addition to a noteworthy trend of increasing abundance of IGFBP-3 regardless of GH treatment. After 4 days, Holstein primary cells cultured with GH had decreased IGFBP-3 mRNA. After 7 days, primary cells isolated from Holstein and Jersey mammary tissue showed a slight response to GH. Mammary cells from milk mirrored the responses to GH observed in primary cells: MEC isolated from Holsteins had decreased IGFBP-3 mRNA after 4 days of treatment with GH and MEC isolated from Jerseys showed the same trend of increasing IGFBP-3 abundance between 2 and 4 days, but with no difference between GH treatments. These results indicate that the effect of GH may differ between breeds and indicate GH has a direct effect on mammary epithelial cells, possibly including effects on the abundance of IGFBP-3 mRNA.

No description available.

No description available.

The overall objective of this project was to investigate the role of locally derived growth factors and extracellular matrix proteins in regulating prepubertal heifer mammary development. In the first experiment, short-term treatment of heifers with GH or E increased proliferation of mammary epithelial cells. Coinciding with increased epithelial cell proliferation, IGF-I protein increased and IGFBP-3 protein decreased within mammary tissues. Thus, proliferation was associated with an apparent net increase in the biological availability of IGF-I within the mammary gland. In the second experiment, decreased mammary development and epithelial cell proliferation in response to ovariectomy coincided with decreased mammary expression of IGF-I mRNA and decreased binding of IGF-I to mammary microsomes. Taken together, these results imply an important role for locally derived IGF-I in regulating heifer mammary development. However, in contrast to our hypothesis, IGF-I mRNA did not differ between cleared or intact mammary fat pad, suggesting that expression of IGF-I mRNA is not regulated by epithelial:stromal interactions. Neither ovariectomy or epithelial:stromal interactions influenced the mRNA expression of IGFBP-3 or IGFBP-5 within mammary tissues. Ovariectomy increased the proportion of ERa positive mammary epithelial cells. In contrast, GH administration to prepubertal heifers did not influence the proportion of ERa-positive epithelial cells. Interestingly, mammary development was more severely affected in heifers ovariectomized before six weeks of age than heifers ovariectomized at three months of age, implying a critical period of ovarian stimulation during the first six weeks of age. Localization of laminin, fibronectin, and collagen in mammary parenchyma suggested specific roles for extracellular matrix proteins in regulating mammary development and ductal morphogenesis. Laminin was decreased and fibronectin was increased by ovariectomy, suggesting a possible role for interactions between the ovary and extracellular matrix proteins within the heifer mammary gland. Finally, the mitogenic capacities of mammary tissue extracts from control and ovariectomized heifers did not differ in their ability to stimulate in vitro proliferation of MAC-T cells. In conclusion, the overall results support the hypothesis that locally derived IGF-I regulates prepubertal heifer mammary development. However, ERa expression and extracellular matrix proteins also appear to be important regulators of heifer mammary development. / Ph. D.

O estudo teve como objetivo avaliar as respostas hormonais, fisiológicas e comportamentais de vacas Holandesas frente a situações de conforto ou estresse térmico ambiental. O experimento desenvolvido entre os verões de 2007 e 2008 no Campus Administrativo da Universidade de São Paulo (USP), Pirassununga, SP, utilizou 28 fêmeas de 1ª a 3ª lactações com produção média de 20 kg/dia divididas em dois grupos experimentais após a parição, com e sem disponibilidade de climatização em galpão do tipo free-stall. Os parâmetros ambientais foram avaliados através do cálculo do índice de temperatura e umidade (ITU). As colheitas de dados fisiológicos (temperatura retal, temperatura de superfície corporal do dorso e base da cauda, frequência respiratória), hormonais (cortisol e IGF-I), comportamentais e de produção e qualidade do leite foram realizadas em cinco condições climáticas no ano (outono, inverno, primavera, verão seco e verão chuvoso) caracterizadas pela temperatura, umidade relativa e radiação solar. No verão os animais foram submetidos ao Teste de Capacidade Termolítica (CT) e a um estudo comparativo de um período de sete dias sob estresse calórico em câmara climática e desafio com aplicação de ACTH. No experimento 1 o teste de capacidade termolítica foi validado. A CT foi igual para animais em lactação ou secos (P>0,05), e maior para vacas mantidas sob sistema de climatização ao longo do ano (P<0,01). Houve influência da exposição ao sol sobre todas as variáveis fisiológicas (P<0,01). Os níveis plasmáticos de cortisol foram maiores antes da exposição ao sol e depois do repouso por 1 hora à sombra para vacas em lactação (P=0,03) e para as sem disponibilidade de climatização (P=0,03). O IGF-I foi maior nas vacas secas em final de gestação (P<0,01). No experimento 2 a temperatura retal não teve influência da climatização, com os dois grupos apresentando valores abaixo de 38,56 ºC ao longo do ano (P=0,11). Observou-se uma tendência de alta (P<0,01) nas concentrações plasmáticas de cortisol entre outono e inverno, começando o decréscimo até o início do verão seco e um novo aumento durante o verão chuvoso, e um comportamento inverso para o IGF-I. Temperatura retal mostrou uma correlação moderada e positiva (P<0,01) com a temperatura superficial (0,46) e frequência respiratória (0,35). A temperatura do ar e ITU apresentaram correlações positivas de moderada à alta com as temperaturas retal, da base da cauda e superficial, e também com a frequência respiratória (P<0,01). No experimento 3 as vacas passaram a maior parte do dia na sombra em pé (84,2 %) independente da estação do ano. O ambiente climatizado proporcionou maior frequência de alimentação e produção de leite durante o verão (P<0,05), assim como teor de gordura 17,9 % maior (P<0,01). No experimento 4 os animais foram submetidos ao estresse pontual causado pelo uso do ACTH e ao estresse calórico prolongado em câmara climática. Tanto a administração de ACTH quanto a exposição ao calor prolongado em câmara climática aumentaram os níveis de cortisol plasmático. Durante o estresse calórico houve diminuição do IGF-I e produção leiteira e aumento das variáveis fisiológicas ligadas a termorregulação. / The study aimed to evaluate the hormonal, physiological and behavioral responses of Holstein cows in situations of a comfort or heat environment. The experiment was conducted between the summers of 2007 and 2008 in the University of São Paulo (USP), Pirassununga, SP, and used 28 females from 1st to 3rd lactations with average production of 20 kg/day divided into two experimental groups after birth, with and without an evaporative cooling system in a free-stall. Environmental parameters were evaluated by temperature and humidity index (THI). Collection of physiological data (rectal temperature, body surface, internal base of tail, respiratory rate), hormonal (cortisol and IGF-I), behavioral and production and milk quality were conducted in five climatic conditions (autumn, winter, spring, dry summer and rainy summer) characterized by air temperature, relative humidity and solar radiation. In summer the animals were subjected to Thermolysis Capacity Test (CT) and a comparative study of a period of seven days under heat stress in climatic chamber and challenged with ACTH administration. In experiment 1 the Thermolysis Capacity Test was validated. The CT was the same for dry or lactating animals (P>0.05) and higher for cows kept in evaporative cooling system throughout the year (P<0.01). The results showed influence of sun exposure on all physiological variables (P<0.01). Plasma levels of cortisol were higher before sun exposure and after the one hour rest under shade for lactating cows (P=0.03) and for no cooled animals (P=0.03). IGF-I was higher in dry cows in late gestation (P<0.01). In experiment 2 the evaporative cooling system did not show influence on rectal temperature, with both groups having values below 38.56 ºC throughout the year (P=0.11). It was observed an upward trend (P<0.01) in plasma cortisol concentrations between autumn and winter, starting the decline until the beginning of dry summer and a further increase during rainy summer, and an opposite pattern for IGF- I. Rectal temperature showed a moderate and positive correlation (P<0.01) with the body surface temperature (0.46) and respiratory rate (0.35). The air temperature and THI showed moderate to high positive correlations with rectal temperatures, and the internal base of tail, and also with the respiratory rate (P<0.01). In experiment 3 cows spent most of the day standing in the shade (84.2%) regardless of season. Cooled cows had a higher feeding frequency and milk production during summer (P<0.05) and fat content 17.9% higher (P<0.01). In experiment 4, cows were subjected to the short stress caused by the ACTH administration and prolonged heat stress in climatic chamber. Both the administration of ACTH as prolonged exposure to heat in climatic chamber increased the levels of plasma cortisol. During heat stress a decrease in plasma IGF-I and milk production was observed, and an increase in physiological variables related to thermoregulation.

A Distrofia Muscular de Duchenne (DMD) é uma doença recessiva ligada ao cromossomo ?X?, causada pela ausência da proteína distrofina que ocorre em vários tecidos, sendo caracterizada por uma severa disfunção da musculatura esquelética ocasionando morte prematura do paciente. Embora controverso, alguns autores reportaram que o GH (hormônio do crescimento) estaria implicado no desenvolvimento da doença e poderia ser utilizado no tratamento da mesma. Desta forma, neste estudo a dosagem sérica de IGF-I (Fator I de crescimento Similar à insulina) um peptídeo GH-dependente que regula as ações do hormônio do crescimento, foi realizada no intuito de verificar se existe correlação ou não entre o desenvolvimento da doença e a concentração sérica de IGF-I. As variações nos níveis deste hormônio foram demonstradas com o decorrer da idade sendo que, nos três primeiros meses todos os animais apresentaram comportamento semelhante com aumento dos níveis de IGF-I, porém no quarto mês os animais Distróficos benignos apresentaram redução média de 34% deste hormônio, enquanto ocorreu aumento de 1% no animal não Distrófico. Ainda, as células granulares adenohipofisárias relacionadas à produção do hormônio de crescimento em cães Golden Retriever com Distrofia Muscular apresentam-se maiores do que nos cães Golden Retriever não distróficos quanto aos seguintes parâmetros (eixo longo, área seccional e volume celular). À microscopia eletrônica de transmissão observamos que as células estudadas apresentam grânulos elétron-densos de parede dupla e distribuídos por todo o citosol. Estes grânulos apresentaram-se maiores nos animais Distróficos quando comparados aos animais não Distróficos. / The Duchenne Muscular Dystrophy (DMD) is a X linked recessive disease, caused by the absence of dystrophin which is found in a variety of tissues and characterized by a severe disfunction of the skeletal musculature that results in a premature death of the patient. Theoretically, the growth hormone (GH) is considered to be associated to the development of Muscular Dystrophy and that could be used in its treatment. Hence in this study, a IGF-I (Insulin like growth factor-I) seric dosage was performed to verify whether or not there might be a between the evolution of the disease and IGF´s seric concentration. IGF-I is a GH-peptide dependent that regulates the GH actions during the growth. Changes in IGF-I levels were recorded during the dog?s post-natal development. On the first trimester, all animals presented similar IGF-I levels, although in the fourth month, a stark 34% decrease was observed in the dystrophic animals whereas a 1% increase was seen in the healthy dog. Furthermore, the GRMD´s granule-containing cells were larger when compared to the healthy animals. The following parameters in this comparison: long axis, cross-sectional area and cell volume. The ultrastructural study showed electron-dense granules composed by a double membrane and homogeneously distributed through the cell. These granules were larger in the dystrophic animals then in healthy dogs.

A distrofia muscular de Duchenne (DMD) é uma doença de caráter hereditário recessivo ligado ao cromossomo X, que determina a ausência da distrofia, a responsável pela progressiva degenaração muscular observada no DMD. Embora não expresse o fenótipo, a camundongo mdx apresenta a ausência da distrofia e o mais comum modelo animal experimental para estudar as repercursões da DMD em muitos orgãos. Este estudo foi realizado na cartilagem do processo condilar da mandíbula de mdx, um importante sítio de crescimento craniofacial. Assim, o PC dos mdx de 4 (G1) e 10 (G2) semanas e dos respectivos controles (camundongos c57BL/10 mice) foram avaliados usando as técnicas de microscopia de luz (H.E, Picrosirius e Safranina-O) e de imunohistoquímica (IGF e IGF-IR). Em ambos os grupos, não houve diferença estatísticamente significante na área do PC e comparação aos controles. O número de e a área dos condrócitos, bem como, a quantidade de matriz extracelular (MEC) forma menores nos grupos mdx. A imunorreatividade para ambos, IGF-I e IGF-IR, proporcionalmente maiores nos grupos mdx. Os dados quantitativos e predominância do colágeno tipo I nos grupos mdx, sugere um processo precoce de envelhecimento na cartilagem do PC desses animais. / The duchenne muscular dystrophy (DMD) is a recessive hereditary disease linked to X-chromossome that determines teh dystrophin abstence, the responsible for progressive muscle degeneration observed in DMD. Although not exhibit the phenotype, the MDX mouse reveal abstence of dystrophin and is the most common experimental animal model for DMD studies in many organs. This study was performed in the articular cartilage of the mandibular condylar process (PC) of MDX, an important site of craniofacial growth. Thus the PC of MDX and respective controls (C57BL/10 mice) were evaluated at the ages of 4 (G1) and 10 (G2) weeks using ligth microscopy (H.E, Picrosirius e Safranin-O) and immunohistochemical (IGF-I e IGF-IR) tecniques. In both groups, there was no statistical significant difference in PC area of the mdx and the respective controls. The number and area of the chondrocytes, as well as the amout of extracellular matrix (MEC) were lower in MDX groups. The immunoreactivity for both, IGF-I and IGF-IR, were propostionally higher im MDX groups. The quantitative data and the predominance of collagen type i fibers in the MDX groups suggest a premature aging process of the PC in these animals.

Avaliou-se neste estudo o efeito do tratamento com somatotrofina recombinante bovina (bST) na população folicular, na taxa de recuperação e qualidade dos oócitos e na produção in vitro de embriões bubalinos. A hipótese foi de que o bST aumenta o número de folículos recrutados e a qualidade oocitária, melhorando a eficiência da técnica de aspiração folicular em fêmeas bubalinas. Foram utilizadas 10 novilhas bubalinas, sendo que o grupo bST (n=5) recebeu 500mg de bST em intervalos regulares, enquanto o grupo Controle (n=5) não recebeu tratamento adicional. Ambos os grupos foram submetidos a 10 sessões de aspiração duas vezes por semana. Foram quantificados o número total de folículos e o tamanho destes a cada sessão. Os oócitos recuperados foram quantificados e classificados de acordo com a sua qualidade (A, B, C, D, E), sendo os A+B+C considerados de boa qualidade. Os embriões produzidos foram vitrificados, e uma parte destes, transferidos em tempo fixo. Foram colhidas amostras de sangue para mensuração de IGF-I plasmático durante o estudo. O bST aumentou o número de folículos observados (12,2 vs. 8,7; p&lt;0,05) e o número de oócitos recuperados (5,2 vs. 4,1; p=0,07), no entanto não afetou a qualidade destes, bem como a sua capacidade de desenvolvimento (p&gt;0,05). Observou-se efeito significativo (p&lt;0,05) de sessão de aspiração no número de folículos observados, de folículos aspirados e de oócitos recuperados. As concentrações plasmáticas de IGF-I não apresentaram efeito de tratamento (p&gt;0,05), no entanto sofreram efeitos de sessão e também da interação entres sessão e tratamento (p&lt;0,05). Obteve-se taxa de concepção de 10,53 % (2/19) após a inovulação dos embriões, gerando o nascimento de dois bezerros normais. Os resultados são sugestivos de que o tratamento com bST aumenta o número de folículos recrutados por onda de crescimento folicular, demonstrando seu potencial em aumentar a eficiência da técnica de aspiração folicular na espécie bubalina / The aim of this experiment was evaluated the effect of recombinant bovine somatotropin (bST) on follicular population, oocyte recovery rates and quality, and on in vitro buffaloes embryo production. The hypothesis was that bST improves the number of follicles per follicular growth wave and oocyte quality, enhancing the results in buffalos females submitted to ovum pick-up programs. A total of ten heifers were assigned in two experimental groups (bST Group that received 500mg of bST in regular intervals and Control Group that did not received any additional treatment). Both groups were submitted to 10 OPU sessions twice weekly (every 3 or 4 days). The number of follicles and its diameters were recoded in all OPU. The oocytes harvest were counted and classified in five categories (A, B, C, D, E). The A+B+C categories were considered as good quality oocyte. The embryos produced were vitrified and some of these embryos were transferred of fixed time. Blood samples for IGF-I were obtained every once weekly. The bST improved the follicular population (12.2 vs. 8.7; p&lt;0.05) and the number of oocytes per session (5.2 vs. 4.1; p=0.07), however the treatment did not affect the oocyte quality and its in vitro development capacity (p&gt;0.05). A significant effect (p&lt;0.05) of OPU session was observed in follicular population, number of aspirated follicles and number of oocyte recovered. The plasma IGF-I was not affected (p&gt;0.05) by treatment, however presented a significant effect of OPU session and also of the treatment by OPU session interaction (p&lt;0.05). The conception rate was 10.5% (2/19) and two healthy and normal calves were born from transferred embryo. These results indicate the viability of bST treatment to improve the follicular recruitment and its potential application in buffaloes donors submitted to OPU programs