Thesis (Ph. D.)--School of Chemical Engineering, Georgia Institute of Technology, 2004. Directed by Athanassios Sambanis. / Includes bibliographical references (leaves 125-135).

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Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Dr. Athanassios Sambanis; Committee Member: Dr. Barbara Boyan; Committee Member: Dr. Peter Thule.

O objetivo deste estudo foi a avaliação histopatológica e radiográfica da resposta pulpar e periapical de dentes de cães após pulpotomia e utilização da Proteína Morfogenética Óssea Recombinante Humana 7. Foram utilizados 60 dentes (120 raízes) de 6 cães, divididos em 8 grupos, nos períodos experimentais de 7 dias (Grupos I, II, III, IV) e 70 dias (Grupos V, VI, VII, VIII). Após a pulpotomia, o remanescente pulpar foi recoberto com os seguintes materiais: Grupos I e V - Proteína Óssea Morfogenética Recombinante Humana 7 (rHuBMP-7) associada ao Colágeno Recombinante Humano (rHuCollagen); Grupos II e VI - Colágeno Recombinante Humano (rHuCollagen); Grupos III e VII (Controle Negativo) - Hidróxido de Cálcio p.a. e soro fisiológico e Grupos IV e VIII (Controle Positivo) - Óxido de Zinco e Eugenol. Decorridos os períodos experimentais, os animais foram mortos, as peças removidas e submetidas ao processamento histológico. A avaliação histopatológica foi realizada subjetivamente em microscópio óptico. A avaliação radiográfica foi realizada considerando-se a integridade da lâmina dura, presença de áreas de rarefação óssea periapical, de reabsorções radiculares (interna e externa) e de ponte de dentina, sendo os resultados submetidos à análise estatística utilizando-se o teste exato de Fisher. Nos espécimes que apresentavam áreas de rarefação periapical, as medidas radiográficas das lesões foram comparadas entre os grupos por meio do teste de Kruskall-Wallis. Os achados histopatológicos evidenciaram que no período de 7 dias, nos Grupos I e II havia um infiltrado inflamatório severo e intensa proliferação vascular no tecido pulpar, no Grupo IV um infiltrado inflamatório moderado enquanto no Grupo III foi observado um infiltrado inflamatório leve, estando o tecido pulpar íntegro. Em todos os grupos não havia formação de ponte de dentina e a região periapical apresentava aspectos de normalidade. No período de 70 dias, nos Grupos V, VI e VIII não houve formação de ponte de dentina, o tecido pulpar apresentava áreas de necrose com presença de células inflamatórias na região periapical e reabsorção cementária e óssea. Por outro lado, no Grupo VII, foi observada presença de ponte de dentina, ausência de processo inflamatório e ausência de reabsorção dos tecidos mineralizados. Com relação aos achados radiográficos, no período de 7 dias, todos os espécimes dos Grupos I, II, III e IV apresentavam integridade da lâmina dura, ausência de rarefação óssea periapical, ausência de reabsorção radicular (interna e externa) e ausência de ponte de dentina. No período de 70 dias, nos Grupos V, VI e VIII não houve formação de ponte de dentina em nenhum espécime sendo observadas áreas de rarefação óssea periapical em 100% das raízes do Grupo VI, 60% das raízes do Grupo VIII e 40% das raízes do Grupo V, sendo as maiores lesões encontradas no Grupo VI, seguida pelos Grupos V e VIII (p<0,05). No grupo VII, foi observada presença de ponte de dentina em 60% dos casos, integridade da lâmina e ausência de rarefação óssea periapical em 100% dos casos. Pode-se concluir que a Proteína Óssea Morfogenética Recombinante Humana 7 quando associada ao Colágeno Recombinante Humano não apresentou resultados satisfatórios. / The purpose of this study was to evaluate, both histopathologically and radiographically, the pulpal and periapical response of dogs’ teeth after pulpotomy and use of recombinant human bone morphogenetic protein-7 (rHuBMP-7). For such purpose, 60 teeth (120 roots), obtained from 6 dogs, were divided in 8 groups and evaluated in two experimental periods: 7 days (Groups I, II, III, IV) and 70 days (Groups V, VI, VII, VIII). After pulpotomy, pulp remnant was covered with the following materials: Groups I and V - recombinant human bone morphogenetic protein-7 (rHuBMP-7) associated to recombinant human like collagen (rHuCollagen); Groups II and VI - recombinant human like collagen (rHuCollagen); Groups III and VII (negative control) – calcium hydroxide and sodium chloride solution; and Groups IV and VIII (positive control) – zinc oxide and eugenol. At the established experimental periods, the animals were sacrificed and the anatomic pieces were obtained and histologically processed. The histopathologic evaluation was realized subjectively in a light microscope. The radiographic evaluation was performed considering the integrity of the lamina dura, presence of areas of periapical bone rarefaction, root resorption (internal and external) and dentin bridge formation. The results were analyzed statistically using Fisher\'s exact test. In the specimens presenting periapical bone rarefaction areas, the lesions’ radiographic measurements were compared among the groups using the Kruskall-Wallis test. The histopathologic findings in the 7-day period revealed that Groups I and II presented a severe inflammatory infiltrate and intense vascular proliferation in the pulp tissue, Group IV presented a moderate inflammatory infiltrate while Group III presented a mild inflammatory infiltrate and intact pulp tissue. In all groups, there was no dentin bridge formation and the periapical region had normal appearance. In the 70-day period, Groups V, VI and VIII showed no dentin bridge formation and pulp tissue presented necrotic areas with inflammatory cells in the periapical region as well as bone and cemental resorption. On the other hand, in Group VII, there was dentin bridge formation, absence of inflammatory process and absence of resorption of mineralized tissues. Regarding the radiographic findings, in the 7-day period, all specimens in Groups I, II, III and IV present intact lamina dura, absence of periapical bone rarefaction, absence of root resorption (internal and external) and absence of dentin bridge formation. In the 70-day period, Groups V, VI and VIII did not present dentin bridge formation in any specimen. Periapical bone rarefaction areas were observed in 100% of the roots in Group VI, 60% of the roots in Group VIII and 40% of the roots in Group V. The largest lesions were found in Group VI, followed by Groups V and VIII (p<0.05). In Group VII, there was dentin bridge formation in 60% of the cases, intact lamina dura and absence of periapical bone rarefaction in 100% of the cases. Based on these results, it may be concluded that recombinant human bone morphogenetic protein-7 associated to recombinant human like collagen did not present satisfactory results.

O objetivo deste estudo foi a avaliação histopatológica e radiográfica da resposta pulpar e periapical de dentes de cães após pulpotomia e utilização da Proteína Morfogenética Óssea Recombinante Humana 7. Foram utilizados 60 dentes (120 raízes) de 6 cães, divididos em 8 grupos, nos períodos experimentais de 7 dias (Grupos I, II, III, IV) e 70 dias (Grupos V, VI, VII, VIII). Após a pulpotomia, o remanescente pulpar foi recoberto com os seguintes materiais: Grupos I e V - Proteína Óssea Morfogenética Recombinante Humana 7 (rHuBMP-7) associada ao Colágeno Recombinante Humano (rHuCollagen); Grupos II e VI - Colágeno Recombinante Humano (rHuCollagen); Grupos III e VII (Controle Negativo) - Hidróxido de Cálcio p.a. e soro fisiológico e Grupos IV e VIII (Controle Positivo) - Óxido de Zinco e Eugenol. Decorridos os períodos experimentais, os animais foram mortos, as peças removidas e submetidas ao processamento histológico. A avaliação histopatológica foi realizada subjetivamente em microscópio óptico. A avaliação radiográfica foi realizada considerando-se a integridade da lâmina dura, presença de áreas de rarefação óssea periapical, de reabsorções radiculares (interna e externa) e de ponte de dentina, sendo os resultados submetidos à análise estatística utilizando-se o teste exato de Fisher. Nos espécimes que apresentavam áreas de rarefação periapical, as medidas radiográficas das lesões foram comparadas entre os grupos por meio do teste de Kruskall-Wallis. Os achados histopatológicos evidenciaram que no período de 7 dias, nos Grupos I e II havia um infiltrado inflamatório severo e intensa proliferação vascular no tecido pulpar, no Grupo IV um infiltrado inflamatório moderado enquanto no Grupo III foi observado um infiltrado inflamatório leve, estando o tecido pulpar íntegro. Em todos os grupos não havia formação de ponte de dentina e a região periapical apresentava aspectos de normalidade. No período de 70 dias, nos Grupos V, VI e VIII não houve formação de ponte de dentina, o tecido pulpar apresentava áreas de necrose com presença de células inflamatórias na região periapical e reabsorção cementária e óssea. Por outro lado, no Grupo VII, foi observada presença de ponte de dentina, ausência de processo inflamatório e ausência de reabsorção dos tecidos mineralizados. Com relação aos achados radiográficos, no período de 7 dias, todos os espécimes dos Grupos I, II, III e IV apresentavam integridade da lâmina dura, ausência de rarefação óssea periapical, ausência de reabsorção radicular (interna e externa) e ausência de ponte de dentina. No período de 70 dias, nos Grupos V, VI e VIII não houve formação de ponte de dentina em nenhum espécime sendo observadas áreas de rarefação óssea periapical em 100% das raízes do Grupo VI, 60% das raízes do Grupo VIII e 40% das raízes do Grupo V, sendo as maiores lesões encontradas no Grupo VI, seguida pelos Grupos V e VIII (p<0,05). No grupo VII, foi observada presença de ponte de dentina em 60% dos casos, integridade da lâmina e ausência de rarefação óssea periapical em 100% dos casos. Pode-se concluir que a Proteína Óssea Morfogenética Recombinante Humana 7 quando associada ao Colágeno Recombinante Humano não apresentou resultados satisfatórios. / The purpose of this study was to evaluate, both histopathologically and radiographically, the pulpal and periapical response of dogs’ teeth after pulpotomy and use of recombinant human bone morphogenetic protein-7 (rHuBMP-7). For such purpose, 60 teeth (120 roots), obtained from 6 dogs, were divided in 8 groups and evaluated in two experimental periods: 7 days (Groups I, II, III, IV) and 70 days (Groups V, VI, VII, VIII). After pulpotomy, pulp remnant was covered with the following materials: Groups I and V - recombinant human bone morphogenetic protein-7 (rHuBMP-7) associated to recombinant human like collagen (rHuCollagen); Groups II and VI - recombinant human like collagen (rHuCollagen); Groups III and VII (negative control) – calcium hydroxide and sodium chloride solution; and Groups IV and VIII (positive control) – zinc oxide and eugenol. At the established experimental periods, the animals were sacrificed and the anatomic pieces were obtained and histologically processed. The histopathologic evaluation was realized subjectively in a light microscope. The radiographic evaluation was performed considering the integrity of the lamina dura, presence of areas of periapical bone rarefaction, root resorption (internal and external) and dentin bridge formation. The results were analyzed statistically using Fisher\'s exact test. In the specimens presenting periapical bone rarefaction areas, the lesions’ radiographic measurements were compared among the groups using the Kruskall-Wallis test. The histopathologic findings in the 7-day period revealed that Groups I and II presented a severe inflammatory infiltrate and intense vascular proliferation in the pulp tissue, Group IV presented a moderate inflammatory infiltrate while Group III presented a mild inflammatory infiltrate and intact pulp tissue. In all groups, there was no dentin bridge formation and the periapical region had normal appearance. In the 70-day period, Groups V, VI and VIII showed no dentin bridge formation and pulp tissue presented necrotic areas with inflammatory cells in the periapical region as well as bone and cemental resorption. On the other hand, in Group VII, there was dentin bridge formation, absence of inflammatory process and absence of resorption of mineralized tissues. Regarding the radiographic findings, in the 7-day period, all specimens in Groups I, II, III and IV present intact lamina dura, absence of periapical bone rarefaction, absence of root resorption (internal and external) and absence of dentin bridge formation. In the 70-day period, Groups V, VI and VIII did not present dentin bridge formation in any specimen. Periapical bone rarefaction areas were observed in 100% of the roots in Group VI, 60% of the roots in Group VIII and 40% of the roots in Group V. The largest lesions were found in Group VI, followed by Groups V and VIII (p<0.05). In Group VII, there was dentin bridge formation in 60% of the cases, intact lamina dura and absence of periapical bone rarefaction in 100% of the cases. Based on these results, it may be concluded that recombinant human bone morphogenetic protein-7 associated to recombinant human like collagen did not present satisfactory results.

Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Addenda held in pocket pasted onto back end paper. / Bibliography: leaves 137-165. / xvi, 165 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis involves the retrospective investigation of the insulin-like growth factors and their binding proteins in the serum of children with chronic renal failure (CRF) and transplantation, before and after treatment with recombinant human growth hormone (rhGH). IGF-IGFBP complexes in pooled serum from prepubertal and pubertal children of both sexes with CRF and renal transplantation, before and after treatment with rhGH, are analysed by fast protein liquid chromatography under neutral conditions. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1997?

In this study, it was aimed to investigate the effects of different co-carbon sources on therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by Pichia pastoris by designing feeding strategies which were applied in the production phase of the bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions, recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the by-products organic acid concentrations were analyzed. In this context, firstly, laboratory scale air filtered shake bioreactor experiments were performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial concentration of co-substrates was found more affordable and appropriate for cell concentration and recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the production phase together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system with different feeding strategies together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1 mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy (MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h, containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1 at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and YP/St=2.23 mg g-1 in the highest rHuEPO production case.

In this study, the effects of pH on therapeutically important protein, recombinant human erythropoietin (rhuEPO), production by Pichia pastoris was investigated at pH=4.0, 4.5, 5.0, 5.5 and 6.0. rHuEPO production was started by methanol induction in fed-batch mode. The highest cell concentration was obtained at pH=4.5 as 81.4 g L-1. The co-substrate substrate sorbitol, which was added batch-wise, was consumed at t=15 h of the operations at pH=4.0, 4.5 and 5.0. However as the pH increases above pH=5.0 the sorbitol consumption rate decreases. The highest rHuEPO concentration was achieved at pH=4.5 as 0.158 g L-1 which was 1.43-, 1.24-, 1.95- and 1.23-fold higher than those obtained at pH=4.0, 5.0, 5.5, and 6.0, respectively. Also at pH=4.5 overall cell yield on substrate was 0.51 g g-1 and overall rHuEPO yield on substrate was 1.45 mg g-1. rHuEPO concentration was decreased in the last 3-6 hour of the operation due to proteolysis. Therefore extracellular protease concentrations in the medium were determined. As expected, since the investigated pH range was acidic, the amount of acidic proteases was found to be higher than neutral and basic proteases. Furthermore the total protease concentration increased linearly in the fermentation broth, having close values at different pH values. Thus, pH did not have a significant effect on extracellular protease activity. Alcohol oxidase (AOX) activities showed similar behavior at different pH. The highest specific AOX activity was attained at pH=4.5, at which the highest rHuEPO concentration was achieved, as 110.1 U g-1 CDW. Keywords:

In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased. Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrations / expression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.