No description available.

In title, [beta] is represented by the Greek letter. Copies of author's previously published articles inserted. Errata pages pasted onto back end-paper. Bibliography: leaves 104-137. Studies milk TGF-[beta] and its receptors in the post-natal gut using a rat model to investigate a link between milk TGF-[beta] and the development of the infant gut and gut mucosal immune system. Finds maternal milk may be a major source of TGF-[beta] to the immature gut and may react with receptors on the cells of the mucosal immune system along the gastro-intestinal tract, modulating infant mucosal immune responses in the transition to the post-natal enteral feeding.

Regenerative medicine has the potential to establish or restore normal function in defective tissues and organs. The realisation of such therapies is restricted due to costs, lack of scalability and inefficient manufacturing process controls. A major contributor to cost is the use of expensive growth factors supplemented into media at high concentrations. In vivo, growth factors exist in soluble, immobilised and transmembrane forms, expressed in a spatiotemporal fashion within the stem cell niche. In comparison to soluble equivalents, immobilised growth factors exhibit increased potency, distinct functional activities, improved cell phenotypic control and act in synergy with other soluble and immobilised ligands. To date, most research into immobilised growth factors has been restricted to planar cell culture surfaces such as tissue culture plastics which have limited scalability. To address the scalability limitations, a novel growth factor immobilisation technology was developed using magnetic microparticles which can be scaled with respect to surface area to volume ratio in standard stirred tank bioreactors. Three clinically relevant growth factors, SCF, TPO and GM-CSF were immobilised and were shown to remain functionally active where surface concentration could be manipulated in a number of ways. Through a series of experiments, it was demonstrated that immobilised growth factors exhibited ~10-fold increase in potency compared with soluble equivalents and remain stable for up to 192 hours following recycling during multiple media passages. Immobilised growth factors were able to expand more cells over a longer period of time after transient exposure and finally, the immobilisation technique was successfully applied to the expansion of umbilical cord derived haematopoietic stem cells using immobilised SCF. The immobilisation method described here has the potential to significantly reduce media costs in large scale cell manufacturing processes.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T18:50:23Z : No. of bitstreams: 1 donato_pm_me_botfm.pdf: 1472808 bytes, checksum: dd94c6d1ccf0834bf27a62051a637836 (MD5) / Ministério da Saúde / As plaquetas são fragmentos do citoplasma do megacariócito e possuem papel fundamental na manutenção da hemostasia. Ao aderirem umas às outras, ativam-se, mudam de forma e liberam grânulos que contêm fatores de crescimento, proteínas que atuam na divisão celular. Os grânulos α- plaquetários secretam fatores de crescimento que regulam eventos, tais como, síntese de DNA, quimiotaxia e citodiferenciação. A estocagem prolongada das plaquetas a 22ºC é prejudicial à viabilidade e à função, mesmo quando estocadas em condições adequadas nos bancos de sangue. Observa-se diminuição da concentração do glutation intraplaquetário, representando diminuição da proteção contra o stress oxidativo, do qual resulta a perda do grupo sulfidril. As plaquetas desempenham funções com consumo de energia, como exemplo a diapedese, que pode ser recuperada após cinco dias de armazenamento pelo azul de metileno. Durante a divisão celular há necessidade de bloqueio das funções citoplasmáticas com aumento da produção de proteínas de adesão para que ocorra a fixação da célula que vai se dividir. Os resultados mostraram que quando adicionamos azul de metileno, a quantidade de células viáveis diminui com aumento de apoptose e necrose, muito provavelmente devido à liberação de energia / Platelets are megakaryocyte cytoplasmic fragments that play a fundamental role in maintaining hemostasis. When they adhere to each other, they become activated, change shape and release granules containing growth factors, proteins involved with cell division. Platelet α-granules secrete growth factors that regulate events such as DNA synthesis, chemotaxis and cytodifferentiation. Long platelet storage at 22ºC is harmful to their viability and function, even when they are stored under proper conditions at blood banks. A reduced concentration of intraplatelet glutathione is observed, representing decreased protection against oxidative stress, from which the loss of the sulfhydryl group results. Platelets perform actions with energy consumption, such as diapedesis, which can be recovered after five storage days by methylene blue. During cell division, cytoplasmic functions must be blocked by increased adhesion protein so that the fixation of the dividing cell can occur. Results showed that when methylene blue is added, the number of viable cells decreases with apoptosis and necrosis increase, which is very likely due to energy release

Doctor of Philosophy / Department of Biology / A. Lorena Passarelli / Baculoviridae is the only known virus family that encodes genes with homology to vertebrate and invertebrate fibroblast growth factors (fgfs), key regulators of developmental processes affecting cell growth, differentiation, and motility. The role of viral fgfs during infection is not known. In this study, we investigated gene regulation and function of the Autographa californica M nucleopolyhedrovirus (AcMNPV) fgf during infection of permissive insect cells. We demonstrated that the AcMNPV fgf, vfgf, was transcribed as a 0.6-kb mRNA at early times post infection, but as part of a 1.4-kb bicistronic mRNA at late times. To determine its function, we examined common characteristics between vFGF and other well-characterized FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate chemokinesis of different types of insect cells. We also constructed a recombinant of AcMNPV lacking a functional vfgf and analyzed it in two insect cell lines. The kinetics of budded virus production were similar in the parental and vfgf-deficient viruses in two cell lines and at both high and low multiplicities of infection. In addition, we observed no obvious differences in the viral DNA synthesis and the protein kinetic profiles of cells infected with the mutant and parental viruses. Finally, coinfection of vfgf-containing and -deficient viruses and their passage for several generations did not reveal a consistent growth advantage for either virus. We propose that vFGF is the signal that directs the motility of uninfected tracheal or blood cells to infected tissues, enabling the virus to infect additional cells and spread systemically in the insect host. This proposal may explain a dispensable role for vfgf during virus infection in cell culture; nonetheless, we expect a distinct phenotypic difference between vfgf-deficient and vfgf-containing viruses during infection in the insect host.

No description available.

Microalgae invite the attention of scientists due to their unique properties, including their quick growth, accumulation of lipids and other valuable substances, fixation of carbon dioxide and treatment of wastewater. This master´s thesis is focused on the study of microalgae. The main goal is to understand and describe the process of microalgae cultivation, in order to optimize it. The theoretical part of this thesis deals with microalgae (mainly Chlorella sp.) characterization, its practical use and cultivation optimization in order to obtain the highest concentration of biomass. The experimental part is divided into three tasks. Aim of the first task was the comparison of the course of autotrophic and heterotrophic cultivation of various strains of Chlorella and Coccomyxa microalgae, using three different cultivation media – synthetic medium for chlorella cultivation and natural fertilizer, Florium, used in two different concentrations (diluted 50 and 20 times). The highest Chlorella sp. biomass concentration of 7,10 g/l was achieved in the synthetic heterotrophic medium. Second task was focused on monitoring of the growth of algae Coccomyxa and Chlorella strain C1A, with respect to temperature and light intensity, using various combinations of these two important growth factors. Chlorella achieved its highest concentration of 11,46 g/l when grown at temperature of 33,5 °C and light intensity of 320 µE.m2.s1. The third and final task was to observe the growth of Dictiosphaerium chlerelloides microalgae on a flat cascade bioreactor. The experiment led to the discovery that these algae were able to grow at temperatures of around 10 °C, at which many well-known commercial algae, such as Chlorella sp. or Arthrospina sp., simply wouldn’t grow.

BACKGROUND: Cells, growth factors and scaffold are the 3 fundamental factors currently proposed necessary for tissue regeneration. The use of these components has to be orchestrated precisely for ideal functional tissue formation. Growth factors enhance cellular activities that may lead to angiogenesis, cell proliferation and extracellular matrix biosynthesis. Due to the complexity of biochemical reactions a single growth factor may have limited effect. In order to explore a mixed profile of growth factors, a new biomaterial containing multiple growth factors derived from human Amniotic Membrane was chosen to compare with a known single growth factor (rhPDGF-BB). AIM: To compare the potential for enhanced bone formation by a morselized amniotic membrane suspension (AmnioSpark) with a known single cytokine PDGF-BB (GEM21,Lynch) under ex-vivo calvaria culture conditions. MATERIALS AND METHODS: 45 Calvaria from 7-9 day neonatal CD-1 mice were surgically harvested under sterile conditions. The calvaria were split through the mid sagittal suture to create 90 test specimens. A 2mm diameter critical size defect was created by biopsy punch thru the center of each calvarial specimen. This defect was bridged with a non-crosslinked type I collagen membrane of the same diameter to act as a scaffold. To compare AmnioSpark (AGF) potential for tissue regeneration against a known single cytokine PDGF-BB, the calvarial specimen were divided into six experimental groups: 1) Defect only, 2) Defect + scaffold, 3) Defect + scaffold + a single dose of (rhPDGF-BB ) a known bone stimulant, 4) Defect + Scaffold + 4 doses (day 0,3,5,7) of ( rhPDGF-BB), 5) Defect + scaffold + a single dose of (AGF) and 6) Defect + scaffold + 4 doses (day 0,3,5,7) of (AGF). Each test group had (N=5). A unique static tissue culture method was used with DMEM medium supplemented with ascorbic acid (150 ug/ml) and bovine serum albumin (5 mg/ml) without fetal calf serum to enhance bone formation for up to 7 weeks. Culture medium was changed every 2 days after day 3 and the harvested media was used for the following analyses: A) Alkaline phosphatase (ALP) as an osteoblastic activity indicator, and B) Tartrate Resistant Acid phosphatase (TRAP) as an osteoclastic bone remodeling activity indicator1. Macro photography and Scanning electron microscope (SEM) image analysis at different magnifications was performed to evaluate surface conditions. Histological analysis was performed with light microscope images on standard 4 um sections using H&E, Tri chrome, Picrosirius red and a fluorescence stain for RUNX2 as an osteoblast marker. RESULTS: With a single dose of test material ALP activity in the AGF group was significantly higher at 5 and 7 days. In addition ALP activity was significantly higher compared to all groups for up to 3 weeks post-application in the multiple dose AGF group (P<0.05). In contrast there was a dramatic decline in ALP in all other groups within the first week. TRAP activity was not detectable in any group. SEM images showed that osteoblast like cells accumulated and new tissue formation occurred over the surface of the scaffold obliterating the defect/membrane interface at 21 days with the AGF stimulus while in the PDGF-BB group the scaffold was still distinguishable from surrounding bone with no new tissue formation or cell migration . Histologic images confirmed an organized distribution of cells along the surface of the scaffold and new bone formation around the periphery of the defect in the AGF group (FIG42), while no bone formation or cell migration occurred in PDGF-BB group (FIG 35-38). Further diagnostic stains confirmed the presence of active osteoblasts (RUNX2)and the production of collagens I and II ( Masson Tri Chrome and PSR). CONCLUSION: Our results indicate that growth factors from amniotic extract (AGF) have the potential to enhance calvarial bone regeneration under an ex-vivo culture condition. These findings suggest that AGF could be a candidate for use as a new type of therapeutic material for regenerative medicine.

The adipose-derived stromal vascular fraction (SVF) is a promising new therapy for equine flexor tendonitis. This heterogeneous population of cells may improve tendon healing via the production of growth and chemotactic factors capable of recruiting endogenous stem cells and increasing extracellular matrix production by tendon fibroblasts (TFBL). The purpose of this study was to evaluate the ability of adipose-derived cells (ADC) culture expanded from the SVF to act as trophic mediators in vitro. We hypothesized that ADCs would produce growth and chemotactic factors important in tendon healing and capable of inducing cell migration and matrix protein gene expression. Superficial digital flexor tendons and adipose tissue were harvested from eight adult horses and processed to obtain SVF cells, ADCs and TFBLs. Adipose-derived cells and TFBLs were grown in monolayer culture for growth factor quantification, to produce conditioned media for microchemotaxis, and in co-culture for quantification of matrix protein gene expression by TFBLs. Growth factor gene expression by SVF cells was significantly greater than in ADCs or TFBLs. Co-culture of TFBLs and ADCs resulted in modest up-regulation of matrix protein expression (collagen types I and III, decorin, and cartilage oligomeric matrix protein) by TFBLs. Media conditioned by ADCs induced ADC migration in a dose dependent manner. These findings support the role of both SVF and ADCs as trophic mediators in tendon regeneration. The differences detected in gene expression between SVF cells and ADCs indicate that additional studies are needed to evaluate the changes that occur during culture of these cells. / Master of Science

Both VEGF and its receptor proteins were localized by immunostaining technique in the luminal epithelium, smooth muscle cells and blood vessels within the oviduct. Moreover, by means of semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) techniques, it has been demonstrated that mRNA of VEGF and its receptors in both healthy and diseased oviduct is expressed preferentially at the time and place where the amount of oviduct fluid is prominent. This supports the notion that VEGF may be a regulator of oviductal secretion. This thesis has consistently demonstrated a modulation pattern of flt-1 expression that is similar to its ligand VEGF in both physiological and pathological conditions. This suggests that flt-1 may be the main receptor responsible for the action of VEGF in the oviduct. As illustrated in both the in-vivo and in-vitro models, the expression of VEGF and flt-1 in the human oviduct is stimulated directly by gonadotropins without the influence of ovarian sex hormones. / Increased knowledge on the regulatory mechanisms of oviductal fluid formation, the first environment that human embryos are exposed to, will be valuable from the clinical management point of view. / Oviductal fluid is a complex mixture of plasma-derived constituents and proteins synthesized by the oviduct epithelium. It has been postulated that vascular endothelial growth factor (VEGF), a known permeability promoter, may be an important regulator of oviductal fluid secretion by stimulating vascular permeability and so serum transudation. However, little is known about the expression of VEGF in the human oviduct. This thesis investigated the modulation of VEGF and its receptors (flt-1 and KDR) in the healthy oviducts, from fertile women undergoing tubal sterilization for unwanted fertility or hysterectomy for benign gynecological conditions, as well as in the hydrosalpinges from sterile women undergoing salpingectomy before the treatment of in-vitro fertilization and embryo transfer. / Lam Po Mui. / Adviser: Christopher J. Haines. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.