This thesis describes a novel approach for cell encapsulation in alginate gel microbeads. The main aim of the thesis was to optimise a microfluidic setup and chip to encapsulate cells in monodisperse alginate gel microbeads. A number of cytotoxicity tests were therefore carried out to determine the effect of formulations used for the production, degradation and gelation of calcium alginate gel beads. Results from these tests revealed that the formulations used had little or no significant effect on cell growth, and therefore, alginate was deemed to be a suitable cell encapsulating material for further investigations. Alginate gel microbeads were produced using hydrodynamic focusing techniques. For this purpose two different microfluidic setups were constructed. Fluids (oil, acidified oil and samples) were driven through the microfluidic setup by gravity. However, a number of drawbacks using this setup arose, such as polydispersity and reproducibility. Syringe pumps were introduced into the design of the second microfluidic setup as a means of driving fluids through the setup. In addition three different microfluidic chips were fabricated with the aim of producing the ideal alginate gel microbead. The first microfluidic chip (PMMA MC1) was fabricated from PMMA and involved producing alginate gel microbeads that were internally gelified. This chip suffered from a number of drawbacks such as continuous blockages within the microfluidic channels, which led to the development of the second microfluidic chip. This chip was also fabricated from PMMA (PMMA MC2) but in contrast to PMMA MC1, gelification occurred externally, i.e. gelation took place off chip, and in this case the alginate microdroplets were dropped into a well containing 1 mL of acidified oil. This encapsulating procedure caused immediate cell death, which indicated that the internal gelation of alginate gel microbeads was favoured. These results also indicated that the design of the microfluidic chip needed developing in order to produce the ideal microbeads that can be used for cell encapsulation. This led to the fabrication of a novel microfluidic chip (PC MC3) which was fabricated from polycarbonate (PC) and involved internal gelation of the calcium alginate gel microbeads. The combination of using the optimised microfluidic setup and PC MC3, in addition to alternations in some of the solutions used to make the alginate microbeads, resulted in the production of the desired ideal gel microbeads containing cells. Snap shots of the encapsulated cells obtained using fluorescence microscopy after 24 hours of encapsulation, revealed that the cells showed some characteristics of living cells, yet at the same time they also showed some characteristics of dead cells. These findings demonstrate the potential use of the optimised microfluidic setup and PC MC3 chip for many biological and medical applications.

Cell cultivation in bioreactors would benefit from developed monitoring systems with online real-time imaging to evaluate cell culture conditions and processes. This opportunity can be provided with the newly developed in situ Microscope also called ISM. The ISM probe is mounted into the wall of a bioreactor and consists of a measurement zone with an illuminating light source to obtain real-time images of moving cells in suspension. The instrument is linked to advanced imaging analysis software which can be specifically adapted for the objects in study. The aim of this project is to analyze the T lymphocyte cell line Jurkat T cells using the ISM equipment and identify specific features of the cells that can be obtained. The results show that the equipment and linked software are suitable for monitoring cell density, cell size distribution and cell surface analysis of the Jurkat cells during cultivation. The ISM could also detect induced changes in cell size caused by osmotic shifts and the course of an infection occurring in the cell suspension using a developed software for online real-time monitoring.

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Made available in DSpace on 2017-07-21T18:53:02Z (GMT). No. of bitstreams: 1 Flavia Roberta Marenda.pdf: 1506229 bytes, checksum: 7e85c48845e1fc15cca9fa308082955b (MD5) Previous issue date: 2015-02-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The passion fruit industrial use reach only 25% of the total fruit and passion fruit peel, representing 50% of the total, are discarded, but can be used in the extraction of pectin. Recent studies indicate that pectin, modified so as native, has antitumor activity. This study aimed to evaluate the cytotoxicity of pectin of passion fruit peel in tumor cell lines. Extraction has been made and the chemical modification of pectin of passion fruit peel, subjected to three different treatments. Both the raw material and pectin were characterized by specific analyzes. The cytotoxicity of untreated pectins, modified autoclaved and autoclaved were evaluated in tumor lines Jurkat, HeLa and HRT-18. The peel of passion fruit represented 54.7% of total fruit. The passion fruit peel flour bleached untreated and shown to be rich in soluble and insoluble fibers. The flour autoclaved pectin showed higher content of phenolic compounds; however, bleached pectin retained more phenolic compounds from the raw material. The modified pectins had a lower degree of methoxylation and lower molecular weight compared to native pectins, confirming the modification process. The best effect Cytotoxicity in Jurkat cells treated with autoclaved pectin modified with IC50 of 2.63 mg mL-1. The calculation of the selectivity index indicated that the modified autoclaved pectin has a low toxicity to healthy cells. In conclusion, the results demonstrate a potential antitumor promising for autoclaved modified pectin, that needs to be further exploited. / O aproveitamento industrial do maracujá atinge apenas 25% do total do fruto e as cascas do maracujá, que representam 50% desse total, são descartadas, mas podem ser utilizadas na extração de pectina. Estudos recentes indicam que a pectina, tanto nativa quanto modificada, apresenta atividade antitumoral. Assim, este trabalho teve por objetivo avaliar a citotoxicidade de pectinas do albedo do maracujá em linhagens tumorais. Foi feita a extração e a modificação química da pectina do albedo de maracujá, submetido a três diferentes tratamentos. Tanto a matéria-prima quanto a pectina foram caracterizadas por análises específicas. A citotoxicidade das pectinas sem tratamento, autoclavada e autoclavada modificada, foram avaliadas em linhagens tumorais Jurkat, HeLa e HRT-18. A casca do maracujá representou 54,7% do total do fruto. As farinhas do albedo do maracujá sem tratamento e branqueada mostraram-se ricas em fibras solúveis e insolúveis. A farinha e a pectina autoclavada apresentaram maior teor de compostos fenólicos; no entanto, a pectina branqueada reteve mais compostos fenólicos provenientes da matéria prima. As pectinas modificadas apresentaram menor grau de metoxilação e menor massa molar, comparada às pectinas nativas, confirmando o processo de modificação. O melhor efeito de citotoxicidade foi obtido em células Jurkat tratadas com pectina autoclavada modificada, com IC50 de 2,63 mg mL-1. O cálculo do Índice de Seletividade (IS) indicou que que a pectina autoclavada modificada apresenta baixa toxicidade para células saudáveis. Em conclusão, os resultados demonstram promissor potencial antitumoral para a pectina autoclavada modificada, que precisa ser melhor explorado.

cAMP-elevating agents such as adenosine and epinephrine (after binding to beta-adrenergic receptor) contribute to local vascular dilation and some of these dilations are endothelium-dependent. Previous intracellular Ca 2+ imaging studies in mouse microvessel endothelial cells reported that addition of adenosine or epinephrine induced a Ca2+ influx which is blocked by CNG channel blockers such as L-cis-diltiazem or LY83583. Inside-out patch clamp studies confirmed the existence of a cAMP-activated current in endothelial cells, strongly suggesting a functional role of CNG, in particular CNGA2, channels in endothelial cells. The current study went further to show that similar Ca2+ influx in response to adenosine or epinephrine occurred in endothelial cells in freshly isolated mouse aortic strips and was again blocked by L-cis-diltiazem. By measuring the isometric force developed in mouse aortic strips, we showed that CNGA2 channel-mediated Ca2+ influx in endothelial cells contributed to the endothelium-dependent vascular dilatation in response to adenosine and epinephrine. / In conclusion, cyclic nucleotides playa vital role in the regulation of intracellular Ca2+ concentration in vascular cells and Jurket T cells. / In Jurkat T cells, cyclic nucleotides regulated Ca2+ mobilization in a different way. Fluorescence-imaging studies showed that cGMP inhibited store-operated Ca2+ influx and histamine-induced Ca 2+ rise in Jurkat T cells through activation of PKG. / Thromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can partly be attributed to TxA2-induced Ca2+ influx, which activates the Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells. Application of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relied on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by a L-type Ca2+ channel inhibitor nifedipine (0.5-1 muM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner, Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+, which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence Of CNGA2 protein in vascular smooth muscle cells, These data suggest a functional role of CNG channels in U-46619-induced Ca 2+ influx and contraction of smooth muscle cells. / Leung, Yuk Ki. / "August 2010." / Adviser: Yao Xiaoxiang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-132). / 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. / Abstract also in Chinese.

Natural Killer (NK) cellterapi är en lovande kandidat för cancerbehandling på grund av dess förmåga att känna igen och döda cancerceller. Adoptiv överföring av expanderade autologa eller allogena NK-celler har visat sig förbättra patientresultaten, särskilt i fall av akut myeloid leukemi (AML) efter stamcellstransplantation. En stor utmaning förknippad med NK-cellterapi är dock att få en tillräcklig mängd NK-celler för att uppnå meningsfulla terapeutiska resultat. Nuvarande metoder för att expandera NK-celler innebär ofta att T-celler avlägsnas från blodet, eftersom T-celler utgör över 50 % av blodets cellpopulation, medan NK-celler endast utgör cirka 10 %. Strategin för att ta bort T-celler involverar vanligtvis användningen av immunomagnetiska metoder, som kräver utbildad personal, är dyra och kräver god tillverkningssed (GMP) för att säkerställa metodens giltighet. Detta projekt syftade till att ta itu med denna fråga genom att etablera ett samodlingssystem mellan NK- och T-celler för att fungera som en modell för NK-expansion från patientens blod, vilket skulle kunna förbättra effektiviteten av cancerbehandling. Projektet syftade också till att undersöka det metaboliska beteendet (skillnader i näringsbehov och biprodukttolerans) mellan de två celltyperna. De två första experimenten med Jurkat gjordes med användning av en modifierad DMEM/RPMI och RPMI 1640-mediet under varierande glukos- och glutaminmålförhållanden. Resultatet från dessa två experiment visar att det modifierade DMEM/RPMI-mediet stödjer tillväxten av Jurkat-celler. Dessutom var produktionen av biprodukter inklusive laktat och ammoniak lägre i detta medium. Emellertid var glukos och glutamin avgörande för Jurkat-celltillväxt eftersom uppenbar konsumtion observerades under odlingsperioden. Det tredje experimentet syftade till att bedöma den negativa/reducerande effekten av glukos- och glutamintillstånd på Jurkat-celler. Resultaten från detta experiment applicerades sedan på NK-92-cellexpansion (fjärde experimentet). Det femte experimentet involverade samodling av båda celltyperna, med början med ett förhållande på 10% NK-celler till 90% Jurkat-celler, en ny celldiameterbaserad distributionsmetod användes för att förutsäga procentandelen NK-92-celler under samodlingen. Från dag 3 till dag 4 var det en ökning av andelen NK-celler, särskilt inom cellstorleksintervallet där de vanligtvis förekommer (17,4 µm). NK-cellerna utökades från 10 % på dag 0 till 52 % (i tillstånd med 2 mM glukos, 2 mM glutamin) på dag 3 och 45 % i tillstånd med 2 mM (glukos), 0,15 mM glutamin på dag 2. Sammantaget uppnådde denna studie framgångsrikt projektets mål att utveckla en samodlingsmodell genom att studera de enskilda cellinjernas metaboliska beteende. Ytterligare studier behövs för att undersöka effekterna av Interleukin 2 (IL-2) som produceras av Jurkat-celler på NK-celler. Dessutom skulle experiment med fler glukos- och glutaminmålkoncentrationer under längre odlingsperioder erbjuda en mer omfattande förståelse av samodlingssystemet, inklusive dess långsiktiga livskraft och celltillväxten av dessa cellinjer. / Natural Killer (NK) cell therapy is a promising candidate for cancer treatment due to its ability to recognize and kill cancer cells. The adoptive transfer of expanded autologous or allogenic NK cells has shown to improve patient outcomes, especially in cases of Acute Myeloid Leukemia (AML) following stem cell transplantation. However, a major challenge associated with NK cell therapy is obtaining a sufficient amount of NK cells to achieve meaningful therapeutic outcomes. Current methods for expanding NK cells often involve the removal of T cells from the blood, as T cells constitute over 50% of the blood's cell population, while NK cells make up only about 10%. The strategy to remove T cells typically involves the use of immunomagnetic beads, which require trained personnel, are expensive, and necessitate good manufacturing practices (GMP) to ensure the method's validity. This project aimed to address this issue by establishing a coculture system between NK and T cells to serve as a model for NK expansion from the patient blood which could improve the effectiveness of cancer treatment. The project also aimed at investigating the metabolic behavior (differences in nutrient demands and byproduct tolerance) between the two cell types. The first two experiment with Jurkat was done using a modified DMEM/RPMI and the RPMI 1640 media under varying glucose and glutamine target conditions. The result from these two experiments shows that the modified DMEM/RPMI media support the growth of Jurkat cells. In addition, the production of byproducts including lactate and ammonia were lower in this media. However, glucose and glutamine were crucial for Jurkat cells growth as evident consumption was observed during the culture period. The third experiment aimed to assess the negative/reducing impact of glucose and glutamine conditions on Jurkat cells. The findings from this experiment were then applied to NK-92 cells expansion (fourth experiment). The fifth experiment involved coculturing both cell types, starting with a ratio of 10% NK cells to 90% Jurkat cells, a new cell diameter based distribution method was used to predict the percentage of NK-92 cells during the coculture. From day 3 to day 4, there was an increase in the percentage of NK cells, particularly within the cell size range where they typically occur (17,4 µm). The NK cells were expanded from 10% on day 0 to 52% (in condition with 2mM glucose, 2mM glutamine) on day 3 and 45% in condition with 2mM(glucose), 0,15mM glutamine on day 2. Overall, this study successfully achieved the project's aim of developing a coculture model through studying the metabolic behavior of the individual cell lines. However, further studies are needed to investigate the effects of Interleukin 2 (IL-2) produced by Jurkat cells on NK cells. Moreover, conducting experiments with more glucose and glutamine targets concentrations over extended culture periods would offer a more comprehensive understanding of the coculture system, including its long-term viability and the cell growth of these cell lines.

<p>Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca²⁺ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. </p><p>A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.</p>

Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca2+ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.