No description available.

Tübingen, Univ., Diss., 2008.

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.

Das Masernvirus (MV) kann in Erkrankten eine schwere, langanhaltende Immunsuppression verursachen, wodurch Infektionen mit opportunistischen Pathogenen begünstigt werden. Diese basiert auf einer Paralyse der hämatopoetischen Zellen, welche das Virus durch Kontakt eines viralen Glykoproteinkomplexes zu einem unbekannten RezeptorX auf der Zell- Oberfläche induzieren kann. Kerncharakterisitika hiervon sind unter anderem die Herabregulation der Akt-Kinase-Phosphorylierung, die Inhibition der zellulären Proliferation und die Aktivierung der neutralen Sphingomyelinase 2 (NSM2). In einem kinetischen Phosphoproteom konnten zwei potentielle Interaktionsrezeptoren des MV identifiziert werden: CD43 und P2X3. Das hochglykosylierte Oberflächenmolekül CD43 ist auf hämatopoetischen Zellen ubiquitär exprimiert und reguliert in T-Zellen deren Überleben, Proliferation, Aktivierung, Migration und Adhäsion. P2X3 wird in hämatopoetischen Zellen nur in geringem Maße exprimiert. Seine funktionelle Bedeutung ist in diesem Kompartiment nicht bekannt. Beide Kandidaten wurden mittels CRISPR/Cas9 Verfahren einzeln oder kombiniert aus Jurkat-T-Zellen ablatiert, welche nachfolgend nach MV-Kontakt hinsichtlich der oben erwähnten MV-modulierten Parameter getestet wurden. Zusätzlich wurden iso- und allosterische P2X3-Inhibitoren an primären und Jurkat-T-Zellen verwendet, um dessen Rolle in Ca2+-Mobilisierung und Proliferation nach T-Zell-Rezeptor Co-Stimulation zu analysieren. Die genetische Depletion beider Rezeptor-Kandidaten verringerte die Effekte des MV auf alle getesteten Parameter signifikant, was darauf hindeutet, dass beide Proteine entscheidend an der T-Zell-Suppression beteiligt sind. Während die isosterische Inhibition von P2X3 keinen Effekt hatte, wurde die Proliferation primärer T-Zellen durch dessen allosterische Inhibition vor Co-Stimulation fast verdoppelt und die Effizienz der Ca2+-Mobilisierung in Jurkat- und primären T-Zellen signifikant erhöht. In P2X3-depletierten Jurkat-Zellen hingegen war die Ca2+-Mobilisierung nach Stimulation signifikant geringer als in WT-Zellen. In dieser Arbeit konnten zwei wichtige Mediatoren der MV induzierten T-Zell-Suppression identifiziert werden. Vor allem P2X3, dessen Expression, Regulation und funktionelle Bedeutung im hämatopoetischen Kompartiment noch nicht erforscht wurde, könnte ein vielversprechender Kandidat für eine antivirale Therapie darstellen, da ein klinisch getesteter P2X3-Inhibitor bereits verfügbar ist. / Measles virus (MV) infection induces a severe and long-lasting immunosuppression in patients resulting in infections through opportunistic pathogens. T cell paralysis is a major contributor to MV induced immunosuppression. This can be achieved through contact of a viral glycoprotein complex with an uncharacterized receptor X on the surface of hematopoietic cells. Contact-mediated downregulation of Akt-kinase phosphorylation, inhibition of proliferation and activation the neutral spingomyelinase 2 (NSM2) are key characteristics of T cell inhibition in vitro. Using a kinetic phosphoproteomic approach, two potential interaction receptor candidates were identified: CD43 and P2X3 receptor. The highly glycosylated surface protein CD43 is ubiquitously expressed on hematopoietic cells and is known to regulate T cell survival, proliferation, activation, migration and adhesion. The expression of P2X3 in this compartment is low and its functional importance unknown. It is widely expressed in neuronal cells where it is a major effector in the pathogenesis of chronic neuropathic pain. Using the CRISPR/Cas9 method both candidates were knocked down singly or combined in Jurkat T cells. Cells were then tested for the MV modulated parameters mentioned above upon MV challenge. In addition to that, iso- and allosteric functional inhibitors for P2X3 were employed on primary and Jurkat T cells to determine its role in calcium influx and proliferation after T cell receptor stimulation. The knockdown of both CD43 and P2X3 significantly decreased MV effects on all analyzed parameters (Akt-kinase phosphorylation, proliferation, NSM2 activation) indicating that both proteins play a major role in MV-induced T cell suppression. While isosteric inhibition of receptor P2X3 had no effect, its allosteric inhibition prior to stimulation increased proliferation of primary T cells almost twofold and significantly increased Ca2+ influx in Jurkat cells and primary T cells. In contrast, genetic depletion of P2X3 significantly reduced calcium influx after stimulation as compared to wildtype cells. In this study two important mediators of MV induced T cell suppression were identified. Amongst those, P2X3 whose expression, regulation and functional importance in the hematopoietic compartment has not been investigated yet, may represent a promising candidate for anti-viral therapy due to the existence of a clinically tested inhibitor.

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.

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.

In this research work, an innovative method for measurement of thermal conductivity of a small volume of liquids, microsphere, and the single cancer cell is demonstrated using a micro-pipette thermal sensor (MPTS). The method is based on laser point heating thermometry (LPHT) and transient heat transfer. When a single pulse of a laser beam heats the sensor tip which is in contact with the surrounding liquids or microsphere/cells, the temperature change in the sensor is reliant on the thermal properties of the surrounding sample. We developed a model for numerical analysis of the temperature change using the finite element method (FEM) in COMSOL. Then we used MATLAB to fit the simulation result with experiment data by multi-parameter fitting technique to determine the thermal conductivity. To verify the accuracy in the measurement of the thermal conductivity by the MPTS method, a 10µl sample of de-ionized (DI) water, 50%, and 70% propylene glycol solution were measured with deviation less than 2% from reported data. Also, to demonstrate that the method can be employed to measure microparticles and a single spherical cell, we measured the thermal conductivity of poly-ethylene microspheres with a deviation of less than 1% from published data. We estimated the thermal conductivity of two types of cell culture growth media for the first time and determined the thermal conductivity of cancerous Jurkat Clone E6-1 to be 0.538 W/m.K ± 2%. Using the sensor of 1-2μm tip size, we demonstrated the MPTS technique as a highly accurate technique for determining the thermal conductivity of microfluidic samples, microparticles, biological fluids, and a non-invasive method for measuring the thermal conductivity of single cancer cell. This MPTS technique can be beneficial in developing a diagnosis method for the detection of cancer at an early stage. We also compared three effective thermal conductivity models for determining the weight percentage of Jurkat cell, considering water and protein as the major constituents. We discovered that a combination of Maxwell-Euken and effective medium theory model provides the closest approximation to published data and, therefore, recommend for the prediction of the cell composition.

Irma Stašiauskaitė Citochalazino E ir deoksinivalenolio veikimo mechanizmų tyrimas MH-22A ir Jurkat linijų ląstelėse SANTRAUKA Šiame darbe buvo analizuota dviejų mikotoksinų - citochalazino E ir deoksinivalenolio - vaidmuo skirtingos kilmės ląstelių proliferacijos bei žūties procesuose ir galimų signalinių kelių aktyvacija atsakant į CE ir DON poveikį. Yra žinoma, kad mikotoksinų sukeliamas toksiškumas, ląsteliniame lygyje gali pasireiškti skirtingais mechanizmais ir tai yra susiję su jų chemine struktūra [Duxbury, 2004]. Abu tirti mikotoksinai savo struktūroje turi epoksido grupes, kurios, manoma, lemia jų toksiškumą. Deoksinivalenolio veikimas literatūroje yra žymiai plačiau išnagrinėtas. Nustatyta, kad 4.5 - 41 µM šio mikotoksino koncentracijos 50% gali slopinti ląstelių proliferaciją. Šiuo atveju DON veikimas priklauso nuo ląstelių rūšies. Yra duomenų, kad mažos DON koncentracijos indukuoja mitogenų aktyvinamas kinazes - p38, JNK1/2 ir ERK [Pestka ir kt., 2005]. Šių MAP kinazių vaidmuo ląstelėje yra nevienareikšmis ir gali varijuoti priklausomai nuo poveikio ar ląstelių tipo. Apie tai byloja daug literatūros šaltinių [Minden ir kt, 1997; Finley ir kt, 2003; Huh ir kt., 2004]. Savo darbe modeline sistema pasirinkome dvi skirtingos kilmės ląstelių linijas: kepenų kilmės pelės hepatomos MH-22A ląstelių liniją ir kraujo kilmės Jurkat ląstelių liniją. Tyrimams naudojome 0.1 μg/ml, 1 μg/ml, 10 μg/ml CE ir DON koncentracijas. Stipriausiu antiproliferaciniu... [toliau žr. visą tekstą] / Irma Stašiauskaitė Study of mechanisms underlying cytochalasin E and deoxynivalenol effects in MH-22A and Jurkat cell lines SUMMARY In recent years, a great deal of interest has been generated regarding the study food safety. The Food and Agriculture Organization estimates that at least 5-10% of the world food supply is lost annually to fungi and mycotoxins – poisonous chemicals produced by microfungi during their growth on food, feeds and various agricultural commodities. The effects of mycotoxins on humans and animals are dose-related and include acute and chronic effects, in the case of some mycotoxins, carcinogenicity, mutagenicity and supression of the immune system. Deoxynivalenol, one of mycotoxines, is produced by several Fusarium genus. It can be found as natural contaminant in variuos cereal crops and in processed grains. Besides its acute and chronic toxicity and effects on immune function, deoxynivalenol contribute to gastrointestinal diseases in exposed humans. Cytochalasin E – a toxin synthesized by Aspergillus clavatus, that inhibit cell division and protein synthesis, is nephrotoxic and considered carcinogenic. The aim of this study was to assess the effects of deoxynivalenol and cytochalasin E on cell viability and to relate apoptosis to activation of signaling molecules of stress activated MAPK pathway. In our study we determined, that cytochalasin E and deoxynivalenol mechanism of action depends on cell type, concentration and time of exposure... [to full text]

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.

Opiates have been shown to inhibit cell growth and trigger apoptosis, but the underlying molecular mechanisms remain unclear. We have previously shown that morphine induces Fas expression and promotes Fas-mediated apoptosis. Here, we investigated the mechanisms by which morphine modulates apoptosis in human Jurkat cells. Morphine-induced apoptosis was inhibited by transfection with a dominant negative Fas-associated death domain (FADD) plasmid, revealing that morphine-induced apoptosis is dependent on FADD. Furthermore, suppression of endogenous p53 expression by RNA interference technology considerably attenuated the morphine-induced apoptosis. In addition, morphine-induced apoptosis seems to be dependent on the activation of phosphatidylinositol 3-kinase (PI3K), as PI3K inhibition by the PI3K inhibitor LY294002 significantly enhanced morphine-induced apoptosis. Moreover, inhibition of Akt or nuclear factor-kappaB (NF-κB) expression by RNA interference technology also dramatically increased morphine-induced apoptosis. Our study thus demonstrates that morphine induces Jurkat cell apoptosis through FADD/p53, anti-apoptotic PI3K/Akt and NF-κB pathways.