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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
101

Modulation of inflammatory process and tissue regeneration in calvaria mouse models

Al-Hashemi, Jacob Yousef 17 June 2019 (has links)
MicroRNAs (miRNAs) are short, non-coding RNAs involved in the regulation of several processes associated with inflammatory diseases and infection. Bacterial infection modulates miRNA expression to subvert innate immune response. In this study, we analyzed bacterial modulation of miRNAs in bone-marrow-derived macrophages (BMMs), in which activity was induced by infection with Porphyromonas gingivalis (Pg) through a microarray analysis. Several miRNA expressions levels were modulated 3 hours post infection (at a multiplicity of infection (MOI) of 25). A bioinformatics analysis was performed to further identify pathways related to the innate immune host-response pathways that are under the influence of the selected miRNAs. To assess the effects of the identified miRNAs on cytokines secretion (pro inflammatory TNF-α and anti-inflammatory IL-10), BMMs were transfected with selected miRNAs mimics or inhibitors. Transfection with mmu-miR-155 and mmu-miR- 2137 did not modify TNF-α secretion while their inhibitors increased it. Inhibitors of mmumiR-2137 and mmu-miR-7674 increased the secretion of the anti-inflammatory IL-10. In Pginfected BMMs, mmu-miR-155-5p significantly decreased TNF-α secretion while inhibitor of mmu-miR-2137 increased IL-10 secretion. In vivo, in a Pg-induced calvarial bone resorption mouse model, injection of mmu-miR-155-5p or anti-mmu-miR-2137 reduced the size of the lesion significantly. Furthermore, anti-mmu-miR-2137 significantly reduced inflammatorycell infiltration, osteoclast activity and bone loss. Bioinformatics analysis demonstrated that pathways related to cytokines and chemokines related pathways but also osteoclast differentiation may be involved in the observed effects. The study highlights the potential therapeutic merits of targeting mmu-miR-155-5p and mmu-miR-2137 to control inflammation induced by Pg infection. To assess the regenerative process in the same animal model, we aimed to compare the effect of Bone Morphogenic Protien 2 (BMP2), Platelets Rich Plasma (PRP), Leukocyte-Platelets Rich Fibrin (L-PRF), and Polygucosamine (PGIcNAc) on bone formation in critical size bone defects in mice. One-hundred-thirty-eight mice were divided into 23 groups (n=6), negative control, different combinations of the PGIcNAc with or without of BMP2, Collagen Sponge (SurgiFoam), PRP, and L-PRF. The 5mm defect, then, was allowed to heal. After six weeks, samples were analyzed for bone formation utilizing radiographs, H&E staining, alkaline phosphatase staining. Our results show that BMP2 were able to produce 90-95% healing of critical size defects after six weeks histologically and radiographically. However, SurgiFoam, PRP and L-PRF with or without PGIcNAc were able to close 60% of the original defect. This study supports that BMP2 is more effective for bone regeneration than SurgiFoam, PRP, L-PRF and PGIcNAc.
102

Vergleich von konstanter und periodischer Trenngrenze bei der Thrombozytapherese mit dem Blutzellseparator Fresenius AS 104

Balke, Bettina 28 October 2002 (has links)
Vergleich zweier Thrombozytaphereseprotokolle des kontinuierlich arbeitenden Zellseparators FRESENIUS AS 104 mit dem Ziel einer regelmäßigen Senkung der Leukozytenkontamination (LK) in Thrombozytapheresekonzentraten unter die kritische Grenze für eine Alloimmunisierung von 1 x 106 Leukozyten pro Transfusionseinheit ("critical immunological load of leukocytes" (cill)). Die Bestimmung der Leukozytenzahl in diesem niedrigen Konzentrationsbereich stellt ein bekanntes Problem dar, weshalb drei Methoden dazu miteinander verglichen wurden. Bei je 50 Thrombozytapheresen von gesunden Spendern wurde das Standardprogramm mit konstanter Trenngrenze bzw. die Programm-Modifikation mit periodischer Trenngrenzenposition eingesetzt. Der Vergleich der beiden Trenngrenzeneinstellungen erfolgte auf der Basis der ermittelten LK in den Thrombozytapheresekonzentraten sowie den errechneten Qualitätsparametern Plättchenausbeute und Separationseffizienz. Zur Bestimmung der LK wurden neben der mikroskopischen Auszählung der Zellen mit Hilfe der modifizierten Nageotte-Kammer zum einen ein neuartiger Blutbildautomat (Abbott CD 3500), zum anderen durchflußzytometrische Methode (FACS) verwendet. Die Effektivitätsparameter beider Thrombozytaphereseverfahren wichen signifikant voneinander ab: Der Thrombozyten-Ertrag ist bei Anwendung der konstanten Trenngrenze war im Mittel mit 3,6 x 1011/TE signifikant höher als bei Anwendung der periodischen Trenngrenze mit 3,3 x 1011/TE (t-Test:p = 0,017). Dementsprechend sind bei vergleichbaren Mittelwerten für Separationsdauer (63min vs. 64min) und Separationsvolumen (3588 ml vs. 3737 ml) sowohl die Separationsleistung (5,9 x 109/min vs. 5,2 x 109/min; t-Test: p = 0,018), als auch die Separationseffizienz (48,0% vs. 43,3%; t-Test: p = 0,005) signifikant niedriger bei der Anwendung der periodischen Trenngrenzenposition. Die Leukozytenkontamination ist signifikant niedriger bei Anwendung der periodischen Trenngrenze: Je nach angewandter Methode liegt die mittlere Leukozyten-Kontamination bei Anwendung der konstanten Trenngrenze bei 4,9 x 106/TE, bei Anwendung der periodischen Trenngrenze bei 2,1 bzw. 1,2 x 106/TE (U-Test: p < 0,0001). Bei der Bestimmung mittels FACS lag die Leukozytenkontamination bei 46 von 50 Thrombozytapheresen mit periodischer Trenngrenze unter dem angestrebten Wert von 1 x 106/TE (vs. konstante Trenngrenze: 9 / 50) . Beim Vergleich zwischen Nageotte-Kammer-Zählung als Goldstandard und Blutbildautomat zeigt der Blutbildautomat Abbott CD 3500 bei Leukozyten-Konzentrationen unter 50 bis 80 pro Mikroliter eine unzureichende Genauigkeit (r = 0,546). Zwischen Nageotte-Kammer-Zählung und FACS-Methode findet sich hingegen eine gute Korrelation (r = 0,930). Die Modifikation des Thrombozytenseparationsverfahrens mit periodischer Verschiebung der Trenngrenze bewirkt eine signifikante Senkung der Leukozytenkontamination. Der Thrombozytenertrag und die Separationseffizienz werden um etwa 10% gegenüber dem Standardverfahren mit konstanter Trenngrenze gesenkt. Dieser Thrombozytenverlust ist jedoch bedeutungslos, da die zum Erreichen des gesetzlich vorgeschriebenen cill-Wertes notwendige Filterung des Standard-Thrombozytapheresekonzentrats ebenfalls zu einem Thrombozytenverlust führt, der in derselben Größenordnung liegt. / Comparison of two different separation protocols for plateletpheresis with the Fresenius Blood Cell Separator AS104. Aim was to achieve a low white blood cell (WBC) contamination of the resulting platelet concentrate below the critical immunological load of leukocytes responsible for alloimmunisation. Furthermore comparison of three different methods to determine the exact count of WBC in this low range of WBC concentration. 50 healthy donors each underwent platelet apheresis with the Fresenius Blood Cell Separator AS104 using the periodically alternating interface position (PAIP) or a standard interface position (SIP). To evaluate the influence, WBC contamination of the platelet concentrate and separation efficiency (SE) were investigated. WBC were counted either microscopically (modified Nagotte chamber) or dermined on a whole blood counter Abbott CD 3500 or on a FACScan flowcytometer. SE with PAIP is lower than with SIP ( 43.3% vs. 48%; t-test: p=0.005). WBC contamination with PAIP is lower than with SIP: depending on the method of counting 2.1 resp. 1.2 x 106/TE for PAIP vs. 4.9 x 106/TE for SIP (u-test: p < 0.0001). Correlation between counting the WBC by the modified Nagotte chamber as a ´gold standard´ methode and the Abbott CD 3500 was poor (r = 0.546), whereas counting by modified Nagotte chamber and by FACScan showed a good correlation (r = 0.93). Modification of the separation protocol for platelet apheresis with the Fresenius Blood Cell Separator AS104 by PAIP results in a lower WBC contamination of platelet concentrates. The lower SE and total amount of platelets in the concentrates with the PAIP method compared to SIP make no difference in the end because the concentrates produced with SIP have additionally to be filtered in order to achieve a WBC conatmination below the cill and undergo thereby a reduction of platelets in about the same range.
103

Interação anlodipina/diclofenaco sobre o comportamento leucocitário em ratos espontaneamente hipertensos (SHR) e ratos wistar normotensos. / Amlodipine and diclofenac interaction on leukocyte behavior in spontaneously hypertensive rats (SHR) and normotensive wistar rats.

Rodrigues, Stephen Fernandes de Paula 30 April 2008 (has links)
Anlodipina e diclofenaco podem ser combinados de forma que o potencial de interação é alto. Investigamos se anlodipina interfere com o efeito antimigratório do diclofenaco em ratos espontaneamente hipertensos (SHR) e ratos wistar. Os ratos foram tratados (15 dias) com diclofenaco e anlodipina, juntos ou separadamente. A migração leucocitária foi estudada por microscopia intravital e a expressão de moléculas de aderência por imunohistoquímica. A anlodipina reduziu migração leucocitária em SHR e ratos wistar por reduzir a expressão de ICAM-1 em células endoteliais venulares. A redução da migração leucocitária em ratos wistar pode decorrer também da redução da expressão de PECAM-1 em células endoteliais. A combinação reduziu o efeito do diclofenaco sobre a migração em SHR e wistar. Em SHR esse efeito envolveu a redução de ICAM-1. A redução da PA causada pela anlodipina em SHR parece contribuir para a redução da migração leucocitária. Interação farmacocinética foi excluída. / Amlodipine and diclofenac may be combined so the potential for interaction of both is high. We determined if amlodipine interferes with the antimigratory effect of diclofenac in spontaneously hypertensive rats (SHR) and wistar rats. Rats were treated with either diclofenac or amlodipine alone or combined (for 15 days). Leukocyte migration was evaluated by intravital microscopy and cell adherence molecules expression by immunohistochemistry. Amlodipine reduces migration in both SHR and wistar rats by reducing venular endothelial cell ICAM-1 expression. In wistar the antimigratory effect of amlodipine may also be due to reduction in PECAM-1 expression. Diclofenac effect was reduced by amlodipine in both SHR and wistar. In SHR amlodipine reduces the effect of diclofenac by impairing its reducing effect on ICAM-1. Reduction of the BP levels seems to contribute to reduction of the leukocyte migration caused by amlodipine in SHR. Pharmacokinetic interaction was excluded.
104

Análise de polimorfismos dos genes KIR e HLA classe I em pacientes com câncer colorretal

Silva, Pamela Portela da January 2016 (has links)
O câncer colorretal (CCR) pode ocorrer em qualquer parte do cólon ou do reto e representa o terceiro câncer mais comum no mundo em ambos os sexos. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). O objetivo deste estudo foi avaliar a associação entre os genes KIR e os ligantes HLA em pacientes com câncer colorretal e controles saudáveis. Examinamos o polimorfismo de 16 genes KIR e seus ligantes HLA em 154 pacientes caucasóides com CCR e 216 controles saudáveis pela técnica de PCR-SSO e PCR-SSP. Quando comparamos os dois grupos, não foram encontradas diferenças significativas para os ligantes HLA e os genes KIR após correção de Bonferroni. Entretanto, o grupo de genótipos Bx (heterozigoto e homozigoto para o haplótipo B) foi mais frequente nos controles, quando comparados com os pacientes. Estes achados sugerem que altos níveis de ativação de sinais KIR aparecem como proteção para o câncer colorretal. / Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 healthy controls by PCR-SSO and PCR-SSP. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx group genotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx genotypes could have some protection to colorectal cancer. These findings suggest that higher levels of activating KIR signals appear as protective to colorectal cancer.
105

Impacto da terapia mecânica não cirúrgica em pacientes com periodontite crônica e agressiva generalizadas / Impact of non-surgical periodontal therapy on generalized chronic and aggressive periodontitis

Karina Schittine Bezerra Lomba 26 February 2010 (has links)
O objetivo deste estudo foi avaliar o impacto do tratamento periodontal não cirúrgico sobre a atividade de elastase e o volume de fluido gengival nos pacientes portadores de periodontite crônica e agressiva generalizadas. Foram avaliados 18 pacientes com periodontite crônica (idade média 48,6 DP 7,5 anos), e 11 com periodontite agressiva (idade média 27,9 DP 6,54 anos). Foram utilizados os parâmetros clínicos de avaliação de profundidade de bolsa à sondagem (PB) (mm), nível de inserção (NI) (mm) e sangramento à sondagem (SS). As medidas clínicas e as amostras de fluido gengival foram colhidas a partir dos cinco sítios mais profundos (P) e de cinco sítios rasos com gengivite (G) de cada paciente, antes e 90 dias após o término do tratamento. As etapas clínicas obedeceram ao seguinte cronograma: seleção e exame periodontal; coleta de fluido gengival; tratamento periodontal; reavaliação, compreendendo o exame periodontal e coleta de fluido gengival. O tratamento levou, em média, 4 sessões de 40 minutos cada por paciente, com intervalo de 1 semana entre elas. As consultas para reavaliação foram feitas 90 dias após o término do tratamento. O teste de Wilcoxon foi utilizado para comparar os dados antes e depois do tratamento e o teste não pareado de Mann-Whitney U-test foi utilizado para comparar os grupos de periodontite crônica e agressiva. A amostra analisada antes e após o tratamento não apresentou diferenças significativas entre o grupo com periodontite crônica e agressiva, que responderam de forma similar a todos os indicadores avaliados, exceto para a profundidade de bolsa à sondagem nos sítios rasos com gengivite (p = 0,039) e para o sangramento à sondagem (p = 0,021) nos sítios profundos, ambos mais reduzidos na periodontite crônica após o tratamento. A elastase apresentou, após o tratamento, redução significativa nos sítios profundos, para a periodontite crônica (p = 0,012) e agressiva (p = 0,02). Em relação ao volume de fluido gengival, houve significativa redução após o tratamento nos pacientes com periodontite crônica e agressiva, tanto nos sítios rasos (p = 0,03 e p = 0,03) como nos profundos (p &#706; 0,001 e p = 0,003), respectivamente. Concluindo, os grupos com periodontite crônica e agressiva generalizadas comportaram- se de maneira semelhante frente à terapia mecânica não cirúrgica. Ainda, a terapia periodontal mecânica não cirúrgica mostrou redução significativa do volume de fluido gengival em todos os sítios analisados, e da atividade neutrofílica, nos sítios profundos, associada a reduções significativas em todos os indicadores clínicos analisados após o tratamento. / The aim of this study was to investigate the impact of non-surgical mechanical treatment on elastase activity and gingival crevicular fluids (GCF) volume of patients with untreated generalized chronic and aggressive periodontitis. Eighteen patients with generalized chronic periodontitis (mean age 48,6 SD 7,5 years old) and eleven with generalized aggressive periodontitis (mean age 27,9 SD 6,54 years old) were evaluated. The clinical parameters adopted were pocket probing depth (PPD) (mm); attachment level (AL) (mm) and bleeding on probing (PB). Clinical measures and GCF were collected from the 5 deepest sites (P) and from 5 shallow sites with gingivitis (G) from each patient of each group, before and 90 days after non- surgical periodontal treatment. There were adopted the following clinical steps: patients selection and periodontal exam; GCFs collection; periodontal treatment; re- evaluations, comprising periodontal exams and GCFs collection. The periodontal treatment lasted about about 4 sections with 40 minutes each, 1 section per week. Re-evaluations were performed 90 days after completion of the treatment. Wilcoxons statistic test compared the data before and after treatment and Mann-Whitneys non parametric U-test was used to compared chronic and aggressive periodontitis groups. There were no significant differences for all clinical parameters analysed before and after treatment,except for pocket probing depth on shallow sites (p = 0,039) and for bleeding on probing (p = 0,021) on deep sites, both in lower levels in chronic periodontitis after treatment. The elastases activity showed, after treatment, a significant reduction on deep sites for chronic (p = 0,012) and aggressive (p = 0,02) periodontitis. In concern to GCFs volume, there was significant reduction on both chronic and aggressive periodontitis, in shallow (p = 0,03 e p = 0,03) and in deep pockets (p &#706; 0,001 e p = 0,003), respectively, after treatment. In conclusion, both groups responded simmilarly to the mechanical non- surgical treatment. Moreover, non- surgical periodontal treatment showed significant reductions on GCFs volume on all analysed sites and reduction on neutrophilics activity, on deep sites followed by the significant reduction on clinical indicators after treatment.
106

Molecular Approaches To understand Cellular Differentiation - A Study Using BeWo Choriocarcinoma Cells

Neelima, P S 08 1900 (has links)
Cellular differentiation is a complex but fascinating process in all multicellular organisms. Differentiation can involve changes in numerous aspects of cell physiology; size, shape, polarity, metabolic activity, responsiveness to signals, and changes in gene expression profiles. These changes form the basis for differentiation to occur. The human hemochorial placenta is an intricate apposition of fetal and maternal tissues that is strategically juxtaposed at the interface, with its widespread ‘villous’ or tree-like projections, directly in contact with maternal blood. It is therefore, ideally suited to perform life-sustaining functions such as exchange of nutrients, respiratory gases and metabolic wastes, with the maternal supply. It also plays a central role in the maintenance of the immunologically privileged status of the fetal semi-allograft. Placental development is directed towards the establishment of a continuous nutrient supply to the developing fetus. This requires efficient access of maternal blood to a transporting surface, the multinucleate syncytiotrophoblast layer. This is made possible by the rapid proliferation and ensuing invasion of mononuclear trophoblasts into the maternal uterus and remodeling of the spiral arteries therein. It is interesting to note that in early pregnancy, it is the placenta that first engages its active growth and proliferation and only then, permits the logarithmic growth phase of the embryo. As a developing organ, the placenta undergoes constant tissue remodeling, which is characterized by the functional loss of trophoblast cells by apoptosis. Most of these changes occur at the trophoblast layer of the placental villous that is composed of two cell types: cytotrophoblasts (CT) and syncytiotrophoblasts (ST). The mononuclear cytotrophoblast cells, which are located between the syncytiotrophoblast layer and its basement membrane, proliferate and fuse during trophoblast differentiation to form the overlying multinucleated syncytium. CT are highly proliferating and invasive cells, in contrast to the ST which are non proliferative less invasive and functionally very active. Syncytiotrophoblast cells form the continuous, uninterrupted, multinucleated, epithelium-like surface of the placental villous that separates maternal blood from the villous interior. ST performs a crucial role in feto-maternal exchanges and serves as an endocrine tissue by its ability to synthesize and secrete a variety of hormones such as GnRH, chorionic gonadotrophin (CG), placental lactogen (PL) and steroid hormones involved in the homeostasis during pregnancy. Thus, differentiation of CT into ST serves as an ideal model to study cellular differentiation as morphologically and functionally these cells exhibit highly contrasting features. The molecular basis of cytotrophoblast differentiation has been studied using primary cultures of human trophoblast cells as a model system. Highly purified preparations of mononucleated cytotrophoblast cells can be isolated from preterm and term placental tissue by enzymatic dispersion. The isolated cells from term placental tissue aggregate spontaneously in culture and fuse to form a multinucleated syncytiotrophoblast which synthesizes and secretes placental lactogen (hPL), chorionic gonadotropin (hCG) and other syncytiotrophoblast-specific protein and steroid hormones . These in vitro changes, which recapitulate important activities accomplished by normal cytotrophoblast cells during in vivo maturation, implicate a critical relationship between the differentiation of cytotrophoblast cells into syncytiotrophoblast cells. Though primary cell culture is an ideal model to study these changes, it comes inherently associated with various problems like health risk of handling human tissues, time involved, variability in each placental samples depending on health status of the subject and quite often lack of history of the subject which makes the results from these experiments difficult to reproduce and assess. One way to overcome this is the cell culture model which is a reproducible experimental system and permits the direct observation of time-dependent processes and their experimental manipulation. BeWo cells, the cells which we have used in our study, were derived from human gestational choriocarcinoma. These cells are the highly invasive malignant counterparts of the normal human trophoblast wherein, the limited capacity for cell proliferation is far exceeded. However, they still retain important features of their normal counterpart, like the potential of hormone production and induced differentiation. Differentiation of CT to ST is precisely controlled by different agents such as transcription factors, hormones, growth factors, cytokines and oxygen levels. BeWo cells have been used by other investigators as well as by us and it has been shown that these cells can be induced to differentiate with the agents mentioned above and terminally differentiate into cells which express typical characteristics of the normal differentiating trophoblast; like morphological transition from cytotrophoblast to syncytiotrophoblast-like cells, increased production of protein and steroid hormones (hCG, hPL, estrogens, progesterone); increased activity of cellular alkaline phosphatase and arrested cell proliferation. Since these cells can be triggered by external agents to differentiate, they serve as a useful model for the study of changes that occur during differentiation. Using primary cells and various cell lines including BeWo cells, various groups have attempted to study trophoblast differentiation and the regulators that control this process. The results of such study have only come out with a list of genes or proteins which might be having a role in this process and no functional correlation has been drawn so far from these studies. The members of the syncytin protein family, ADAM (a disintegrin and metalloprotease) proteins may well be some of the main players in the process of trophoblast fusion; some of the requisites of trophoblast fusion being redistribution of phosphatidylserine to the outer leaflet of the plasma membrane and activity of certain intracellular proteases. Clearly, further studies on trophoblast differentiation are needed to answer the question of the precise identity of regulatory proteins and role of these proteins during differentiation. The present study is aimed at gaining insights into the process of trophoblast differentiation and the molecular events which occur during this process. Our aim is also to study the regulated process of differentiation using BeWo cell model and identify the differentially expressed genes and relate the known function of these gene products to changes seen during differentiation process. We have employed the Differential Display Reverse Transcriptase Polymerase Chain Reaction (DD-RTPCR) and Microarray analysis to monitor the changes in gene expression. In CHAPTER 1, a brief account of morphological, biochemical and physiological changes which occur during placentation and trophoblast differentiation is discussed. Various aspects of placental function are discussed in brief, with special reference to the many unique abilities of trophoblast cells that contribute to a successful pregnancy. Detailed accounts of molecular mechanism of cellular differentiation, the models used in these studies and the advantages and drawbacks have been highlighted. The results of the previous studies from our laboratory using different model system and the outcome of the study are also outlined in this chapter. The advantages and disadvantages of the primary cell lines and the ease of handling of continuous cell culture model, BeWo is also presented in this chapter. The aim and objective of our study is to understand the molecular mechanisms underlying the trophoblast differentiation and the literature available is reviewed in the light of the objective and the aims and scope of the present study. The details regarding the materials used and the techniques employed during the entire study are outlined in CHAPTER 2-‘Materials and Methods’. The conditions for culture of BeWo human choriocarcinoma cell line are described and details of procedures employed for the validation of BeWo cells as a model system for monitoring the process of cellular differentiation are mentioned in this chapter. The details of the procedures employed for isolation of RNA, Reverse Transcriptase Polymerase Chain Reaction (RTPCR), Differential Display RT-PCR (DD-RT-PCR), Microarray analysis, Northern Blot analysis and Western Blot analysis are also described. The principle of the MTT assay used for verifying the viability of cells following various treatments is provided along with the working protocol. This chapter also includes protocols of the in vivo studies in rat, the methods employed for rat uterine mince cultures and isolation of rat uterine epithelial cells and dose and duration of the various treatments with steroid hormones and their inhibitors, treatment with protein kinase inhibitors in cell culture system are also described. In addition, this chapter also describes the procedures for transfection of hTERT, silencing of SLPI gene using SiRNA approach, gelatin zymography, MAP Kinase assay, FACS, cloning and expression of SLPI protein and procedure employed for raising antibodies to SLPI in rabbit. Finally, details of statistical tests employed fro anlaysis of data are presented. The results obtained in the present study are presented in 4 chapters(Chapters 3-6), CHAPTER 3 describes the characterization and validation of model system employed- BeWo cells to study human trophoblastic differentiation. BeWo cells under normal culture conditions resemble cytotrophoblasts like cells and when treated with various effectors of differentiation can be induced ot differentiate into syncytiotrophoblasts. We used 10 µM Forskolin to induce differentiation in BeWo cells. Forskollin is known to induce characteristic changes associated with human trophoblast differentiation in these cells. Incubation of BeWo cultures in the presence of 10 µM Forskolin resulted in dramatic morphological biochemical changes intheir cytotrophoblast-like phenotype. Mononuclear cells were seen to fuse to form multinucleate syncytial structures over a period of 72-96 hours in culture. This process was also associated with an increased production of β-hCG, Endoglin and hTERT thereby validating this model system for study of human trophoblastic differentiation. Analysis of cell cycle genes in this system established the arrest of proliferation thus further validating the system. The viability of these cells, during the entire period of culture, was verified using the MTT assay. This chapter discusses the importance of in vitro cell culture systems in the study of human placental development, and also addresses the suitability of these model systems for the study of human trophoblast proliferation and differentiation. One of the important finding of our earlier studies was that arrest of proliferation was a prerequisite for trophoblast differentiation to occur. This conclusion was based on the fact that telomerase expression which is a hallmark of all proliferating cells was down regulated in BeWo cells by 48h as assessed by TRAP (Telomere Repeat Amplification Protocol) assay or RT-PCR analysis for hTERT which is the catalytic subunit of telomerase. Telomerase activity was undetectable by about 96th by which time syncytium formation is normally completed after the addition of differentiation inducing agents like Forskolin, TGF β etc. Although the telomeric holo enzyme consists of many components the subunits which are critical for enzyme action are hTERT and hTR; hTR; hTR which is the RNA component of telomerase is ubiquitously expressed in most cell types including telomerase negative cells such as differentiated somatic cells. Since the BeWo cells can be induced to differentiate into multinucleated ST by addition of Forskolin and periodically the aged ST are eliminated by apoptosis. It is very well documented that the life span of ST is very limited and the ST have to be replaced by the freshly formed ST out of fusion of CT. Considering this, it was of interest to test whether differentiation can be prevented or delayed by extending the expression of telomerase activity. This would further validate our system that one of the requisites for cells to differentiate is down regulation of hTERT in BeWo cells. This was achieved by transfection of BeWo cells with hTERT expression vector. The results of the study clearly established that we were able to over express hTERT in BeWo cells; we also noticed an increase in the proliferation of BeWo cells as assessed by BrdU incorporation. In agreement with this observation is the fact that, in contrast to the empty vector transfected cells, in hTERT transfected group, the cell density appeared to be clearly more at 72 h. That the decrease in the hTERT expression in the control (empty vector transfected) is not due to cell death was established by MTT assay, which indicated that there was no difference in the viability between control and hTERT transfected cells. Further more, results of analysis for a variety of cell proliferation and differentiation markers by RT-PCR and Western blot analysis clearly supports the conclusion that hTERT over expression delays syncytium formation. Although reports are available on the differential expression of genes during differentiation of CT to ST with both primary cell lines as well as BeWo cell line, relatively less is known about the functional importance of differentially expressed genes. In CHAPTER 4, results of our studies to profile the differentially expressed genes during Forskolin induced differentiation in BeWo cells by two approaches DD-RTPCR and microarray analysis and relate the known functions of these genes to changes that occur during the differentiation of CT to ST are presented. We identified several genes that had robust change during differentiation by DD RTPCR and the differential expression of ten transcripts was confirmed by Northern blot analysis. The genes which we identified were SLPI, Elongation factor-1 alpha -1, Prolyl hydroxylase beta, LIMO-4 etc. These genes were either shown to have a role during differentiation of cells or have functional role in the syncytiotrophoblasts. Secretory Leucocyte Protease Inhibitor was one of the differentially expressed transcripts which were significantly up regulated during Forskolin induced differentiation of BeWo cells. SLPI which is a 12 KDa protein reported to exhibit a variety of activities which include inhibition of proteases and elastase, in addition to antibacterial and anti inflammatory activities. It was chosen for our further studies because of its multifunctional role in placenta and also during implantation. Micro array analysis revealed the up-regulation of hCG, hCS, and Endoglin thus validating the experimental system. Several candidate genes that could influence trophoblast differentiation, cell adhesion and cellular proliferation were identified. Genes involved in cellular proliferation include cyclin M3, replication factor 3, signal-induced proliferation-associated gene 1, osteonectin, clusterin, etc clearly indicating a growth-arrested phenotype for the differentiating BeWo cells. Trophoblastic differentiation associated genes included adipose differentiation-related protein, GADD45A, PPAR binding protein, galectin 3, tubulins, collagen, stathmin, etc. The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest or DNA repair or apoptosis. Although we did not observe any change in the p53 mRNA levels, the total protein level as well the phosphorylation status of p53 was up regulated upon differentiation. We confirmed the down regulation of Cyclin D1, D2 and PCNA in differentiated cells and up regulation of CDK inhibitors, P27kip1, P21cip1which are p53 induced genes by RT-PCR and Western blot analysis. Phosphorylation of ser 20 leads to reduced interaction of p53 with its negative regulator MDM2. MDM2 inhibits the accumulation of p53 by targeting it for ubiquitination and proteosomal degradation. Analysis for the phosphorylated status of p53 revealed that specifically the ser 20 phosphorylated p53, was increased upon differentiation. Phosphorylation of ser-392 has been reported to influence the growth repressor function, DNA binding and transcriptional activation of p53 and in agreement with this, western blot analysis revealed an increase in the ser-392 phosphorylated p53. These results suggest that p53, a nuclear protein regulating several genes involved in proliferation and differentiation is playing a pivotal role in growth arrest during trophoblast differentiation. We also noticed that several components of the apoptotic cascade are differentially expressed in cytotrophoblast and the syncytiotrophoblasts layer, and these changes appear to be associated with the stage of apoptosis. Apoptosis is involved in the removal of aging syncytiotrophoblasts and it also promotes cytotrophoblast fusion and formation of the syncytial layer. We found that various apoptosis related genes are up regulated and anti apoptotic genes suppressed following differentiation in our micro array analysis. We identified the involvement of p53 in this process also and chapter 4 deals with this aspect. Genes which regulate the invasive behaviour of trophoblasts which include MMP2, cathepsin K, cystatin N, SLPI and cysterine-rich angiogenic inducer 61, etc. were found to be up regulated following differentiation in our micro array analysis, which establishes these differences in gene expression reflects the physiological changes that occur during placentation. The Co-ordinated regulation of ptoteases and protease inhibitors I (for example SLPI, cystatin B and MMP2) suggests that these genes play an important role in the regulation trophoblast invasion at the uterine-placenta interface in vivo. Our studies revealed that one of the transcripts,namely, SLPI(Secretory leukocyte protease inhibitor) was robustly up regulated as assessed in DDRT-PCR, micro-array, Northern blot and RT-PCR analysis. Considering its importance in implantation, placentation and maintenance of pregnancy several aspects of this multifunctional protein were studied in detail and the results are presented in CHAPTER 5. Studies on the regulation of this transcript in Be-Wo cells revealed that SLPI mRNA is regulated by progesterone in Be-Wo cells. The up regulation of SLPI mRNA by progesterone was specifically inhibited by Progesterone receptor antagonist, RU 486 and estradiol 17β did not have any effect on the expression of SLPI mRNA expression in BeWo cells. The absence of regulation of SLPI by estradiol in BeWo cells was also established by the fact that simultaneous addition of progesterone and aromatase inhibitor, fadrazole did not block the increase in SLPI expression. Interestingly in vivo and in vitro studies using rat uterine minces and rat epithelial cells revealed that SLPI mRNA is regulated by Estradiol 17β and the effect is specifically inhibited by estrogen receptor antagonists such as ICI 182780, Tamoxifen, and Centchorman. Promoter analysis of rat and human SLPI revealed the absence of a consensus progesterone responsive element (PRE) in human and estrogen responsive element (ERE) in rat, suggesting the possibility of a non-genomic action of progesterone or estrogen in the induction of SLPI mRNA. This was confirmed by the observation that induction of SLPI mRNA could be effectively blocked by the addition of Staurosporine, an inhibitor of protein kinase C along with progesterone and estrogen to either BeWo cells or rat uterine epithelial cells. These results suggest that the non-genomic action of steroid hormones may be involved in the induction of SLPI. In the present study, we have also identified the intracellular signaling pathway that regulates SLPI gene expression by using various protein kinase inhibitors. We have also shown that activation of MAP kinase pathway upon progesterone treatment and the involvement of protein kinases in this activation, permitting us to conclude the non genomic action of progesterone in induction of SLPI mRNA in BeWo cells. The results of these studies are presented in detail in Chapter 5. The observation that SLPI expression is markedly increased during differentiation and differentially regulated by progesterone and estradiol, and induction by non genomic pathway prompted us to undertake studies to investigate its role during differentiation. This was accomplished by using SiRNA to silence the expression of SLPI in Forskolin induced differentiating BeWo cells and the results of this study are presented in CHAPTER 6. Different concentrations and combinations of oligos were used to silence the SLPI gene and we found that effective knockdown (>80%) was achieved with SiRNA concentrations ranging from 5-25nM. A combination of oligos also increased the knockdown from 50% to 90% as assessed by RT-PCR and western blot analysis for mRNA and protein levels of SLPI respectively. We found that inhibition of SLPI expression by SiRNA also inhibited the morphological differentiation of BeWo cells. Functionally this was reflected, by increase in the protease activity as assessed by gelatin zymography. It should be noted that SLPI is a protease inhibitor; it inhibits a variety of proteases, including proteases from neutrophils, pancreatic acinar cells and mast cells and SLPI present in the syncytiotrophoblast may have a crucial role in controlling protease activity associated with invasiveness and differentiation. Inhibition of differentiation by silencing the expression of SLPI provides an opportunity to monitor the changes in gene expression where in a single gene has been silenced in contrast to the model employed in chapter 4. We carried out microarray analysis using control (Forskolin treated) and SLPI silenced (Forskolin treated) samples. The results revealed that proliferation and differentiation, apoptosis and inflammatory pathways genes are affected due to SLPI silencing and the results of this study are presented in CHAPTER 7. We confirmed the changes in gene expression by semi quantitative RT-PCR analysis of the some important genes in each pathway. A comparison of the results obtained with that of our earlier microarray analysis which is described in chapter 4 revealed that the changes in levels of expression of the genes involved in cell proliferation, differentiation, apoptosis and inflammation were completely reversed after silencing the expression of SLPI. We have presented in chapter 5 the importance of MAP kinase pathway in Forskolin induced differentiation and the activation of this pathway when SLPI expression is increased following progesterone treatment. Interestingly after silencing the expression of SLPI we found that MAP kinase pathway is affected. It was observed that silencing of SLPI expression resulted in inhibition of activation of MAP kinase as assessed by the phosphorylation status by ELISA and no activation of MAP kinase was observed in SLPI silenced Forskolin treated cells. CHAPTER 8 provides a general discussion of the results obtained in the present study in the light of current understanding the type of genes involved, changes during human trophoblastic proliferation and differentiation and the key players during this process. This chapter also brings out the importance of SLPI during trophoblastic differentiation, placentation, implantation and its regulation by steroid hormones. The highlights and salient features of the present study are summarized in this chapter. In CONCLUSION, the present investigation has led to the identification of specific genes involved in trophoblast differentiation, human placental growth and development. Also evident from this study is the usefulness of the trophoblastic cell culture system for the study of cellular differentiation. We have attempted to relate the gene expression changes to physiological changes that occur during placentation, implantation and pregnancy. Many of the regulatory events that we have described during human trophoblastic differentiation, may not only be restricted to these cells, but may represent common principles/features of cellular differentiation in general. Loss of differentiation is a wide-spread feature of tumor progression, and frequently accompanies aggressive neoplastic behavior. Our studies provide unequivocal evidence to support cellular differentiation as a natural barrier to malignant transformation. Most importantly we have shown that silencing of a single gene can disrupt this differentiation process and the importance of SLPI during differentiation process perse.
107

Asymptomatic Bacteriuria in the Elderly

Rodhe, Nils January 2008 (has links)
The aim of this thesis was to explore the features of asymptomatic bacteriuria (ASB) in elderly people living in the community, and to seek diagnostic tools to discriminate between ASB and symptomatic urinary tract infection (UTI). All men and women aged 80 and over living in an urban district of Falun, Sweden, were invited to participate. Urine samples were obtained together with information on symptoms and on health indicators. The same cohort was surveyed again after 6 and 18 months. Urinary cytokines were analysed in 16 patients with UTI, in 24 subjects with ASB and in 20 negative controls. ASB occurred at baseline in 19.0% of women and 9.4% of men, and was found at least once in 36.9% of women and in 20.2% of men. ASB in women was associated with reduced mobility and urge urinary incontinence. Of those with ASB at baseline, 60% still had bacteriuria at 6 and 18 months, but among those with repeated findings of ASB with E. coli, only 40% had the same bacterial strain after 18 months. In women, the risk of developing a UTI within 24 months was higher among those with ASB at baseline than in those without. Urinary levels of cytokines (CXCL1, CXCL8 and IL-6) and leukocyte esterase were higher in patients with UTI than in patients with ASB. There is convincing evidence that ASB is harmless and should not be treated with antibiotics. However, such treatment is still often given, thereby unnecessarily contributing to the increased number of bacteria resistant to common antibiotics. This study confirms the high prevalence of ASB in elderly people living in the community. In order not to be misled by a urinary test showing bacteria, it is important to restrict urinary testing for bacteria to patients where there is a suspicion of UTI. In elderly patients with diffuse symptoms or in patients who are unable to communicate their symptoms, further diagnostic help could possibly be obtained by evaluating the urinary levels of leukocyte esterase and/or IL-6.
108

Análise de polimorfismos dos genes KIR e HLA classe I em pacientes com câncer colorretal

Silva, Pamela Portela da January 2016 (has links)
O câncer colorretal (CCR) pode ocorrer em qualquer parte do cólon ou do reto e representa o terceiro câncer mais comum no mundo em ambos os sexos. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). O objetivo deste estudo foi avaliar a associação entre os genes KIR e os ligantes HLA em pacientes com câncer colorretal e controles saudáveis. Examinamos o polimorfismo de 16 genes KIR e seus ligantes HLA em 154 pacientes caucasóides com CCR e 216 controles saudáveis pela técnica de PCR-SSO e PCR-SSP. Quando comparamos os dois grupos, não foram encontradas diferenças significativas para os ligantes HLA e os genes KIR após correção de Bonferroni. Entretanto, o grupo de genótipos Bx (heterozigoto e homozigoto para o haplótipo B) foi mais frequente nos controles, quando comparados com os pacientes. Estes achados sugerem que altos níveis de ativação de sinais KIR aparecem como proteção para o câncer colorretal. / Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 healthy controls by PCR-SSO and PCR-SSP. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx group genotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx genotypes could have some protection to colorectal cancer. These findings suggest that higher levels of activating KIR signals appear as protective to colorectal cancer.
109

Estudo de polimorfismos dos genes KIR e HLA em pacientes com câncer de próstata

Silva, Pamela Portela da January 2011 (has links)
O câncer de próstata é o segundo câncer mais comum entre homens, uma vez que tanto a incidência como a mortalidade aumentam exponencialmente após a idade de 50 anos. As células Natural Killer (NK) fazem parte do sistema imune inato e reconhecem moléculas de HLA de classe I na célula alvo, através de seus receptores de membrana, chamados killer immunoglobulin-like-receptors (KIR). O objetivo desse estudo foi avaliar a associação entre os genes KIR e HLA em pacientes com câncer de próstata e grupo controle. Genotipamos 200 pacientes com diagnóstico de câncer de próstata e 185 pacientes saudáveis para os genes KIR e HLA classe I por PCR-SSP. Quando comparados os grupos, não foram encontradas diferenças significativas para HLA-C do grupo 1 e do grupo 2, HLA-Bw4, HLA-A3 e A11. Nenhuma diferença foi observada na frequência dos genes KIR nos pacientes com câncer de próstata e nos controles. Esses resultados sugerem que não há potencial papel para o sistema dos genes KIR no câncer de próstata. / Prostate cancer is the second most common cancer among men, since both incidence and mortality exponentially increases in men over fifty years of age. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA alleles in patients with prostate cancer and healthy controls. Two hundred prostate cancer patients and 185 healthy controls were typed for HLA class I and KIR genes by PCR-SSP. When both groups were compared, no significant differences were found for HLA-C group 1 and group 2, HLA-Bw4, HLA-A3 and A11. No difference was seen either in KIR frequency between patients with prostate cancer and controls. In conclusion, our data suggests no potential role for the KIR gene system in prostate cancer.
110

Análise de polimorfismos dos genes KIR e HLA classe I em pacientes com câncer colorretal

Silva, Pamela Portela da January 2016 (has links)
O câncer colorretal (CCR) pode ocorrer em qualquer parte do cólon ou do reto e representa o terceiro câncer mais comum no mundo em ambos os sexos. As células Natural Killer (NK) fazem parte do sistema imune inato reconhecendo moléculas de HLA de classe I em células alvo, através de seus receptores de membrana killer cell immunoglobulin-like receptors (KIR). O objetivo deste estudo foi avaliar a associação entre os genes KIR e os ligantes HLA em pacientes com câncer colorretal e controles saudáveis. Examinamos o polimorfismo de 16 genes KIR e seus ligantes HLA em 154 pacientes caucasóides com CCR e 216 controles saudáveis pela técnica de PCR-SSO e PCR-SSP. Quando comparamos os dois grupos, não foram encontradas diferenças significativas para os ligantes HLA e os genes KIR após correção de Bonferroni. Entretanto, o grupo de genótipos Bx (heterozigoto e homozigoto para o haplótipo B) foi mais frequente nos controles, quando comparados com os pacientes. Estes achados sugerem que altos níveis de ativação de sinais KIR aparecem como proteção para o câncer colorretal. / Colorectal cancer (CRC) can occur anywhere in the colon or rectum and represents the third most common cancer in the world in both sexes. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study was to evaluate the association between the KIR genes and HLA ligands in patients with colorectal cancer and healthy controls. We examined the polymorphism of 16 KIR genes and their HLA ligands in 154 caucasoid CRC patients and 216 healthy controls by PCR-SSO and PCR-SSP. When both groups were compared, no significant differences were found for HLA ligands and KIR genes after Bonferroni correction. However, the Bx group genotypes (heterozygous and homozygous for the haplotype B) were more frequent in controls, when compared with patients. These findings suggest that individuals with Bx genotypes could have some protection to colorectal cancer. These findings suggest that higher levels of activating KIR signals appear as protective to colorectal cancer.

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