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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.
691

Extracellular Matrix Based Materials for Tissue Engineering

Aulin, Cecilia January 2010 (has links)
The extracellular matrix is (ECM) is a network of large, structural proteins and polysaccharides, important for cellular behavior, tissue development and maintenance. Present thesis describes work exploring ECM as scaffolds for tissue engineering by manipulating cells cultured in vitro or by influencing ECM expression in vivo. By culturing cells on polymer meshes under dynamic culture conditions, deposition of a complex ECM could be achieved, but with low yields. Since the major part of synthesized ECM diffused into the medium the rate limiting step of deposition was investigated. This quantitative analysis showed that the real rate limiting factor is the low proportion of new proteins which are deposited as functional ECM. It is suggested that cells are pre-embedded in for example collagen gels to increase the steric retention and hence functional deposition. The possibility to induce endogenous ECM formation and tissue regeneration by implantation of growth factors in a carrier material was investigated. Bone morphogenetic protein-2 (BMP-2) is a growth factor known to be involved in growth and differentiation of bone and cartilage tissue. The BMP-2 processing and secretion was examined in two cell systems representing endochondral (chondrocytes) and intramembranous (mesenchymal stem cells) bone formation. It was discovered that chondrocytes are more efficient in producing BMP-2 compared to MSC. The role of the antagonist noggin was also investigated and was found to affect the stability of BMP-2 and modulate its effect. Finally, an injectable gel of the ECM component hyaluronan has been evaluated as delivery vehicle in cartilage regeneration. The hyaluronan hydrogel system showed promising results as a versatile biomaterial for cartilage regeneration, could easily be placed intraarticulary and can be used for both cell based and cell free therapies.
692

One Bead One Compound Screening for Cyclic Peptide Binding Partners

Utterström, Johanna January 2018 (has links)
In recent years a significant research focus has been on the development of biomimicking three-dimensional substrates for cell culturing. Hydrogels mimicking the extracellular matrix is a well-suited scaffold for this purpose and there are many different ways these can be cross-linked to retain their shape. The group of Molecular Materials at IFM, Linköping University, is focusing on the development of physical hydrogels hybridized through peptide-peptide interactions but all peptides used for this today are created using rational design and on top of this very large, making them time-consuming and expensive to fabricate. The aim of this project was to evaluate if One Bead One Compound (OBOC) libraries could be used as an alternative to rational design in the finding of cyclic peptide binding partners used in the hybridization of hydrogels. The results were not very promising though since only seven peptides passed all screening steps and of these only two could be sequenced. Of these two, only one was water soluble enough to enable binding interactions analysis but was then found to be a false hit. Nevertheless, it should be noticed that only a fraction of all possible combinations was screened and the results cannot exclude OBOC libraries as an approach in the quest of finding new cyclic peptide binding partners.
693

Functional characterization of the human adenovirus pVII protein and non-coding VA RNAI

Inturi, Raviteja January 2017 (has links)
Human adenovirus (HAdV) is a common pathogen causing a broad spectrum of diseases. HAdV encodes the pVII protein, which is involved in nuclear delivery, protection and expression of viral DNA. To suppress the cellular interferon (IFN) and RNA interference (RNAi) systems, HAdVs encode non-coding virus-associated (VA) RNAs. In this thesis we have investigated the functional significance of the pVII protein and VA RNAI in HAdV-5 infected cells. We report that the propeptide module is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. We also found that the Cul3-based E3 ubiquitin ligase complex alter the precursor pVII protein stability via binding to the propeptide sequence. In addition, we show that inhibition of the Cul3 protein reduces HAdV-5 E1A gene expression. Collectively, our results suggest a novel function for the pVII propeptide module and involvement of Cul3 in viral E1A gene expression. Our studies show that the cellular E3 ubiquitin ligase MKRN1 is a novel pVII interacting protein in HAdV-5 infected cells. MKRN1 expression reduced the pVII protein accumulation in virus-infected cells and affected infectious virus formation. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the prolonged HAdV-5 infection. Furthermore, the precursor pVII protein enhanced MKRN1 self-ubiquitination, suggesting the direct involvement of pVII in the initiation of MKRN1 degradation. Hence, we propose that the MKRN1 is a novel antiviral protein and that HAdV-5 infection counteracts its antiviral activity. In papers III and IV, we tested the ability of various plant and animal virus encoded RNAi/miRNA and IFN suppressor proteins to functionally substitute for the HAdV-5 VA RNAI. Our results revealed that the Vaccinia virus E3L protein was able to partially substitute for the HAdV-5 VA RNAI functions in virus-infected cells. Interestingly, the E3L protein rescued the translational defect but did not stimulate viral capsid mRNA accumulation observed with VA RNA. Additionally, we show that the HAdV-4 and HAdV-37 VA RNAI are more effective in virus replication compared to HAdV-5 and HAdV-12 VA RNAI. In paper IV, we employed a novel triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinaxline 1,10-phenanthroline (BQQ-OP), to unravel triplex structure formation in 
VA RNAI. The BQQ-OP cleavage of HAdV-4 VA RNAI indicates that a potential 
triplex is formed involving the highly conserved stem 4 of the central domain and side 
stem 7. Further, the integrity of HAdV-4 VA RNAI stem 7 contributes to the virus growth in vivo.
694

Investigating Amyloid β toxicity in Drosophila melanogaster

Jonson, Maria January 2017 (has links)
In this thesis Drosophila melanogaster (the fruit fly) has been used as a model organism to study the aggregation and toxic properties of the human amyloid β (Aβ) peptide involved in the onset of Alzheimer's disease (AD). AD is one of many misfolding diseases where the important event of a protein to adopt its’ specific three-dimensional structure has failed, leading to aggregation and formation of characteristic amyloid fibrils. AD has a complex pathology and probably reflects a variety of related molecular and cellular abnormalities, however, the most apparent common denominator so far is abnormal Amyloid-β precursor protein (APP) processing, resulting in a pool of various Aβ-peptides. In AD, the Aβ peptide misfolds, aggregates and forms amyloid plaques in the brain of patients, resulting in progressive neurodegeneration that eventually leads to death. By expressing the human Aβ protein in the fly, we have studied the mechanisms and toxicity of the aggregation in detail and how different cell types in the fly are affected. We have also used this model to investigate the effect of potential drugs that can have a positive impact on disease progression. In the first and second work in this thesis, we have, in a systematic way, proved that the length of the Aβ-peptide is essential for its toxicity and propensity to aggregate. If the peptide expressed ends at amino acid 42 it is extremely toxic to the fly nervous system. However, this toxicity can be completely abolished by expressing a variant that is shorter than 42 amino acids (1-37 to 1-41 aa), or be significantly reduced by expressing a longer variant (1-43 aa). Toxicity can be partly mitigated in trans by co-expressing the 1-42 variant with a 1-38 variant. This supports the theory that the disease progression could be inhibited if the formation of Aβ 1-42 is decreased. In the third work we demonstrate that amyloid aggregates can be found in various cell types of Drosophila, however, the toxicity seem to be selective to neurons. Our results indicate that the aggregates of glial expressing flies have a more mature structure, which appear to be less toxic. This also suggests that glial cells might spread Aβ aggregates without being harmed. The last work in this thesis investigates how curcumin (turmeric) can affect Aβ aggregation and toxicity. Curcumin appears to shift the equilibrium between the less stable aggregates and mature fibers toward the final stage resulting in an improved lifespan for treated flies. In summary, this thesis demonstrates that the toxicity of Aβ in Drosophila is highly dependent on the Aβ variant expressed, the structure of the protein aggregates and which cell type that expresses the protein. We have also shed light on the potential of using Drosophila when it comes to examining possible therapeutic substances as a tool for drug discovery.
695

New Molecular Approaches to Glioblastoma Therapy

Baskaran, Sathishkumar January 2017 (has links)
Glioblastoma (GBM) is the most common high-grade brain tumor diagnosed in patients who are more than 50 years of age. The standard of care treatment is surgery, followed by radiotherapy and chemotherapy. The median life expectancy of patients is only between 12 to 15 months after receiving current treatment regimes. Hence, identification of new therapeutic compounds and gene targets are highly warranted. This thesis describes four interlinked studies to attain this goal. In study 1, we explored drug combination effects in a material of 41 patient-derived GBM cell (GC) cultures. Synergies between three compounds, pterostilbene, gefitinib, and sertraline, resulted in effective killing of GC and can be predicted by biomarkers. In study 2, we performed a large-scale screening of FDA approved compounds (n=1544) in a larger panel of GCs (n=106). By combining the large-scale drug response data with GCs genomics data, we built a novel computational model to predict the sensitivity of each compound for a given GC. A notable finding was that GCs respond very differently to proteasome inhibitors in both in-vitro and in-vivo. In study 3, we explored new gene targets by RNAi (n=1112) in a panel of GC cells. We found that loss of transcription factor ZBTB16/PLZF inhibits GC cell viability, proliferation, migration, and invasion. These effects were due to downregulation of c-MYC and Cyclin B1 after the treatment. In study 4, we tested the genomic stability of three GCs upon multiple passaging. Using molecular and mathematical analyses, we showed that the GCs undergo both systematic adaptations and sequential clonal takeovers. Such changes tend to affect a broad spectrum of pathways. Therefore, a systematic analysis of cell culture stability will be essential to make use of primary cells for translational oncology. Taken together, these studies deepen our knowledge of the weak points of GBM and provide several targets and biomarkers for further investigation. The work in this thesis can potentially facilitate the development of targeted therapies and result in more accurate tools for patient diagnostics and stratification.
696

Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS

Bahrampour, Shahrzad January 2017 (has links)
The central nervous system (CNS) consists of an enormous number of cells, and large cellular variance, integrated into an elaborate network. The CNS is the most complex animal organ, and therefore its establishment must be controlled by many different genetic programs. Considering the high level of complexity in the human CNS, addressing issues related to human neurodevelopment represents a major challenge. Since comparative studies have revealed that neurodevelopmental programs are well conserved through evolution, on both the genetic and functional levels, studies on invertebrate neurodevelopmental programs are often translatable to vertebrates. Indeed, the basis of our current knowledge about vertebrate CNS development has been greatly aided by studies on invertebrates, and in particular on the Drosophila melanogaster (fruit fly) model system. This thesis attempted to identify novel genes regulating neural cell specification and proliferation in the CNS, using the Drosophila model system. Moreover, I aimed to address how those genes govern neural progenitor cells (neuroblasts; NBs) to obtain/maintain their stemness identity and proliferation capacity, and how they drive NBs through temporal windows and series of programmed asymmetric division, which gradually reduces their stemness identity in favor of neural differentiation, resulting in appropriate lineage progression. In the first project, we conducted a forward genetic screen in Drosophila embryos, aimed at isolating genes involved in regulation of neural proliferation and specification, at the single cell resolution. By taking advantage of the restricted expression of the neuropeptide FMRFa in the last-born cell of the NB lineage 5-6T, the Ap4 neuron, we could monitor the entire lineage progression. This screen succeeded in identifying 43 novel genes controlling different aspects of CNS development. One of the genes isolated, Ctr9, displayed extra Ap4/FMRFa neurons. Ctr9 encodes a component of the RNA polymerase II complex Paf1, which is involved in a number of transcriptional processes. The Paf1C, including Ctr9, is highly conserved from yeast to human, and in the past couple of years, its importance for transcription has become increasingly appreciated. However, studies in the Drosophila system have been limited. In the screen, we isolated the first mutant of Drosophila Ctr9 and conducted the first detailed phenotypic study on its function in the Drosophila embryonic CNS. Loss of function of Ctr9 leads to extra NB numbers, higher proliferation ratio and lower expression of neuropeptides. Gene expression analysis identified several other genes regulated by Ctr9, which may explain the Ctr9 mutant phenotypes. In summary, we identified Ctr9 as an essential gene for proper CNS development in Drosophila, and this provides a platform for future study on the Drosophila Paf1C. Another interesting gene isolated in the screen was worniou (wor), a member of the Snail family of transcription factors. In contrast to Ctr9, whichdisplayed additional Ap4/FMRFa neurons, wor mutants displayed a loss of these neurons. Previous studies in our group have identified many genes acting to stop NB lineage progression, but how NBs are pushed to proliferate and generate their lineages was not well known. Since wor may constitute a “driver” of proliferation, we decided to study it further. Also, we identified five other transcription factors acting together with Wor as pro-proliferative in both NBs and their daughter cells. These “drivers” are gradually replaced by the previously identified late-acting “stoppers.” Early and late factors regulate each other and the cell cycle, and thereby orchestrate proper neural lineage progression.
697

Investigation of Chromatin Organization and mRNA Expression in Drug Treated Human Erythroleukemia Cells / Undersökning av Kromatinorganisation och mRNA-uttryck i Läkemedelsbehandlade Humana Erytroleukemiceller

Minhas, Anam January 2022 (has links)
Syftet med detta projekt var att undersöka hur vanligt använda cancerläkemedel påverkar mRNA-uttryck och kromatinorganisation i humana erytroleukemiceller. Som modell användes K562-celler från en patient i blastocystkris (2), för att utvärdera leukemicellernas svar på cancerläkemedel vinblastin och doxorubicin. Vinblastin och doxorubicin valdes på grund av deras distinkta mekanismer i cancercellen: medan doxorubicin interkaleras i DNA, hämmar topoisomeras II-aktivitet vilket orsakar celldöd, riktar vinblastin sig mot mikrotubuli för att stoppa mitotisk delning och proliferation. Uttryck av mRNA undersöktes i celler vid 0-timmar, 6-timmar och 24-timmar drogbehandling, samt efter en veckas återhämtning från 24-timmars drogbehandling. Kromatintillgänglighet med ATAC-seq undersöktes i K562-celler vid 0- timmar, 1-timmar, 6-timmar, 24-timmar och 24-timmar + en veckas återhämtning. Därefter utfördes DNA (ATAC-seq) och RNA (mRNA-seq) extraktion och biblioteksberedning på tre biologiska replikat, och öppna DNA-regioner samt mRNA expression undersöktes via sekvensering. Resultaten visade en stark korrelation mellan de biologiska replikaten, vilket indikerar att resultaten var upprepbara. Differentiellt uttryck av mRNA vid doxorubicin- och vinblastinbehandlingar utfördes genom att jämföra mRNA-nivåerna i läkemedelsbehandlade prover med obehandlade (0-timmar). Uppreglerade och nedreglerade gener identifierades och MA-grafer genererades för att visuellt analysera de differentiellt uttryckta generna vid olika tidpunkter efter läkemedelsbehandling och en veckas återhämtning. För att hitta anrikningar av funktionella genkategorier bland de läkemedelsinducerade eller -undertryckta generna, utfördes genontologianalyser. Slutligen användes verktyget Integrative Genomics Viewer (IGV) för att visuellt utforska mRNA-nivåerna och deras differentiella uttrycksmönster under läkemedelsbehandlingar. För ATAC-seq utfördes inte detaljerad dataanalys på grund av tidsbegränsning, men genomets öppenhet undersöktes visuellt genom IGV. Sammantaget inducerade doxorubicinbehandling en långsamt men långvarig förändring av genuttrycket, vilket involverade flera olika biologiska processer. Doxorubicinbehandlade K562-celler ändrade genuttryck att stöda kemoresistens snarare än att inducera apoptos eller celldöd. Behandlingen hade en långvarig inverkan på mRNA-nivåer som sträckte över återhämtningsveckan. Den totala uttrycksförändringen i återhämtningsproverna var förknippad med återhämtning av tumörigena egenskaper och återställning av mekanismener som stöder cellernas tillväxt. Vinblastine förorsakade snabb ökning av mRNA involverade i cytoskelettet. Vid 24-timmars vinblastinbehandling upplevde tumörcellerna stress på grund av grovt elongerad struktur, och de inducerade gener som stöder tumörbildning. En ökning av totala mRNA-nivåer detekterades i vinblastinbehandlade K562-leukemiceller, vilket var särskilt tydligt under återhämtningen. Resultaten visade att cellerna som överlevde vinblastinbehandling fokuserade på att återställa sin strukturella form. Sammantaget visade resultaten att monoterapi inte fungerar effektivt mot leukemiceller eftersom K562-leukemiceller inte bara överlevde läkemedelsbehandlingarna utan också inducerade mRNA som är involverade i resistens mot läkemedelsbehandlingar. / The primary objective of this project is to investigate how commonly used cancer drugs affect mRNA expression and chromatin organization in human erythroleukemia cells. As a model, K562 cells derived from a patient in blastocyst crisis (2) were utilized, evaluating the leukemia cells’ cellular responses to cancer medicines vinblastine and doxorubicin. Vinblastine and doxorubicin were chosen due to the distinct pathways they target in the cell: while doxorubicin intercalates into DNA and inhibits topoisomerase II activity, which eventually cause cell death, vinblastine targets microtubules to stops mitotic division and excessive proliferation. Expression of mRNA was investigated in cells harvested at 0h, 6h, 24h and 24h + one week recovery. Chromatin accessibility with ATAC-seq was investigated in K562 cells harvested at 0h, 1h, 6h, 24h and 24h + one week recovery. Then DNA (ATAC-seq) and RNA (mRNA-seq) extraction and library preparation were performed on three replicates, and the genome-wide results was investigated via sequencing. The results showed a strong correlation between the biological replicates, indicating that the experimental conditions were sustained in these biological variables. Differential Expression of mRNA upon doxorubicin and vinblastine treatments was performed by comparing the mRNA levels in drug-treated samples to non-treated (0h) upregulated and down regulated genes were identified and MA plots generated to visually analyze the differentially expressed genes at different time points after drug treatment and one week recovery. To find enrichments of functional gene categories among the drug-induced or -repressed genes, gene ontology analyses were performed. Finally, the Integrative genomics viewer (IGV) tool was used to visually explore the mRNA levels and their differential expression pattern during drug treatments. For ATAC-seq, detailed data analysis was not performed due to limitation of time, and data was only visually explored through IGV. Taken together, doxorubicin treatment showed slow initial response within 6h followed by an extensive change in gene expression in 24h, involving several different biological processes. The response was more inclined towards chemoresistance rather than inducing apoptosis or cell death. There was a sustained increase in mRNA levels of doxorubicin treated leukemia cells during recovery week. The overall expression change in the recovery samples was majorly linked with not only gaining back the tumourigenic properties and restoring the mechanism which were affected by doxorubicin action but, based on changes in mRNA expression, it looks like doxorubicin treatment made the tumour cells more aggressive. The initial, 6h, response to vinblastine increases mRNAs involved in cytoskeleton. Upon 24h vinblastine treatment the tumour cells experienced stress due to shear force and structural deformity, and they induced genes supporting tumourigenesis. An increase in total mRNA levels was detected in vinblastine-treated K562 leukemia cells, which was particularly evident during recovery. The results indicated that the cells that survived vinblastine treatment focused on recovering its structural form. Overall, the results indicated that monotherapy does not effectively work against leukemia cells as K562 leukemia cells not only survived the drug treatments but also induced mRNAs involved in resistance against drug treatment.
698

Influence of Nrf2 Activators and Keap1 Inhibitors on Antioxidative Phenotypes of THP-1-Derived M1 and M2 macrophages: Therapeutic Potential for Systemic Lupus Erythematosus

Svahn, Leo January 2023 (has links)
POPULAR SCIENTIFIC SUMMARY Systemic lupus erythematosus (SLE) is not your average disorder. It behaves like a mischievous troublemaker, wreaking havoc throughout the body, causing inflammation that affects multiple organs. SLE presents a puzzle that keeps health care professionals worldwide intrigued, searching for answers amidst its complex of immunologic manifestations and clinical symptoms. While we’ve made progress in understanding SLE, its specific cause remains a mystery. What we do know is that SLE triggers a fascinating interplay between genetic, hormonal, and environmental factors in susceptible individuals. Macrophages, specialized white blood cells, can be likened to moody actors on a stage wearing different masks and wielding functional props. Among them are M1 macrophages, fiery troublemakers who provoke pro-inflammatory responses, and M2 macrophages, peacemakers striving for balance by generating anti-inflammatory responses. Then there is NRF2, the vigilante, normally held by its captor, KEAP1. However, when cells stress NRF2 manages to break free from KEAP1 and spring into action, embarking on a crucial journey into the cell nucleus where DNA is stored. Once inside, NRF2 binds specific regions of the DNA, promoting genes associated with protective activities, including antioxidative responses and detoxification processes, thereby shielding cells from further harm. Now, let us envision a therapeutic strategy that utilizes this; if we can deliberately unleashNRF2 on command, triggering a powerful cascade of antioxidative responses throughout the body,such a treatment would offer tremendous promise and serve as a paradigm for patients sufferingfrom chronic inflammation. But the question remains: Is it possible? In this study, we investigated the effects of certain chemicals on macrophages in a controlledlab environment. Our goal was to explore their potential for therapeutic purposes. Excitingly, wediscovered that these chemicals can indeed influence macrophages to produce a stronger antiinflammatory and antioxidant response. These findings could be promising for developing futuretreatments, especially in patients diagnosed with conditions such as SLE. / ABSTRACT Systemic lupus erythematosus (SLE) is a multifaceted, chronic autoimmune disorder that leads to inflammation and affects various organs. A wide range of immunologic manifestations and clinical symptoms characterizes SLE. While the specific cause remains unknown, it is thought to result from a combination of genetic susceptibility and the intricate interplay between environmental and hormonal factors. A significant subset of SLE patients also experience renal manifestation, lupus nephritis (LN), characterized by distinct inflammatory responses in which macrophages play a role. Macrophages exhibit different functional characteristics depending on their environment, and generally display two contrasting phenotypes; M1, which elicits proinflammatory responses, and M2, which generates anti-inflammatory responses Homeostasis is vital, yet environmental stress is inevitable. NRF2, a transcription factor known for its involvement in oxidative stress response, plays a pivotal role. Under basal conditions, NRF2 resides in the cytoplasm and is targeted for degradation by the protein KEAP1. However, during cellular stress, the NRF2-KEAP1 complex dissociates, allowing NRF2 to translocate into the nucleus where it binds specific regulatory regions of genes that promote cytoprotective activities. The NRF2 pathway has gained attention as a potential target for therapeutic strategies in inflammatory conditions, including SLE. This study aimed to assess the effects of certain chemical NRF2 activators and a KEAP1 inhibitor on an in vitro model of M1 and M2 macrophage polarization. The objective was to investigate whether these compounds could enhance antioxidative response. To evaluate this, key genes and proteins involved in antioxidative pathways were analyzed. Gene expression was assessed using quantitative real-time PCR (qPCR), and protein presence was determined through immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). The findings of this study indicate that stimulation of macrophage subgroups with the selected compounds promotes a shift towards anti-inflammatory and antioxidative response. / <p>Rektor tilldelade Leo Svahn stipendie Österby för <em>välartade obemedlade studier</em>.</p>
699

Kinetic studies of NS3 and NS5B from Hepatitis C virus : Implications and applications for drug discovery

Dahl, Göran January 2009 (has links)
The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.   Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.
700

Molecular Genetic Analysis in B-cell Lymphomas : A Focus on the p53 Pathway and p16INK4a

Zainuddin, Norafiza January 2010 (has links)
The presence of TP53 mutations has been associated with inferior outcome in diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In DLBCL, the impact of the TP53 codon 72 polymorphism and MDM2 SNP309 has not been clearly elucidated, whereas MDM2 SNP309 was suggested as a poor-prognostic marker in CLL. In addition, p16INK4a promoter hypermethylation has been implicated as a negative prognostic factor in DLBCL. The aim of this thesis was to further evaluate these molecular markers in well-characterised materials of DLBCL and CLL. In paper I, we investigated the prognostic role of TP53 mutation, codon 72 polymorphism and MDM2 SNP309 in DLBCL (n=102). The presence of TP53 mutations (12.7%) correlated with a poor lymphoma-specific and progression-free survival, and a particularly pronounced effect was observed in the germinal center subtype. Neither the MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. In paper II, we applied pyrosequencing to measure the level of p16INK4a methylation in DLBCL (n=113). Thirty-seven percent of cases displayed p16INK4a methylation; however, no clear association could be observed between degree of methylation and clinical characteristics or lymphoma-specific survival. In papers III–IV, we investigated the prognostic role of MDM2 SNP309 (n=418) and TP53 mutation (n=268) in CLL. No correlation was observed between any particular MDM2 SNP309 genotype and time to treatment and overall survival. Furthermore, no association was found between the different MDM2 SNP309 genotypes and established CLL prognostic markers. TP53 mutations were detected in 3.7% of CLL patients; where the majority showed a concomitant 17p-deletion and only three carried TP53 mutations without 17p-deletion. We confirmed a significantly shorter overall survival and time to treatment in patients with both TP53 mutation and 17p-deletion. Altogether, our studies could confirm the negative prognostic impact of TP53 mutations in DLBCL, whereas MDM2 SNP309 and TP53 codon 72 polymorphisms appear to lack clinical relevance. We also question the role of p16INKa methylation as a poor-prognostic factor in DLBCL. Finally, the presence of TP53 mutation in CLL appears to be rare at disease onset and instead arise during disease progression.

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