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- Xu, Ning, 1980-
(author)
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Adenoviral Control of RNAi/miRNA Pathways in Human Cells
- 2008
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Doctoral thesis (other academic/artistic)abstract
- RNA interference (RNAi) is a diverse, conserved regulatory mechanism in eukaryotic cells, which silences the target gene expression in a homology-dependent manner. Although it has been well documented that RNAi is an antiviral mechanism in plants and insects, it is still unclear whether RNAi naturally limits viral infections in vertebrates. Viruses are masters of adopting strategies to subvert cellular defense mechanisms. Not only can viruses use elaborate strategies to suppress the effects of defensive RNAi, but they can also redirect or interfere with cellular functions orchestrated by endogenous small RNAs. In our work we have focused on studying the relationship of human adenovirus type 5 (Ad5) infection and the RNAi/miRNA pathways. We show that Ad5 infection inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). For Dicer inhibition, the virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional small RNAs that are incorporated into active RISC. Furthermore, by cloning small RNAs, we found that approximately 80% of Ago2-containing RISC immunopurified from late infected cells was associated with VA RNA-derived small RNAs (mivaRNAs). Interestingly, the small RNAs derived from VA RNAII, the minor VA RNA species, appear to be the major mivaRNAs occupying RISC and associate with polyribosomes, which indicates their potential roles as miRNAs regulating translation of cellular mRNAs. During our previous work, we observed that the strand bias of VA RNAI derived small RNA (mivaRI) incorporating into active RISC varied in the different viable Ad5 mutant viruses infected cells. It has been reported that Ad5 VA RNAI had two transcription initiation sites, which produced two clusters of VA RNAI with 3 nt difference at their 5’ end. Our data show that this heterogeneity resulted in a dramatic difference in mivaRI guide strand selection. Collectively, our data contributes to understanding the interplay between virus and host. This study would be beneficial in designing optimal adenovirus vectors for therapeutic RNAi application.
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| 2. |
- Xu, Ning
(author)
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Assessment of rat spinal cord injury models
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Traumatic spinal cord injury (SCI) is a complicated and devastating condition, causing different extents of motor, sensory and autonomic dysfunctions. In addition, there is a risk for secondary complications after SCI including posttraumatic syringomyelia (PTS) that can cause further functional loss. Since there is no available effective treatment, tremendous efforts have been made to develop new therapeutic strategies to promote functional recovery after SCI. In experimental research, it is important to utilize model systems, including in vivo models that mimic the clinical situation to develop new treatments. Furthermore, multiple tests should be applied to comprehensively evaluate the models and novel treatments. We developed two assessment tools for swimming and wading in rats with SCI, with the sensitivity to injury severities from the most severe to the very mildest. Both the Karolinska Institutet Swim Assessment Tool (KSAT) and the wading scale consist of six parameters, reflecting different functional aspects. KSAT and Wading scores for four experimental groups of different injury severity consistently displayed the functional improvement after injury. The internal consistency, inter-rater and test-retest reliability were all very high. We also found a high correlation between KSAT/Wading score and spared white matter at injury epicenter, and between the KSAT, wading and BBB scores. In addition, we studied kinematic analysis of swimming in two SCI models (mild contusion and compression SCIs) and a control group by a simple two-dimensional system representing the hindlimbs with 3 segments and 2 angles, analyzing six parameters. The results showed that three parameters Swim Speed, Stroke Time and Extension time/Flexion time changed significantly between week 2 and 8. The results of Swim Speed, Angular Velocity and Stroke Time at week 8 were highly correlated with spared tissue at injury epicenter, particularly in contusion SCI. However, for these parameters there was overlap between very mildly injured rats and controls, not achieving the same sensitivity as the KSAT score. To study neural cell therapy of PTS, we developed a novel rat model mimicking the clinical situation. We used a combination of mild low thoracic contusion trauma and subarachnoid injection of autologous venous blood. The injured rats developed cysts that were extracanalicular, mainly rostral to the injury and lined with astrocytes. T2-weighted magnetic resonance imaging (MRI) scanned 20 weeks after injury showed hyperintense fluid-filled cysts and hypointense areas of tissue degeneration with iron-laden macrophages/microglia. However, the functional analysis did not reveal deterioration coinciding with cyst expansion. Under the guidance of MRI, human neural precursor cells (hNPCs) were transplanted into the cysts. The hNPCs survived, covered the surface of the cyst walls and migrated into surrounding tissue. Moreover, the cells partially obliterated the cysts and in some areas merged the walls of the cysts. In conclusion, KSAT and wading scale were found to be reliable tools to assess motor activity in swimming and wading, while kinematic analysis did not prove to be very useful for functional testing. The new rat PTS model closely mimics the pathophysiological and anatomical features of the clinical situation. Using this model, transplantation of hNPCs was shown to be a potential treatment to obliterate cysts in PTS.
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| 3. |
- Xu, Ning
(author)
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Interaction of Triglyceride-rich Lipoproteins with Platelets and Vitamin K-dependent Coagulation Factors
- 2000
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Doctoral thesis (other academic/artistic)abstract
- 1. During incubation of platelets with 3H-arachidonic acid (20:4, n-6) and 14C-cholesterol doubly labelled and colloidal gold labelled chylomicrons (CMs) and chylomicron remnants (CMRs) CMs were taken up more efficiently than CMRs. Addition of unlabelled CMs, VLDLs, LDLs and HDLs decreased the uptake of labelled CMs. Electron microscopic studies demonstrated an accumulation of CM-Au's both in the open canalicular system and cytoplasm of the platelets. There was no evidence for a lipoprotein receptor mediated breakdown of CMRs. 2. Plasma exposed CMs and CM-prothrombin complexes could induce platelet aggregation and enhance the platelet serotonin- and arachidonic acid release. Platelet aggregation induced by plasma exposed CMs could be inhibited in a dose dependent manner by an antiserum against prothrombin. Coagulation factor Xa inhibitor (TenStop) inhibited platelet aggregation and serotonin release that were induced by CM-prothrombin complexes in a dose-dependent manner. Native chyle CMs did not induce platelet aggregation, but decreased ADP and thrombin induced platelet aggregation and serotonin release. Neither did CMRs induce platelet aggregation, but they potentiated the aggregation and serotonin release induced by ADP and thrombin. 3. The ability of chyle CMs to bind human prothrombin was studied in vitro. The binding was Ca2+ and temperature dependent but could not be reversed with EDTA. The metabolism in vivo of CM-125I-prothrombin complexes was compared to that of free 125I-prothrombin injected in saline or together with CMs. The plasma decrease of 125I-prothrombin was faster in the group obtained CM-125I-prothrombin complexes than in the other groups. The radioactivity in the liver was higher in the group that obtained CM-125I-prothrombin complexes. 4. All vitamin K-dependent coagulation proteins (factors VII, IX, and X, prothrombin, proteins C and S) and C4b binding protein (C4BP) were found in TG-rich lipoproteins of human plasma. A relative increase of prothrombin, protein S and C4BP was seen in TG-rich lipoproteins after a fat meal compared to the fasting lipoproteins. There was no association of the coagulation proteins with LDLs and HDLs. Nor were coagulation factor V, serum amyloid P component or thrombomodulin associated with TG-rich lipoproteins in vivo.
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