1) The document examines the effect of mechanical loading on WNT signaling components in cartilage.
2) It finds that physiological loading activates canonical WNT signaling in chondrocytes, as shown by β-catenin nuclear translocation. WNT activity peaks at 2 hours of loading.
3) Higher strain levels and non-physiological loading decrease WNT activity, as seen through reduced β-catenin translocation and upregulation of WNT inhibitors.
4) The results suggest mechanical loading regulates WNT signaling to maintain cartilage tissue integrity, and abnormal loading disrupts this process. Further study is needed to understand the downstream effects and mechanisms involved.
Metabolic investigation of segmental overgrowth: new insights in pathogenic m...BiologInc
[Biolog Webinar] Dr. Luigi Boccuto's presentation of his investigation of the pathogenic mechanisms of segmental overgrowth caused by mosaic mutations in the genes of the PI3K-AKT pathway. He will describe how Biolog Phenotype MicroArray™ Technology is used to identify new treatment targets and test compounds with potential therapeutic effects.
Metabolic investigation of segmental overgrowth: new insights in pathogenic m...BiologInc
[Biolog Webinar] Dr. Luigi Boccuto's presentation of his investigation of the pathogenic mechanisms of segmental overgrowth caused by mosaic mutations in the genes of the PI3K-AKT pathway. He will describe how Biolog Phenotype MicroArray™ Technology is used to identify new treatment targets and test compounds with potential therapeutic effects.
Action research in classroom setting copy (2)susanaparejo39
This Action Research in Classroom Setting is designed by the author in order to help the practitioner write the action research in a comprehensive way.
I received a PhD in April of 2007 from the Schultz Lab at the Scripps Research Institute in La Jolla, CA. Here is a PowerPoint presentation of my primary work - a use of functional genomics tools to probe cellular disease problems, notably in cancer models.
Introduction: Aging-associated vascular stiffening augments cardiovascular disease risk in the elderly. Research to identify targetable cellular and molecular mechanisms is of key interest as no current therapies are available to specifically target vascular stiffening. In this context, enzymes that mediate remodeling of the vascular matrix and those that promote cellular dysfunction are attractive targets. In pre-clinical models, pulse wave velocity (PWV), the gold standard measure of in vivo vascular stiffness, can be measured longitudinally and non-invasively, to make inroads towards the discovery and validation of potential targets.
A novel target and model: We have identified a central role for tissue transglutaminase (TG2) in vascular stiffening during aging. TG2 is a multifunctional protein of the transglutaminase family, whose primary function is to assist in the formation of a strong and stable matrix by catalyzing crosslinking of matrix proteins. Recent studies have shown that TG2 has putative crosslinking-independent functions in aging-associated vascular stiffening and dysfunction. The crosslinking independent mechanisms of TG2 remain incompletely understood due to the lack of pre-clinical models and specific inhibitors that can selectively inhibit a single function of TG2. Thus, we developed a novel knock-in mouse, the TGM2-C277S mouse, by mutating the active site cysteine of TG2 using the CRISPR-Cas9 gene editing technology to selectively target its crosslinking function.
Results and conclusion: We first validated the TGM2-C277S mouse and confirmed that this mutation removes TG2’s crosslinking function but retains its crosslinking independent functions. We next compared PWV wild type (WT), global TG2 knockout (TG2-/-), and the TGM2-C277S mice, to identify the contributions of the crosslinking-dependent and crosslinking-independent functions of TG2 to vascular aging in vivo. PWV increased significantly with age in WT mice, and to a much lower magnitude in the TGM2-C277S mice. TG2-/- mice were further protected against aging associated increase in PWV. Together, these studies show that TG2 contributes significantly to overall vascular stiffening in aging through both crosslinking dependent and crosslinking independent functions.
The learning objectives are:
To understand changes in pulse wave velocity (PWV) with age in mouse models
To determine the specific role of tissue transglutaminase (TG2) in vascular aging
To evaluate the role of vascular matrix vs. VSMCs to overall in vivo stiffness described by PWV
Tumor suppression and inflammation: controlling the senescence associated se...adamfreund
This is the powerpoint presentation from a talk I gave at a conference in October, 2009. It will be hard to follow without the spoken part, but it will hopefully give anyone who is interested a brief introduction to my thesis research.
This is the Powerpoint presentation from my recent presentation at the TTP LabTech US Acumen Users Group Meeting (UGM) held at the British Consulate-General in Cambridge, MA on May 18, 2010
MAPK Signaling pathway (Mitogen-activated protein kinase), how the pathway helps in regulation of mitosis, It's activation and inactivation inside the cell, roles of MAPK pathway in cancerous cell, different classes of MAP kinase in human
5. Hypothesis
Wnt signalling is mechano-regulated in cartilage
Aims
1. Examine the effect of physiological load onWNT signalling
components (activators/inhibitors) in chondrocytes
2. Determine the influence of physiological and non-physiological
load onWNT signalling components in explants.
6. Methods: Mechanical loading
Chondrocytes Explants
Primary chondrocytes released from
cartilage using pronase (1mg/ml) and
collagenase type II (0.4mg/ml) in DMEM F-
12 media.
- Seeded 4×106/well
- 48 hours stabilisation
period
7-day old bovine
Metacarpophalangeal
joint
4mm explants
Full-depth cartilage
explant
- Multiple washes in Hank’s balanced
salt solution (HBSS) with 4× strength
antibiotics
- 48 hours stabilisation period
Bose Electroforce 3200
7. Methods: Post-load analysis
Chondrocytes
o 7.5% strain 1Hz (1hr, 2, 4 hrs)
• Physiological regime
o 7.5%, 10%, 14% 1Hz (2hrs)
• Physiological injurious
Explants
o Physiological load 2.5MPa 1Hz 10mins
o Injurious load 8MPa 1 Hz 10 cycles
o Gene level analysis
• Quantitative PCR
o Protein level analysis
• Immunofluorescence in conjunction with confocal microscopy
Evaluation ofWNT components
8. Results: Effect of loading duration (7.5% strain)
β-catenin nuclear translocation in chondrocytes subjected to tensile strain
o Loading regime: 7.5% strain 1Hz (physiological)
FITC green= β-catenin; DAPI blue
9. Differential expression of WNT signalling components exposed to strain
Most WNT
activity is
detected at
2hrs
Results: Effect of loading duration (7.5% strain)
10. Results: Effect of strain level (1 Hz, 2hrs)
β-catenin nuclear translocation in chondrocytes subjected to tensile strain
FITC green= β-catenin; DAPI blue
11. Results: Explants 2.5MPa 10mins 1 Hz
β-catenin nuclear translocation in chondrocytes subjected to physiological compressive loading.
FITC green= β-catenin; DAPI blue
Canonical
WNT
signalling
activation
14. Dr. Emma Blain
Prof.Vic Duance
Dr.YadanZhang
Paulina Stadnik
Kuwait Cultural Office (UK)
Arthritis Research UK
Biomechanics and
BioengineeringCentre