Friday, March 25, 2016

Research on a Budget, Part II

Just how much CAN a high school student accomplish in several months, working 
1-2 hours per day on a research project?


In her study of extracellular matrix enzymes and neuronal regeneration, 
Claudia Shin, PA '12 was able to:

       1) design, carry-out, and publish a study comprised of several specific aims

       2) collaborate with two professional research teams 

       3) learn statistical analyses required to challenge her data and interpret her observations

       4) submit, revise and publish an article in the Journal of Experimental Secondary Science 

For her study, Claudia utilized unc-70 mutant C. elegans, graciously 
offered by the Hammarlund lab. These worms lack beta-spectrin, resulting in an inability to withstand mechanical strain. Unc-70 worms carrying fluorescent reporters in subsets of motor neurons allow one to continually observe the regeneration of axons broken from the stress of normal movement. This elegant model system, developed by Marc Hammarlund, has launched the field of regeneration research into a new era. An improvement over laser-assisted "axotomies," these cleaner models are derived from a single mutation, thus axonal regrowth can be observed in the context of an otherwise normal environment.
 

Claudia used RNA interference by feeding to silence PXN-2 in wild-type worms as well as unc-70 mutants to examine the effects of a lack of PXN-2 on normal axon development and regeneration. Both her wild-type and unc-70 worms expressed GFP in DD, GABA-ergic motor neurons, for easy observations of axonal commissures.



 Wild-type                                      





Unc-70 mutants                         

                                              Control Bacteria                                    RNAi Feeding Strain (pxn-2)



Matrix enzymes such as PXN-2, a peroxidasin, had previously been linked to regeneration in laser axotomy studies. Claudia asked whether a functional role for PXN-2 would also be observed in the beta-spectrin model. Ultimately, she demonstrated that RNAi-mediated knock-down inhibited regeneration. 




Claudia reached out to Joel Jacob, a mathematics teacher on campus, to learn how to identify outliers, medians and inner quartile ranges within her data sets. Then, after rejecting null hypotheses in 2-sample T-tests and ANOVA, Claudia was able to confidently assert that her observed changes in axonal regeneration were not due to random chance. As RNA interference was the only manipulated variable in her worm cultures, she concluded that knock-down of PXN-2 led the changes in regeneration she observed.


Critical Thinking - Potential Hypotheses for Future Directions  

Taking into account that peroxidasins are thought to reside in the basement membrane and act on type IV collagen, Claudia proposed three potential explanations for her observations:

  1. Peroxidasins affect scaffolding in the basement membrane
  2. Peroxidasins affect the chemorepellant gradient
  3. Peroxidasins affect the chemoattractant gradient

     
                                                                               https://exp1.physgeo.uni-leipzig.de/bip/research/cellular-motility/

                                       




Chemorepellents and chemoattractants are both involved in the extension of axonal commissures away from the ventral nerve cord, circumferentially towards the dorsal muscle wall.



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