Hi guys!
I'm sorry I haven't posted in such a long time, but to be fair, I haven't had much new things to tell you about. I have experienced first hand that research is a lot of data collection, so for the last several weeks I have been doing larval turning, survival curves, and, as always, virgining.
As the project comes to a close, my team is beginning to wrap up the collection and analysis of the high sugar and high protein data. We have a full set of data for the high sugar larval turning, and though the results are currently inconclusive and not statistically significant, we are setting up a couple more tests and crosses to see if we can get results more similar to the ones Dr. Zarnescu obtain when she first did experiments with high sugar and protein. We are also still collecting flies for both the developmental and adult survival curves. For the developmental survival curves, the flies are hatched and raised on the high protein and sugar food, but for the adult survival curve, the flies are hatched on normal food and then transferred to high protein or sugar food. The adult survival curves are more applicable to a potential cure for humans, because ALS doesn't develop until adulthood and therefor wouldn't need to be treated until then.
So that's what I've been up to. I'm also working on my paper and presentation, and looking forward to hearing all about everyone's final products!
Tuesday, April 15, 2014
Thursday, March 20, 2014
Week Six
Happy Spring Break everybody! The University of Arizona is on spring break this week, which means the three undergraduates who with me make up the drug screen team in the lab are currently out of town, leaving me to continue to run the experiments, collect data, and hopefully not mess anything up.
In addition to more larval turning (because you can never turn too many larvae, right? No, actually, I'm still collecting a full data set for the larvae eating both the high protein and high sugar food. A full data set is thirty larvae per condition (specific genotype and concentration)) I have also taken over setting up the survival data for the week. My team is collecting data on how long adult flies that have 'recovered' from ALS (do not have crumpled wings, but instead have straight normal wings) can live. We then compare this data to how long the flies would normally live on their normal food, and see if there is any improvement and increase in their lifespan. So I have been collecting males flies with straight wings every day from the crosses we set up on high sugar and high protein food. Every three days I then check to see how many flies are still alive, and record the number.
The experiments with high sugar and high protein are about half way done at this point, at least with the larval turning. From the data I have collected so far, it seems like the high sugar diet may have some positive outcome on the motor functions of larvae, which shows that it is countering the debilitation that the disease causes. However, there is not noticeable change with the high protein diet. Of course, nothing can be said for sure until all the data is collected. And then if something looks particularly promising, we will probably have to collect a second set of data, to verify the first set.
In addition to more larval turning (because you can never turn too many larvae, right? No, actually, I'm still collecting a full data set for the larvae eating both the high protein and high sugar food. A full data set is thirty larvae per condition (specific genotype and concentration)) I have also taken over setting up the survival data for the week. My team is collecting data on how long adult flies that have 'recovered' from ALS (do not have crumpled wings, but instead have straight normal wings) can live. We then compare this data to how long the flies would normally live on their normal food, and see if there is any improvement and increase in their lifespan. So I have been collecting males flies with straight wings every day from the crosses we set up on high sugar and high protein food. Every three days I then check to see how many flies are still alive, and record the number.
The experiments with high sugar and high protein are about half way done at this point, at least with the larval turning. From the data I have collected so far, it seems like the high sugar diet may have some positive outcome on the motor functions of larvae, which shows that it is countering the debilitation that the disease causes. However, there is not noticeable change with the high protein diet. Of course, nothing can be said for sure until all the data is collected. And then if something looks particularly promising, we will probably have to collect a second set of data, to verify the first set.
Tuesday, March 4, 2014
Week Four
Week four was, as always, very busy in the lab. I virgined, larval turned, and set up crosses of fruit flies. Wednesday was lab meeting, and this week a graduate student, Alyssa, was presenting about a paper she was hoping to publish that had recently been sent to reviews and returned with corrections that should be made before being sent to a scientific journal or magazine. The paper discussed how futsch, a microtubule-associated protein expressed in both central and peripheral nervous system neurons, is a translational target and is neuroprotective. The entire lab contributed on ways to make the paper publishing ready, by explaining and defending the thesis and data presented in the paper. Alyssa discussed what the reviews found flawed in her paper, and what they determined to be not clear enough. The lab also discussed what scientific journal would be best to send this paper. For me, this was a really interesting look into the publishing process of a scientific research paper.
I also began to input my larval turning data that I have been collecting for the last two weeks into excel. I re-learned how to work excel, as it seems I have to every time I use it, and I looked for statistical significance in the data I collected. I don't have a full set of data quite yet, but as soon as I do I will be able to look to see if the diet of high sugar is improving the motor function of the diseased larvae enough to be statistically significant.
I also began to input my larval turning data that I have been collecting for the last two weeks into excel. I re-learned how to work excel, as it seems I have to every time I use it, and I looked for statistical significance in the data I collected. I don't have a full set of data quite yet, but as soon as I do I will be able to look to see if the diet of high sugar is improving the motor function of the diseased larvae enough to be statistically significant.
Tuesday, February 25, 2014
Week Three
The third week of my senior research project I began larval turning for the larvae that were eating the high sugar food my team made the previous week. One interesting thing I noticed throughout larval turning was that the larvae eating the food with lower concentrations of sugar (1X, 2X) developed faster than those who were eating the food with higher concentrations (4X, 8X). I also learned which larvae are appropriate to collect for larval turning. The larvae has several stages of development, known as instars. The first instar is when they have just emerged from their eggs, but are still very small. The larvae that need to be used for larval turning are second instar larvae, when they have begun to grow and develop more. The last stage, the third instar, is right before the larvae begins to pupate, and these larvae are not good for larval turning because they are beginning to move slower and the pupa case is forming.
Another important thing I learned this week is the importance of communication in the lab. The larval turning needs to be done using a GFP (green florescent protein) scope, so that we can check to make sure that the cross was successful. If if was, the larvae will glow due to the GFP marker. But there is only one GFP scope in the lab, and I learned that just about everybody wants to use it, and that it is critical to properly communicate your plans to the rest of the lab so that they can also plan accordingly.
All in all it was a busy, interesting week. Hope everyone else's projects are going well.
Another important thing I learned this week is the importance of communication in the lab. The larval turning needs to be done using a GFP (green florescent protein) scope, so that we can check to make sure that the cross was successful. If if was, the larvae will glow due to the GFP marker. But there is only one GFP scope in the lab, and I learned that just about everybody wants to use it, and that it is critical to properly communicate your plans to the rest of the lab so that they can also plan accordingly.
All in all it was a busy, interesting week. Hope everyone else's projects are going well.
Sunday, February 16, 2014
Week Two
Hello again, and hope everyone has had a great second week with their SRPs. Mine has been very busy. I spent a lot of time collecting virgin D42 females and TDP-43 males, which I talked about in my first post, but I also got to learn and practice some new techniques this week. One of these was larval turning. This is a way for us to check to see if the food we are feeding the fruit flies has any affect on their motor functions. I collected larvae from their containers and put them on an agar plate that contained grape juice. I then would, while looking at the larvae under a microscope, flip them onto their backs. After flipping them, I would record the time it took for them to flip back over and begin moving again. In larvae that contain both the TDP-43 and the D42 genes and therefore the characteristics for ALS, the time it takes to flip back over is significantly longer. When larval turning, two times have to be recorded, the 'freeze time' and the 'turning time'. Often, when a larvae is turned on to its back, it initial reaction is to stop moving and try to assess its situation before flipping over. The time we care more about is the flipping time, so was have to record both the time it take to actually flip and the time it spends frozen on its back. A larvae isn't considered fully flipped until you can see the entire strip on its back and it begins to move in a forward motion. Sometimes larvae will partially flip over and wiggle on their sides to move instead of completely flipping over. I was larval turning with white eyed flies. There are the flies we use as controls to show that they do not have TDP-43 genes or D42 genes. Flies with either of those genes will have red eyes instead of white eyes.
Wednesday was lab meeting for my group, the drug screen team. They went over what we are doing right now, results of past experiments, and what we plan to do in the future. It was cool how the entire lab pitched ideas for reasons behind results, thing that should be standardized or re-done, and things that could be tested in the future. My team also discussed two previous experiments they had been working on that I had been briefly involved with the previous week. They had been using enoxacin (an antibacterial) and different peptides to see if there were increased rescues in the fruit fly population. Both had no statistically significance, and large amounts of error due to inconsistency in the number if rescues in the vials and the variability of the number of flies both produced and rescued. They also commented on how drugs in the past had work only in specific tissues, and that they would often rescue glia and motor phenotypes but not muscle phenotypes. To finish the lab meeting, they outlined their plans for future experiments, which right now include testing fruit flies who ate food with high concentrations of sugar. To set up this experiment, we had to make our own fruit fly food.
The making of the food was a new experience for everyone in my team, not just me. The food is prepared using agar, yeast, several other components, and then the component we are testing, the drug, or in this case, the sucrose, is mixed in. It was quite an adventure making the food, resulting in several overcooked batches and a lab that smelled vaguely of burned marshmallows. We made a batch with the normal concentration of sugar, and ones with two, four and eight times that concentration. We are testing to see if high concentrations of sugar have an effect on the condition of the fruit flies due to past experiments that showed very low larval turning times, meaning that the larvae had greater control over their motor functions and muscles. Additionally, the larvae were all very robust and juicy across multiple phenotypes.
After we made the food, I set up crosses of fruit flies with the flies I had been collecting all week. I put six virgin D42 females and three TDP-43 males in each vial. I set up crosses with three phenotypes for the TDP-43 flies: human wildtype, white (control), and G298S. There were four different concentrations of food, and there were three vials for each phenotype for each concentration, resulting in a total of 36 vials, over 200 virgins female fruit flies, and over 100 male fruit flies.
So that was my week, hope everyone else is having a great time with their SRPs too!
Wednesday was lab meeting for my group, the drug screen team. They went over what we are doing right now, results of past experiments, and what we plan to do in the future. It was cool how the entire lab pitched ideas for reasons behind results, thing that should be standardized or re-done, and things that could be tested in the future. My team also discussed two previous experiments they had been working on that I had been briefly involved with the previous week. They had been using enoxacin (an antibacterial) and different peptides to see if there were increased rescues in the fruit fly population. Both had no statistically significance, and large amounts of error due to inconsistency in the number if rescues in the vials and the variability of the number of flies both produced and rescued. They also commented on how drugs in the past had work only in specific tissues, and that they would often rescue glia and motor phenotypes but not muscle phenotypes. To finish the lab meeting, they outlined their plans for future experiments, which right now include testing fruit flies who ate food with high concentrations of sugar. To set up this experiment, we had to make our own fruit fly food.
The making of the food was a new experience for everyone in my team, not just me. The food is prepared using agar, yeast, several other components, and then the component we are testing, the drug, or in this case, the sucrose, is mixed in. It was quite an adventure making the food, resulting in several overcooked batches and a lab that smelled vaguely of burned marshmallows. We made a batch with the normal concentration of sugar, and ones with two, four and eight times that concentration. We are testing to see if high concentrations of sugar have an effect on the condition of the fruit flies due to past experiments that showed very low larval turning times, meaning that the larvae had greater control over their motor functions and muscles. Additionally, the larvae were all very robust and juicy across multiple phenotypes.
After we made the food, I set up crosses of fruit flies with the flies I had been collecting all week. I put six virgin D42 females and three TDP-43 males in each vial. I set up crosses with three phenotypes for the TDP-43 flies: human wildtype, white (control), and G298S. There were four different concentrations of food, and there were three vials for each phenotype for each concentration, resulting in a total of 36 vials, over 200 virgins female fruit flies, and over 100 male fruit flies.
So that was my week, hope everyone else is having a great time with their SRPs too!
Thursday, February 6, 2014
Week One
Hi everyone!
I guess I should start out describing what exactly I am doing for my project. I am working with a team in a lab in the Molecular and Cellular Biology Department at the University of Arizona. What we are doing is screening potential drug candidates for their ability to counteract the toxic effects of TDP-43 expression in motor neurons. TDP-43 is a multifunctional protein that, among other things, is a telltale marker of ALS. ALS is a neurodegenerative condition in which the motor neurons degrade over time, and they cannot properly communicate with muscles. One of the projects that the lab is undertaking is screening different drugs for their effectiveness against TDP-43 and therefore, also ALS. This is achieved by feeding fruit flies various foods with different drugs in them.
We have to make sure that the fruit flies we harvest actually have the TDP-43 genes and mutation. The way we do this is a bit tricky. We put males with the TDP-43 gene in test tubes with females that have a different protein complex, called GAL4- D42. GAL4 connects to a region of the TDP-43 genes, UAS, and then enable the TDP-43 to be expressed and bind to the D42, which means that the offspring of these flies will express the characteristics of ALS. All of these proteins expressed in these flies are human proteins. In front of the TDP-43 gene on the sequence, there is a Green Florescent Protein (GFP) marker. This is a protein that, as the name implies, will exhibit a green florescent light when put under a ultraviolet light. This is a back-up check, to make sure that the flies actually were the result of a cross with a male with TDP-43 and a female with GAL4-D42. We are working with several different types of TDP-43, several different mutations that occur on that portion of the gene.
So that was a lot of science which pretty much boils down to having males flies with TDP-43, females flies with GAL4-D42, and baby flies with the characteristics of ALS (in this case, crumpled wings, impairing motor function.) These parent flies are put into a test tube to do their business, and after seven days, the parents are taken out. They have, at this point, laid eggs with lots of little baby flies. On day fourteen, we do a screen: we look to see if there are pupa and larvae, and if flies have already hatched, we see if they have straight wings of crumpled wings, and we counted a record the number of flies. If they have straight wings, they are counted as a "rescue", because they have recovered from the symptoms of ALS, crumbled wings. After all the flies are counted, the ones with straight wings are taken and put under a microscope, where we check to make sure they contain the GFP. If they do, they will glow, which is pretty cool. If the flies do not light up, it shows that they were not actually the result of a cross between a male with TDP-43 and a female with GAL4-D42, which means they didn't actually have the characteristics of ALS, which means they aren't a true 'rescue'. On day eighteen, we do the same as day fourteen, but we also count the number of pupa in the container, so we can gain some statistical understanding of how many rescues were in a tube. Three rescues out of thirteen pupa is a lot different than three rescues out of twenty seven pupa.
Glowing fruit flies! Super cool!
So other tasks involved in working towards this end product of rescued flies includes pulling males from the TDP-43 stock and virgin females from the D42 stock. The females have to be virgins because otherwise we don't know if the eggs are the result of a cross between a TDP-43 male or a D42 male. I learned how to identify the differences between male and female fruit flies (you have to look for a darkened abdomen as well as their genitalia) and if the females were virgins (virgins have a little black spot on their abdomen, and they also often have slightly crumpled wings because they have just hatched.) We look at the flies under a microscope, collect the flies we need, put them in tubes, label the tubes, and store them in a 25 °C incubator. The flies we don't need (the female TDP-43 flies and the male and non-virgin D42 flies) are thrown away into this huge erlenmeyer flask filled with vinegar and tons of dead fruit flies. It is just as nasty as it sounds, and I pray that I never accidentally knock over one of those and have to deal with the tidal wave of fruit fly carcasses and vinegar.
Female on the left, male on the right. Lovely looking, eh?
We also "flip" the flies every several days. We have a constant rotation of flies, four rows worth, and when you flip them, it means you throw away the oldest flies, the ones nearest to death anyways, and then transfer the flies from the first row (the newest flies) into a new container, leaving behind the egss they left. The eggs will then hatch, and we can harvest those flies. I flipped for the first time today. The first few tubes I fliped resulted in some escaped flies, but by the end of it I had it more under control.
So that is mostly what I have been doing the last several days. A lot of looking at flies under a microscope. I also attended most of a lab meeting on Wednesday, which was a very interesting experience. A member of the lab was presenting on his work in the lab, which is eventually going to get published. He was talking about translation differences of G298S9 and human wildtype 2 TDP-43. These are just a mutation of the genes and the wildtype, or the version normally found in nature. I could understand most of the beginning of the presentation, but later as it became more statistics focus, I got a bit lost. Eventually it became a group-wide discussion as the head of the lab found a flaw in the reasoning. I thought it was really cool how everyone bounced different ideas back and forth and discussed what would and would not work.
So that had been my week. I hope everyone else has had a great week too. I'm super excited for the upcoming months and really pleased with what I have learned so far.
I guess I should start out describing what exactly I am doing for my project. I am working with a team in a lab in the Molecular and Cellular Biology Department at the University of Arizona. What we are doing is screening potential drug candidates for their ability to counteract the toxic effects of TDP-43 expression in motor neurons. TDP-43 is a multifunctional protein that, among other things, is a telltale marker of ALS. ALS is a neurodegenerative condition in which the motor neurons degrade over time, and they cannot properly communicate with muscles. One of the projects that the lab is undertaking is screening different drugs for their effectiveness against TDP-43 and therefore, also ALS. This is achieved by feeding fruit flies various foods with different drugs in them.
We have to make sure that the fruit flies we harvest actually have the TDP-43 genes and mutation. The way we do this is a bit tricky. We put males with the TDP-43 gene in test tubes with females that have a different protein complex, called GAL4- D42. GAL4 connects to a region of the TDP-43 genes, UAS, and then enable the TDP-43 to be expressed and bind to the D42, which means that the offspring of these flies will express the characteristics of ALS. All of these proteins expressed in these flies are human proteins. In front of the TDP-43 gene on the sequence, there is a Green Florescent Protein (GFP) marker. This is a protein that, as the name implies, will exhibit a green florescent light when put under a ultraviolet light. This is a back-up check, to make sure that the flies actually were the result of a cross with a male with TDP-43 and a female with GAL4-D42. We are working with several different types of TDP-43, several different mutations that occur on that portion of the gene.
So that was a lot of science which pretty much boils down to having males flies with TDP-43, females flies with GAL4-D42, and baby flies with the characteristics of ALS (in this case, crumpled wings, impairing motor function.) These parent flies are put into a test tube to do their business, and after seven days, the parents are taken out. They have, at this point, laid eggs with lots of little baby flies. On day fourteen, we do a screen: we look to see if there are pupa and larvae, and if flies have already hatched, we see if they have straight wings of crumpled wings, and we counted a record the number of flies. If they have straight wings, they are counted as a "rescue", because they have recovered from the symptoms of ALS, crumbled wings. After all the flies are counted, the ones with straight wings are taken and put under a microscope, where we check to make sure they contain the GFP. If they do, they will glow, which is pretty cool. If the flies do not light up, it shows that they were not actually the result of a cross between a male with TDP-43 and a female with GAL4-D42, which means they didn't actually have the characteristics of ALS, which means they aren't a true 'rescue'. On day eighteen, we do the same as day fourteen, but we also count the number of pupa in the container, so we can gain some statistical understanding of how many rescues were in a tube. Three rescues out of thirteen pupa is a lot different than three rescues out of twenty seven pupa.
So other tasks involved in working towards this end product of rescued flies includes pulling males from the TDP-43 stock and virgin females from the D42 stock. The females have to be virgins because otherwise we don't know if the eggs are the result of a cross between a TDP-43 male or a D42 male. I learned how to identify the differences between male and female fruit flies (you have to look for a darkened abdomen as well as their genitalia) and if the females were virgins (virgins have a little black spot on their abdomen, and they also often have slightly crumpled wings because they have just hatched.) We look at the flies under a microscope, collect the flies we need, put them in tubes, label the tubes, and store them in a 25 °C incubator. The flies we don't need (the female TDP-43 flies and the male and non-virgin D42 flies) are thrown away into this huge erlenmeyer flask filled with vinegar and tons of dead fruit flies. It is just as nasty as it sounds, and I pray that I never accidentally knock over one of those and have to deal with the tidal wave of fruit fly carcasses and vinegar.
Female on the left, male on the right. Lovely looking, eh?
We also "flip" the flies every several days. We have a constant rotation of flies, four rows worth, and when you flip them, it means you throw away the oldest flies, the ones nearest to death anyways, and then transfer the flies from the first row (the newest flies) into a new container, leaving behind the egss they left. The eggs will then hatch, and we can harvest those flies. I flipped for the first time today. The first few tubes I fliped resulted in some escaped flies, but by the end of it I had it more under control.
So that is mostly what I have been doing the last several days. A lot of looking at flies under a microscope. I also attended most of a lab meeting on Wednesday, which was a very interesting experience. A member of the lab was presenting on his work in the lab, which is eventually going to get published. He was talking about translation differences of G298S9 and human wildtype 2 TDP-43. These are just a mutation of the genes and the wildtype, or the version normally found in nature. I could understand most of the beginning of the presentation, but later as it became more statistics focus, I got a bit lost. Eventually it became a group-wide discussion as the head of the lab found a flaw in the reasoning. I thought it was really cool how everyone bounced different ideas back and forth and discussed what would and would not work.
So that had been my week. I hope everyone else has had a great week too. I'm super excited for the upcoming months and really pleased with what I have learned so far.
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