Hello Bio Research Class of 2016-17
Welcome to my blog site. So try and add new posts about your research projects. I want to hear from all of you! Please subscribe to this blog Fitz
For my project, we are observing the behavior of mice when injected with ethanol and comparing it to mice injected with saline, our control. The way it's done we are mimicking the cause and effect of pulling an all-nighter and then consuming alcohol the next day. We are working with 2 strains of adult mice (B6 and D2) and we are observing how sleep deprivation affects them based on gender, strain, and age. Only half of them are sleep deprived, but all of them go through the elevated maze before they are placed into the activity box. The activity box runs for about 20mins so it's the most boring part of the day. We run 16 mice in one day, 2 in the maze since the other 2 are broken, and only 4 go into the activity box at once. That's one day down. On the second day of a run, we observe and record their behavior in the activity box for 20mins after being injected with just saline. The rest of the time, it's data collecting; transferring data from a Windows 98 to the first Mac that was ever made. It's only been a week since our first real test run, but I'm already tired and overwhelmed by the data collecting. The first time we turned on/off the PC, it took a whole day for it to shut down. The last time it had been used was 2007. It is also very hot down in the animal lab.
Tomorrow, we are working on a new group of mice (still of the same strains) to measure the effects of long-term alcohol use.
This is an experiment that they gave me when I first started. This lab uses 2 serum additives for the cell growth media- Fetal Bovine Serum (FBS) and Newborn Calf Serum (NBCS). We add enough so that the GM is 10% FBS. NBCS is relatively inexpensive, but the FBS can cost upwards of $300-$500 for a single bottle. 10% NBCS doesn't work well with any of the cell lines, so it can't be used on its own. The lab now wants to know if there is a concentration at which a mix of FBS and NBCS could be used instead of straight FBS. I was given 7 cell lines to maintain: A549, CAMA, DU145, MB231, MB453, MB468, and MCF7. These cells well originally harvested from human tumors. I am growing a portion of each of these cell lines in 10% FBS, 7.5% FBS to 1.5% NBCS, 5% FBS to 5% NBCS, and 10% NBCS. The 10% FBS is my positive control, the 10% NBCS is my negative control, and the 3:1 and 1:1 mixes are my variables. The cells are incubated in hypoxic conditions to mimic those found in a tumor. The initial hypothesis was that the cell lines could be grown in the 1:1 mix at a comparable rate to the 10% FBS. However the cells mostly seem to prefer the 3:1 mix better. I plan on taking each of the cell lines through at least 3 passages to check for changes in growth rates, final cell count and morphology changes. So far, I've finished A549, DU145, and MB231-they grew the fastest, and tolerated the variable mixes pretty well. MB453, MB468 and CAMA GROW SO SLOWWW, and I'm still working with them. MCF7 is on its last passage now, and I expect to be finished with it by the end of the week. I also found out yesterday that I have to make an informal presentation to my lab and mentor about my work so far. Then we have a formal presentation at the end of July where I'll have to present to my lab and another lab. Needless to say, I feel 0% prepared, and I'm really just praying I don't say anything stupid in front of my mentor. For example: "I allowed my itty bitty teeny tiny microscopic tissue cells to grow for a periololically long time at which point I proceeded to perform an Expelliarmus charm in order to change the growth media and perform more science wizardry." Really hoping that doesn't happen....
We have pretty much finished the experiment I was working on. I maintained cell cultures of preterm piglet mesangial cells. We did several experiments on these cells. We treated them with gentamicin, an aminoglycoside antibiotic, at differing concentrations. We also treated one group with the drug and a reactive oxygen species (ROS) inhibitor , N-acteylcysteine (NAC). We preformed the same experiment with doxorubicin/adriamycin, an anticancer drug. We then observed the cells for proliferation and apoptosis over 72 hours in addition to testing for ROS. The gentamicin had no effect on cell proliferation and did not show significant increase in ROS. The doxorubicin caused cell death, but not proportional to the amount of apoptosis, leading us to think necrosis may be involved. Doxorubicin also caused ROS generation. ROS was present to a lesser extent in the sample treated with NAC. Right now we are running the same experiment with lower concentrations of doxorubicin. The idea is that these findings might be important to treating pregnant mothers and preterm babies.
My abstract is due next Tuesday and I present on the last day of my program, July 24.
Last week we started my time study in order to analyze the types of effects that sepsis has on the kidney in a pediatric rat model. The rats used in this study are 17-18 day old rat pups and are used to mimic the model of an infant child that has come down with sepsis. Last week we completed our first round of fourteen surgeries. The surgeries consisted of two sham surgeries and twelve cecal ligation and puncture surgeries (CLP). Pain medications were administered during surgery and a variety of tests were conducted at 4 hrs post surgery in order to obtain data for that part of the time study. The tests that were performed included glomerular filtration rate (GFR), intravital video microscopy (IVVM) to test for vascular perfusion in the outer cortex of the kidney, and a variety of assay kits were used to measure lactate, creatinine, and blood urea nitrogen levels in blood serum from the rat pups that we performed surgery on. This week we will be doing the same tests at 6 hrs post surgery. An Evans blue dye assay will also be used at this time to measure capillary leakage in the kidney.The week after that the same tests will be repeated at 12 hrs and 18 hrs post surgery. After all of the tests, for the time intervals, are complete I will be making a poster and presenting my data to my program on August 8th. The purpose of this experiment is to study the affects of sepsis at different time intervals in order to potentially come up with new therapeutic methods of treating sepsis in infant children.
Today was my first day in lab at University of Memphis. The research is on identifying E.coli with spectroscopy. We are using 4 different strains of E. coli, 1 Streptococcus, and 1 ATCC culture. For the first day we only prepared the nutrient broth medium for the bacteria and inoculated them. It has to incubate for 24 hours and tomorrow we will do the most tedious part. We have to put the bacteria in small aluminum disks. This is done by putting a layer of bacteria and letting it dry and repeat the process until we have a deep, flat surface of bacteria in the disk. This will result in a strong absorbance when we use the infrared. We hope to find a common signal to demonstrate that it is indeed E. coli.
Last week I got to do my first stereotactic surgery on point mutation mice. First, we took measurements of the three dimensions of where the bregma is located on the skull. From there we calculated the measurements of where the basal lateral amygdala (BLA) was located based on the measurements of the bregma. I adjusted the frame so I could drill a small hole where the measurements of the BLA were and inserted a cannula. After both cannula were inserted, I made a cap out of dental cement to hold them in place and placed the mice in a recovery chamber where they could recover for 3-4 days. I also got to observe mice who had previously been through surgery go through training in the startle chamber. In the startle chamber they would go through a series of beeps that were followed by a shock and the reaction of the mouse is measured and saved by a computer. After the mice were trained we made injections into the cannula with diazepam and retested them in the startle chamber. The data was measured and recorded and will be analyzed later on. We will also observe these mice in the elevated plus maze later this week.
I have been doing research at Le Bonheur since March and we are finishing up the project. We have been looking at transthoracic lines. In the past, the only types of catheters that were used were the right internal jugular catheter and the femoral venous catheter. It has surfaced that these can be dangerous to babies because their veins are so tiny and this can lead to obstruction. Dr. Kumar brought transthoracic catheters to Le Bonheur and has since modified the approach in order to improve patient conditions. Our research is a comparative study of the old approach versus the new approach to transthoracic catheters. I was in charge of gathering and organizing all of the data. All data was obtained from patient's charts and x-rays. We looked for date of placement and date of removal along with any problems that were associated with the lines including: pericardial effusion, thrombosis, dislodgment and arrythmias. The old approach to transthoracic line entered the skin in the abdominal region and went to the heart while the new approach originates at the the left clavicular region and moves downward toward the heart. We are starting to interpret all of the data that was collected and are hoping to find that the new approach is safer and more advatageous for young babies.
At the chiropractic clinic, we work with patients who undergo back problems and patients who need spinal adjustments. Currently, I am still trying to figure out what types of questions I should be asking the patients that could potentially lead to my research study. Mostly the patients that come in are hurt by car injuries that can result to whiplash. Other patients, in various age and sizes, come in due to sports injury as well. Different people come in everyday with different type of back pain. Currently I am "following" or "tracking" about 20 different patients and seeing how their physical and behavior changes as time goes by. I will have to talk to more patients and ask them more questions that can possibly open my eyes into what I will be studying for.
For my project, I am working with Drosophila flies. I am observing their courtship behavior. Flies go through steps of courtship when they are trying to mate. I will be using DGRP lines, which is a database of over 200 lines that have been inbred for 20 generations. The reason behind observing courtship behavior is to analyze each line's social interactions. A characteristic of those with autism is the lack of social skills. I will be measuring copulation latency for each line of flies. Copulation latency is the time it takes for the flies to mate after being paired together. Lines that take longer to mate are the lines that show a lack of social skills. After working through all of the lines that I am going to work on, I can see all of the lines that show a longer copulation latency. I will be looking for SNP's that these lines share. SNP's are a change in a single nucleotide in the DNA. They occur throughout the DNA. they can be used as markers because they appear between genes. I will eventually map out the genetic code for the lines with longer latency and find the SNP's that they share. This could show me the genes that could be involved with their lack of social skills. So far i have finished 3 of the lines, and I am currently part way finished with 5 others. To observe their mating behavior, I pair a virgin male and female fly together once they are 3-5 days old. I film them as they interact and go through their courtship steps.
For my project, I am working with Drosophila flies. I am observing their courtship behavior. Flies go through steps of courtship when they are trying to mate. I will be using DGRP lines, which is a database of over 200 lines that have been inbred for 20 generations. The reason behind observing courtship behavior is to analyze each line's social interactions. A characteristic of those with autism is the lack of social skills. I will be measuring copulation latency for each line of flies. Copulation latency is the time it takes for the flies to mate after being paired together. Lines that take longer to mate are the lines that show a lack of social skills. After working through all of the lines that I am going to work on, I can see all of the lines that show a longer copulation latency. I will be looking for SNP's that these lines share. SNP's are a change in a single nucleotide in the DNA. They occur throughout the DNA. they can be used as markers because they appear between genes. I will eventually map out the genetic code for the lines with longer latency and find the SNP's that they share. This could show me the genes that could be involved with their lack of social skills. So far i have finished 3 of the lines, and I am currently part way finished with 5 others. To observe their mating behavior, I pair a virgin male and female fly together once they are 3-5 days old. I film them as they interact and go through their courtship steps.
The project I am working on is looking at the effects of the glucocorticosteroid, dexamethasone, commonly prescribed to patient with acute lymphoblastic leukemia (ALL). Dexamethasome has a side effect seen in about 10% of adolescent patients called osteonecrosis, which is caused by reduction of blood flow to the bone. This leads to cell death majorly in the osteoporotic sites which leads to pain and reduction in joint mobility. If osteonecrosis is not reverses quickly, it mean replacement of the joint entirely. In Dr. Relling's lab, they've been studying genetic predisposition from osteonecrosis based on genotype of the BMP-7 gene for some months now. The project I'm assisting with is focus on drug interaction between dexamethosome and allopurinol, a drug given to patients with ALL to protect kidneys from uric acid buildup occurring during tumor lysis. Right now, I'm running ELISA assays to check for concentration of BMP-7 protein in male and female mice plasma treated with dexamethosome and allopurinol v. dexamethosome only. Essentially, the goal of the overall experiment is to see if different concentrations of BMP-7 protein leads to more or less genetic predisposition to develop osteonecrosis when being treated with dexamethosome. Once this is discovered, hypothetically, you could find the genotype of the patient for the BMP-7 gene before starting drug therapy and plan according with more steroid holidays etc. and lessen the likeliness for the patient to develop osteonecrosis. We've run a couple of BMP-7 ELISA's and are currently doing some troubleshooting as the dexamethosome is interfering with attachment of the immunoglobin found in the mouse plasma producing false blank results. We should have a plan to avoid this beginning next week.
After harvesting IC-21 mouse macrophages and THP-1 macrophages for a couple weeks I had enough cells to set up my experiment. Setting up my experiment consisted of adding 250,000 cells per well to a 24 well plate and adding medium, PGE2, and "clean" and "dirty" (containing LPS) titanium to the wells. Once the plate incubated for 24 hours I was able to take up the medium with the cytokines present in the medium and froze my samples. I then was able to run a cell proliferation ELISA by adding MTS to each well with fresh new medium; the MTS causes the cells to give off a purple/blue color and one can read the optical densities of the samples with a spectrophotometer at the required wavelength. Once the samples were obtained I was able to run my cytokine ELISAs. For the THP-1 cell line samples I ran IL-1ß, IL-6, IL-10, and TNF-α ELISAs and determined their concentrations in pg/mL based on the various concentrations of PGE2. Whether or not the cytokine concentrations increased or decreased depended on if the cytokine has an inflammatory or anti-inflammatory morphology. I have not started on the IC-21 cell line yet, and future experiments will allow me to specify a specific concentration of PGE2 that causes the cytokine level to increase or decrease. It has been a great experience and if I have learned anything it is that micro pipetting all day can wear on you.
I have been working in the Ophthalmology department trying to see if a certain strain of mice (BXD) are a good model for certain human cases of Primary Open Angle Glaucoma. The beginning of the summer was spent researching the Genome Wide Association Study (GWAS). Once we selected genes that have known links to human POAG, we began using statistical analyses in the genenetwork.org site to measure the expression level of these genes in the BXD mouse eyes. We also researched where the genes were expressed in humans and mice as well as their method of action in each. We began to narrow our list of genes down based on expression level, localization, and method of action. We are also measuring which parental strand is more associated with IOP in the BXD mice using a matrix plot. Finally, we are researching the specific SNP’s in humans that lead to POAG in these genes and searching for SNP’s in similar positions in the mouse model.
The project that I am working on is comparing the gait of different phenotypes of dystonic mice to the wild type. Dystonia is a neurological disorder that is characterized by involuntary muscle contractions leading to abnormal postures and movements. However, it is usually included with another neurological disorder. The dystonic gait is described as sustained abnormal posture of the foot or leg and hyper flexion of the hips. The phenotypes that we are comparing are SGCE, CIZ1, and GNAL at the ages of 1 month, 2, month and 6 month. The DigiGait System was used to measure their gait. This system is a composed of a transparent treadmill belt with a camera underneath it that captures the ventral side of animals. It is attached to a computer that will process the videos and record over 40 different measurements of the animals gait including the length of the stance phase in milliseconds, the size of the area of the paw in cm2, and many other measurements. Each mouse ran at a speed of 20 cm/s and five-seconds of that run was saved for processing. After the videos were processed, the three runs for each mouse were averaged together. Then student t tests were used to test for significance of the different phenotypes compared to the wild type at the different ages.
Okay so the project I'm doing involves appetite and obesity. We're studying the neurological basis of obesity. There are a couple of types of neurons that regulate appetite within the hypothalamus: neuropeptide y neurons, agouti-related protein neurons, and pro-opiomelanocortin neurons. The first two are appetite-stimulating neurons and the last is an appetite suppressing neurons. My project is looking at how astrocytes, a type of glial cell that are responsible for supporting the function of neurons, respond to different hormones or cytokines that enter circulation and act on the appetite-altering neurons. In order to do this, I'm making primary cultures of astrocytes from the hypothalamus and the cortex of multiple mice. We're comparing hypothalamic and cortex astrocytes because they've been known to migrate through various parts of the brain, so we're also trying to see if they'll respond the same way to different hormones and cytokines. So after the astrocytes I'm culturing reach a high enough confluency, we'll split them onto different cover disks and change the media from a growth media to something that represents the normal extracellular environment of the brain. We'll then treat the astrocytes with a calcium marker. We're using calcium release as an indicator of astrocytic activation. So then we'll take the cover slips and put them into a calcium imaging microscope while exposing them to different hormones and cytokines. We haven't gotten to that part yet. It will be a couple of weeks.
Okay so the project I'm doing involves appetite and obesity. We're studying the neurological basis of obesity. There are a couple of types of neurons that regulate appetite within the hypothalamus: neuropeptide y neurons, agouti-related protein neurons, and pro-opiomelanocortin neurons. The first two are appetite-stimulating neurons and the last is an appetite suppressing neurons. My project is looking at how astrocytes, a type of glial cell that are responsible for supporting the function of neurons, respond to different hormones or cytokines that enter circulation and act on the appetite-altering neurons. In order to do this, I'm making primary cultures of astrocytes from the hypothalamus and the cortex of multiple mice. We're comparing hypothalamic and cortex astrocytes because they've been known to migrate through various parts of the brain, so we're also trying to see if they'll respond the same way to different hormones and cytokines. So after the astrocytes I'm culturing reach a high enough confluency, we'll split them onto different cover disks and change the media from a growth media to something that represents the normal extracellular environment of the brain. We'll then treat the astrocytes with a calcium marker. We're using calcium release as an indicator of astrocytic activation. So then we'll take the cover slips and put them into a calcium imaging microscope while exposing them to different hormones and cytokines. We haven't gotten to that part yet. It will be a couple of weeks.
For my project, we are observing the behavior of mice when injected with ethanol and comparing it to mice injected with saline, our control. The way it's done we are mimicking the cause and effect of pulling an all-nighter and then consuming alcohol the next day. We are working with 2 strains of adult mice (B6 and D2) and we are observing how sleep deprivation affects them based on gender, strain, and age. Only half of them are sleep deprived, but all of them go through the elevated maze before they are placed into the activity box. The activity box runs for about 20mins so it's the most boring part of the day. We run 16 mice in one day, 2 in the maze since the other 2 are broken, and only 4 go into the activity box at once. That's one day down. On the second day of a run, we observe and record their behavior in the activity box for 20mins after being injected with just saline. The rest of the time, it's data collecting; transferring data from a Windows 98 to the first Mac that was ever made. It's only been a week since our first real test run, but I'm already tired and overwhelmed by the data collecting. The first time we turned on/off the PC, it took a whole day for it to shut down. The last time it had been used was 2007. It is also very hot down in the animal lab.
ReplyDeleteTomorrow, we are working on a new group of mice (still of the same strains) to measure the effects of long-term alcohol use.
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ReplyDeleteThis is an experiment that they gave me when I first started. This lab uses 2 serum additives for the cell growth media- Fetal Bovine Serum (FBS) and Newborn Calf Serum (NBCS). We add enough so that the GM is 10% FBS. NBCS is relatively inexpensive, but the FBS can cost upwards of $300-$500 for a single bottle. 10% NBCS doesn't work well with any of the cell lines, so it can't be used on its own. The lab now wants to know if there is a concentration at which a mix of FBS and NBCS could be used instead of straight FBS.
ReplyDeleteI was given 7 cell lines to maintain: A549, CAMA, DU145, MB231, MB453, MB468, and MCF7. These cells well originally harvested from human tumors. I am growing a portion of each of these cell lines in 10% FBS, 7.5% FBS to 1.5% NBCS, 5% FBS to 5% NBCS, and 10% NBCS. The 10% FBS is my positive control, the 10% NBCS is my negative control, and the 3:1 and 1:1 mixes are my variables. The cells are incubated in hypoxic conditions to mimic those found in a tumor. The initial hypothesis was that the cell lines could be grown in the 1:1 mix at a comparable rate to the 10% FBS. However the cells mostly seem to prefer the 3:1 mix better.
I plan on taking each of the cell lines through at least 3 passages to check for changes in growth rates, final cell count and morphology changes. So far, I've finished A549, DU145, and MB231-they grew the fastest, and tolerated the variable mixes pretty well. MB453, MB468 and CAMA GROW SO SLOWWW, and I'm still working with them. MCF7 is on its last passage now, and I expect to be finished with it by the end of the week.
I also found out yesterday that I have to make an informal presentation to my lab and mentor about my work so far. Then we have a formal presentation at the end of July where I'll have to present to my lab and another lab. Needless to say, I feel 0% prepared, and I'm really just praying I don't say anything stupid in front of my mentor. For example: "I allowed my itty bitty teeny tiny microscopic tissue cells to grow for a periololically long time at which point I proceeded to perform an Expelliarmus charm in order to change the growth media and perform more science wizardry." Really hoping that doesn't happen....
We have pretty much finished the experiment I was working on. I maintained cell cultures of preterm piglet mesangial cells. We did several experiments on these cells. We treated them with gentamicin, an aminoglycoside antibiotic, at differing concentrations. We also treated one group with the drug and a reactive oxygen species (ROS) inhibitor , N-acteylcysteine (NAC). We preformed the same experiment with doxorubicin/adriamycin, an anticancer drug. We then observed the cells for proliferation and apoptosis over 72 hours in addition to testing for ROS. The gentamicin had no effect on cell proliferation and did not show significant increase in ROS. The doxorubicin caused cell death, but not proportional to the amount of apoptosis, leading us to think necrosis may be involved. Doxorubicin also caused ROS generation. ROS was present to a lesser extent in the sample treated with NAC. Right now we are running the same experiment with lower concentrations of doxorubicin. The idea is that these findings might be important to treating pregnant mothers and preterm babies.
ReplyDeleteMy abstract is due next Tuesday and I present on the last day of my program, July 24.
Last week we started my time study in order to analyze the types of effects that sepsis has on the kidney in a pediatric rat model. The rats used in this study are 17-18 day old rat pups and are used to mimic the model of an infant child that has come down with sepsis. Last week we completed our first round of fourteen surgeries. The surgeries consisted of two sham surgeries and twelve cecal ligation and puncture surgeries (CLP). Pain medications were administered during surgery and a variety of tests were conducted at 4 hrs post surgery in order to obtain data for that part of the time study. The tests that were performed included glomerular filtration rate (GFR), intravital video microscopy (IVVM) to test for vascular perfusion in the outer cortex of the kidney, and a variety of assay kits were used to measure lactate, creatinine, and blood urea nitrogen levels in blood serum from the rat pups that we performed surgery on. This week we will be doing the same tests at 6 hrs post surgery. An Evans blue dye assay will also be used at this time to measure capillary leakage in the kidney.The week after that the same tests will be repeated at 12 hrs and 18 hrs post surgery. After all of the tests, for the time intervals, are complete I will be making a poster and presenting my data to my program on August 8th. The purpose of this experiment is to study the affects of sepsis at different time intervals in order to potentially come up with new therapeutic methods of treating sepsis in infant children.
ReplyDeleteToday was my first day in lab at University of Memphis. The research is on identifying E.coli with spectroscopy. We are using 4 different strains of E. coli, 1 Streptococcus, and 1 ATCC culture. For the first day we only prepared the nutrient broth medium for the bacteria and inoculated them. It has to incubate for 24 hours and tomorrow we will do the most tedious part. We have to put the bacteria in small aluminum disks. This is done by putting a layer of bacteria and letting it dry and repeat the process until we have a deep, flat surface of bacteria in the disk. This will result in a strong absorbance when we use the infrared. We hope to find a common signal to demonstrate that it is indeed E. coli.
ReplyDeleteLast week I got to do my first stereotactic surgery on point mutation mice. First, we took measurements of the three dimensions of where the bregma is located on the skull. From there we calculated the measurements of where the basal lateral amygdala (BLA) was located based on the measurements of the bregma. I adjusted the frame so I could drill a small hole where the measurements of the BLA were and inserted a cannula. After both cannula were inserted, I made a cap out of dental cement to hold them in place and placed the mice in a recovery chamber where they could recover for 3-4 days. I also got to observe mice who had previously been through surgery go through training in the startle chamber. In the startle chamber they would go through a series of beeps that were followed by a shock and the reaction of the mouse is measured and saved by a computer. After the mice were trained we made injections into the cannula with diazepam and retested them in the startle chamber. The data was measured and recorded and will be analyzed later on. We will also observe these mice in the elevated plus maze later this week.
ReplyDeleteI have been doing research at Le Bonheur since March and we are finishing up the project. We have been looking at transthoracic lines. In the past, the only types of catheters that were used were the right internal jugular catheter and the femoral venous catheter. It has surfaced that these can be dangerous to babies because their veins are so tiny and this can lead to obstruction. Dr. Kumar brought transthoracic catheters to Le Bonheur and has since modified the approach in order to improve patient conditions. Our research is a comparative study of the old approach versus the new approach to transthoracic catheters. I was in charge of gathering and organizing all of the data. All data was obtained from patient's charts and x-rays. We looked for date of placement and date of removal along with any problems that were associated with the lines including: pericardial effusion, thrombosis, dislodgment and arrythmias. The old approach to transthoracic line entered the skin in the abdominal region and went to the heart while the new approach originates at the the left clavicular region and moves downward toward the heart. We are starting to interpret all of the data that was collected and are hoping to find that the new approach is safer and more advatageous for young babies.
ReplyDeleteAt the chiropractic clinic, we work with patients who undergo back problems and patients who need spinal adjustments. Currently, I am still trying to figure out what types of questions I should be asking the patients that could potentially lead to my research study. Mostly the patients that come in are hurt by car injuries that can result to whiplash. Other patients, in various age and sizes, come in due to sports injury as well. Different people come in everyday with different type of back pain. Currently I am "following" or "tracking" about 20 different patients and seeing how their physical and behavior changes as time goes by. I will have to talk to more patients and ask them more questions that can possibly open my eyes into what I will be studying for.
ReplyDeleteFor my project, I am working with Drosophila flies. I am observing their courtship behavior. Flies go through steps of courtship when they are trying to mate. I will be using DGRP lines, which is a database of over 200 lines that have been inbred for 20 generations. The reason behind observing courtship behavior is to analyze each line's social interactions. A characteristic of those with autism is the lack of social skills. I will be measuring copulation latency for each line of flies. Copulation latency is the time it takes for the flies to mate after being paired together. Lines that take longer to mate are the lines that show a lack of social skills. After working through all of the lines that I am going to work on, I can see all of the lines that show a longer copulation latency. I will be looking for SNP's that these lines share. SNP's are a change in a single nucleotide in the DNA. They occur throughout the DNA. they can be used as markers because they appear between genes. I will eventually map out the genetic code for the lines with longer latency and find the SNP's that they share. This could show me the genes that could be involved with their lack of social skills. So far i have finished 3 of the lines, and I am currently part way finished with 5 others. To observe their mating behavior, I pair a virgin male and female fly together once they are 3-5 days old. I film them as they interact and go through their courtship steps.
ReplyDeleteFor my project, I am working with Drosophila flies. I am observing their courtship behavior. Flies go through steps of courtship when they are trying to mate. I will be using DGRP lines, which is a database of over 200 lines that have been inbred for 20 generations. The reason behind observing courtship behavior is to analyze each line's social interactions. A characteristic of those with autism is the lack of social skills. I will be measuring copulation latency for each line of flies. Copulation latency is the time it takes for the flies to mate after being paired together. Lines that take longer to mate are the lines that show a lack of social skills. After working through all of the lines that I am going to work on, I can see all of the lines that show a longer copulation latency. I will be looking for SNP's that these lines share. SNP's are a change in a single nucleotide in the DNA. They occur throughout the DNA. they can be used as markers because they appear between genes. I will eventually map out the genetic code for the lines with longer latency and find the SNP's that they share. This could show me the genes that could be involved with their lack of social skills. So far i have finished 3 of the lines, and I am currently part way finished with 5 others. To observe their mating behavior, I pair a virgin male and female fly together once they are 3-5 days old. I film them as they interact and go through their courtship steps.
ReplyDeleteThe project I am working on is looking at the effects of the glucocorticosteroid, dexamethasone, commonly prescribed to patient with acute lymphoblastic leukemia (ALL). Dexamethasome has a side effect seen in about 10% of adolescent patients called osteonecrosis, which is caused by reduction of blood flow to the bone. This leads to cell death majorly in the osteoporotic sites which leads to pain and reduction in joint mobility. If osteonecrosis is not reverses quickly, it mean replacement of the joint entirely. In Dr. Relling's lab, they've been studying genetic predisposition from osteonecrosis based on genotype of the BMP-7 gene for some months now. The project I'm assisting with is focus on drug interaction between dexamethosome and allopurinol, a drug given to patients with ALL to protect kidneys from uric acid buildup occurring during tumor lysis. Right now, I'm running ELISA assays to check for concentration of BMP-7 protein in male and female mice plasma treated with dexamethosome and allopurinol v. dexamethosome only. Essentially, the goal of the overall experiment is to see if different concentrations of BMP-7 protein leads to more or less genetic predisposition to develop osteonecrosis when being treated with dexamethosome. Once this is discovered, hypothetically, you could find the genotype of the patient for the BMP-7 gene before starting drug therapy and plan according with more steroid holidays etc. and lessen the likeliness for the patient to develop osteonecrosis. We've run a couple of BMP-7 ELISA's and are currently doing some troubleshooting as the dexamethosome is interfering with attachment of the immunoglobin found in the mouse plasma producing false blank results. We should have a plan to avoid this beginning next week.
ReplyDeleteAfter harvesting IC-21 mouse macrophages and THP-1 macrophages for a couple weeks I had enough cells to set up my experiment. Setting up my experiment consisted of adding 250,000 cells per well to a 24 well plate and adding medium, PGE2, and "clean" and "dirty" (containing LPS) titanium to the wells. Once the plate incubated for 24 hours I was able to take up the medium with the cytokines present in the medium and froze my samples. I then was able to run a cell proliferation ELISA by adding MTS to each well with fresh new medium; the MTS causes the cells to give off a purple/blue color and one can read the optical densities of the samples with a spectrophotometer at the required wavelength. Once the samples were obtained I was able to run my cytokine ELISAs. For the THP-1 cell line samples I ran IL-1ß, IL-6, IL-10, and TNF-α ELISAs and determined their concentrations in pg/mL based on the various concentrations of PGE2. Whether or not the cytokine concentrations increased or decreased depended on if the cytokine has an inflammatory or anti-inflammatory morphology. I have not started on the IC-21 cell line yet, and future experiments will allow me to specify a specific concentration of PGE2 that causes the cytokine level to increase or decrease. It has been a great experience and if I have learned anything it is that micro pipetting all day can wear on you.
ReplyDeleteI have been working in the Ophthalmology department trying to see if a certain strain of mice (BXD) are a good model for certain human cases of Primary Open Angle Glaucoma. The beginning of the summer was spent researching the Genome Wide Association Study (GWAS). Once we selected genes that have known links to human POAG, we began using statistical analyses in the genenetwork.org site to measure the expression level of these genes in the BXD mouse eyes. We also researched where the genes were expressed in humans and mice as well as their method of action in each. We began to narrow our list of genes down based on expression level, localization, and method of action. We are also measuring which parental strand is more associated with IOP in the BXD mice using a matrix plot. Finally, we are researching the specific SNP’s in humans that lead to POAG in these genes and searching for SNP’s in similar positions in the mouse model.
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ReplyDeleteThe project that I am working on is comparing the gait of different phenotypes of dystonic mice to the wild type. Dystonia is a neurological disorder that is characterized by involuntary muscle contractions leading to abnormal postures and movements. However, it is usually included with another neurological disorder. The dystonic gait is described as sustained abnormal posture of the foot or leg and hyper flexion of the hips. The phenotypes that we are comparing are SGCE, CIZ1, and GNAL at the ages of 1 month, 2, month and 6 month. The DigiGait System was used to measure their gait. This system is a composed of a transparent treadmill belt with a camera underneath it that captures the ventral side of animals. It is attached to a computer that will process the videos and record over 40 different measurements of the animals gait including the length of the stance phase in milliseconds, the size of the area of the paw in cm2, and many other measurements. Each mouse ran at a speed of 20 cm/s and five-seconds of that run was saved for processing. After the videos were processed, the three runs for each mouse were averaged together. Then student t tests were used to test for significance of the different phenotypes compared to the wild type at the different ages.
Okay so the project I'm doing involves appetite and obesity. We're studying the neurological basis of obesity. There are a couple of types of neurons that regulate appetite within the hypothalamus: neuropeptide y neurons, agouti-related protein neurons, and pro-opiomelanocortin neurons. The first two are appetite-stimulating neurons and the last is an appetite suppressing neurons. My project is looking at how astrocytes, a type of glial cell that are responsible for supporting the function of neurons, respond to different hormones or cytokines that enter circulation and act on the appetite-altering neurons. In order to do this, I'm making primary cultures of astrocytes from the hypothalamus and the cortex of multiple mice. We're comparing hypothalamic and cortex astrocytes because they've been known to migrate through various parts of the brain, so we're also trying to see if they'll respond the same way to different hormones and cytokines. So after the astrocytes I'm culturing reach a high enough confluency, we'll split them onto different cover disks and change the media from a growth media to something that represents the normal extracellular environment of the brain. We'll then treat the astrocytes with a calcium marker. We're using calcium release as an indicator of astrocytic activation. So then we'll take the cover slips and put them into a calcium imaging microscope while exposing them to different hormones and cytokines. We haven't gotten to that part yet. It will be a couple of weeks.
ReplyDeleteOkay so the project I'm doing involves appetite and obesity. We're studying the neurological basis of obesity. There are a couple of types of neurons that regulate appetite within the hypothalamus: neuropeptide y neurons, agouti-related protein neurons, and pro-opiomelanocortin neurons. The first two are appetite-stimulating neurons and the last is an appetite suppressing neurons. My project is looking at how astrocytes, a type of glial cell that are responsible for supporting the function of neurons, respond to different hormones or cytokines that enter circulation and act on the appetite-altering neurons. In order to do this, I'm making primary cultures of astrocytes from the hypothalamus and the cortex of multiple mice. We're comparing hypothalamic and cortex astrocytes because they've been known to migrate through various parts of the brain, so we're also trying to see if they'll respond the same way to different hormones and cytokines. So after the astrocytes I'm culturing reach a high enough confluency, we'll split them onto different cover disks and change the media from a growth media to something that represents the normal extracellular environment of the brain. We'll then treat the astrocytes with a calcium marker. We're using calcium release as an indicator of astrocytic activation. So then we'll take the cover slips and put them into a calcium imaging microscope while exposing them to different hormones and cytokines. We haven't gotten to that part yet. It will be a couple of weeks.
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