Name: Grisel Paulino

BIOAP 4580 Problem Set #2

Due Friday, 2/27 in class
**Please remember to cite sources used
1. Why is it important to denature your protein sample with SDS before using it for a Western Blot analysis?
SDS sodium dodecyl sulfate denatures the protein by removing its disulfide bonds making it linear (as opposed to a
secondary, tertiary, or quaternary structure) and dependent on its length and mass-to-charge ratio. SDS imparts an evenly
distributed negative charge on the linearized molecule so that it is separated through electrophoresis by mass only. In this
step the larger proteins will be found at the top of the gel and smaller proteins will be found at the bottom. This when you
are separating proteins of varying sizes that can be easily distinguished through PAGE (polyacrylamide gel
electrophoresis). SDS makes it easier for the detection and purification of a specific protein in western blotting. Adding
SDS-page to western blotting resolves the issue of cross-reactivity of antibodies. This simply means that linearizing the
protein and running it through PAGE will make sure the Antibody for that specific protein only detects it because of its
specific antigenic site. This is so the antibody will not confuse a different proteins antigenic site for the protein of
interests. The improper binding to the wrong protein by our antibody will give you an impure detection and analysis of your
protein of interest.
References: Mahmood, Tahrin, and Ping-Chang Yang. Western Blot: Technique, Theory, and Trouble Shooting. North
American Journal of Medical Sciences 4.9 (2012): 429434. PMC. Web. 2 Mar. 2015.
http://en.wikipedia.org/wiki/Western_blot
2. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) are similar techniques in that they both
measure RNA levels, yet they have several differences that make them unique and preferable in certain experiments.
Compare and contrast these two methods. (The best way to do this is to use a table)
Procedure:
qRT-PCR or Quantitative Reverse Transcription Polymerase Chain compared to other RNA quantification methods, such
asRNA sequencing, qRT-PCR is considered to be the most powerful, sensitive, and quantitative assay for the detection of
RNA levels. qRT-PCR Enables reliable detection and measurement of products generated during each cycle of PCR
process.
Applications of real time quantitative RT-PCR: relative and absolute quantification of gene expression, validation of DNA
microarray results, variation analysis, counting bacterial, viral, or fungal loads, etc.
RNA-sequencing (RNA-Seq) has revolutionized the exploration of gene expression. Advances in the RNA sequencing
workflow, from sample preparation through data analysis, enable rapid profiling and deep investigation of the
transcriptome.
Applications of real time quantitative RNA-sequencing: Identify and quantify both rare and common transcripts, with over
six orders of magnitude of dynamic range, Align sequencing reads across splice junctions, and detect isoforms, novel
transcripts and gene fusions, Derive strand information with high precision, and Perform robust whole-transcriptome
analysis on a wide range of samples, including low-quality samples
http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction#cite_note-pmid20204872-9
http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechQPCR.shtml
(NOTE: Besides the obvious differences in their procedures, consider the differences in data that they give, costs and
learning curve associated with the techniques, time to obtain results, and limitations of these techniques)

http://www.tlcr.org/article/viewFile/1029/1680/5658

RNA- sequencing (RNA-seq)

Data

Cost

-High Throughput
(reproducibility)
-One experiment provides info
about all genes.
High cost for one analysis
(>$1000)

Quantitative ReverseTranscription PCR

(qRT-PCR)
-Medium Throughput
-High sequence-specific
-High Sensitivity
Low Cost (<$500)

Learning Curve

Longer period of learning

(harder to learn)

Faster period of learning

(easier to learn)

Time

*Depending on how many

genes

*Depending on how many

genes

Days-to-months
-Bioinformatician needed for
the analysis of the results
-High cost
-Time consuming

Hours to a day
-Analyzes one gene at a time
-Need to know the gene
sequence prior to analysis
-Requires large amounts of
RNA
-Operator variability
-expensive equipments and
reagents

Limitations

3. Glutamate is a ubiquitous neurotransmitter involved in excitatory post-synaptic potentials (EPSPs). In the hippocampus,
these EPSPs are mediated primarily through the NMDA receptor (NMDAR) binding of glutamate (together with a glycine
co-activator and prior depolarization of the post-synaptic membrane) to the NMDAR stimulates opening of the receptor
channel, letting cations (especially Ca2+) into the post-synaptic cell and making it more likely to conduct action potentials.
a) The interactions between NMDAR and glutamate are critical to long-term potentiation (LTP) and memory formation in
the hippocampus.
Describe the changes that occur at the pre-synaptic terminal and the post-synaptic terminal that follow establishment of
LTP.

Long-term Potentiation LTP is said to be an increase in the synaptic response following potentiated pulses of electrical
stimuli that has sustained at a level above the baseline for hours or longer. Interactions between postsynaptic neurons
and the specific presynaptic neurons are involved in LTP. The long-term stabilization of synaptic changes is determined by
an increase of pre- and postsynaptic structures such as axonal button, dendritic spine and postsynaptic density.
Pre-Synaptic
When long-term potentiation occurs there are permanent changes in the presynaptic neuron along with an increase in
AMPA receptor sites. Long-term potentiation causes an increase in the release of the neurotransmitter glutamate.
Post-Synaptic
It is proposed that Ca2+ could act to initiate protein synthesis in the post-synaptic membrane, and to simulate the
diffusion of nitric oxide out of the post synaptic membrane and into the pre synaptic terminal button, where it would
stimulate the production of glutamate, resulting in an increase in the rate of glutamate release. There is a significant
change in the number of receptor sites present on the post-synaptic membranes adding more AMPA glutamate receptors.
This increases the level of excitability in these neurons. Another change that occurs would be the sites doubling their
contact surface by splitting in two. These "perforated synapses" can be associated with the mechanism of millions of
neurons that create billions of connections in our brains every time we learn something new.
http://web.mst.edu/~rhall/neuroscience/05_simple_learning/ltp.pdf
b) The NMDAR is actually the primary site of alcohols effects ethanol inhibits the NMDAR channel from opening and
prevents EPSPs or LTP from happening, especially with acute ethanol dosage (which is why your memory of what
happened that night goes fuzzy). The real damage comes from the hangover you feel the next day neurons respond to
the inhibition of NMDAR by upregulating its expression and downregulating its inhibition (by GABAergic neurons). Ethanol
withdrawal thus leads to overexcitation of the NMDAR, letting too many cations into the post-synaptic cell and causing
cellular damage and neuronal apoptosis.
Assuming abstinence is not an option, describe how you would create a drug to combat this death of neurons, and how
you would assay its efficacy.
There are multiple ways to create a drug that could combat the death of these neurons. The most efficient way to
combat a conflict such as this is through brute force. This technique involves injecting different drugs into the test subject
and seeing which one works. The easiest and fastest way to execute this technique would be by creating an active pool of
different compounds that could potentially work (or not) and testing them as a batch. The batch that does work should
then be tested in smaller batches and the cycle goes on testing the efficacy of each batch until it can be narrowed down to
one compound (or multiple) creating the drug. There are multiple areas of mechanism that could be addressed with
developing a drug like this. We could address the inhibition of NMDAR, the compensated up regulation of NMDAR, The
down regulation of its inhibition by GABAergic neurons, and decreasing the amount of cations being let into the postsynaptic cell. Creating a drug that stops cellular damage or neuronal apoptosis may increase the chances of developing
cancer.

4. Phantom limb pain (PLP) is a mild to extreme pain felt in an area where a limb has been amputated. Patients can
experience different kinds of pain including tingling, throbbing, piercing, and pins and needles sensations. While
sensations can disappear or decrease over time, a constant pain for more than six months has a poor prognosis for
improvement.
There are 3 proposed theoretical mechanisms to explain PLP:
1. Peripheral Mechanism
2. Central Neural Mechanisms
3. Psychogenic Mechanism
a) Describe each mechanism, in detail. (Please site sources)
Peripheral Mechanism- During amputation, peripheral nerves are severed. This results in massive tissue and neuronal
injury causing disruption of the normal pattern of afferent nerve input to the spinal cord. Deaffrrentation is a process that
3

follows this and the proximal portion of the severed nerve sprouts to form neuromas. There is an increased accumulation
of molecules enhancing the expression of sodium channels in these newly formed neuromas Resulting in hype-excitability
and spontaneous release. This abnormal peripheral activity is thought to be a potential source of the stump pain, including
phantom pain. There are studies that suggest that drugs that block the sodium channels reduce phantom pain.
(mechanism of PLP is still being studied for patients with congenital absence of limbs)
Central Neural Mechanism-

Spinal
The axonal sprouts at the proximal section of the amputated peripheral nerve form connections with the receptor neurons
of the spinal cord. Even some neurons that are not responsible for pain transmission also sprout into the Lamina II of the
spinal cord (specifically, dorsal horn- area involved in the transmission of nociceptive afferent inputs). This process is
followed by another process is called central sensitization increased neuronal activity, expansion of the neuronal receptive
field, and hyper-excitability of other regions. During this process, there is also an increase in the activity at NMDA
receptors mediated by neurotransmitters. This is then followed by a phenomenon called the windup phenomenon where
there is an up-regulation of NMDA receptors in the area. This process causes a change in the firing pattern of the central
nociceptive neurons. The target neurons at the spinal level for the descending inhibitory transmission from the supraspinal
centers may be lost. There also may be a reduction in the local intersegmental inhibitory mechanisms at the level of the
spinal cord, resulting in spinal disinhibition and nociceptive inputs reaching the supra spinal centers.

Brain
1. Cortical reorganization is perhaps the most cited reason for the cause of PLP. During reorganization, the cortical areas
representing the amputated extremity are taken over by the neighboring representational zones in both the primary
somatosensory and the motor cortex. Cortical reorganization partly explains why the afferent nociceptive stimulation of
neurons within the stump or surrounding area produces the sensation in the missing limb. The extent of cortical
reorganization has been found to be directly related to the degree of pain and the size of the deafferentiated region.
Multiple imaging studies have correlated greater extent of somatosensory cortex involvement with more intense phantom
limb experience.
2. The body schema concept can be thought of as a template of the entire body in the brain and any change to the body,
such as an amputation, results in the perception of a phantom limb. The body schema concept is the neuromatrix and
neurosignature hypothesis, which is known as a network of neurons within the brain that integrates numerous inputs from
various areas including limbic and somatosensory components. It results in an output pattern that evokes pain or other
meaningful experiences. The deprivation of various inputs from the limbs to the neuromatrix causes an atypical
neurosignature to be produced that results in the generation of PLP (abnormal somatosensory phenomenon). Illusory
perceptions are now being studied to be one of the mechanisms of phantom where it said that the parietal and frontal
lobes are also involved besides the primary somatosensory cortex. Painful sensations, such as PLP, may be related to the
misinterpretation of motor intention and sensory feedback and a corresponding activation of the parietal and frontal brain
areas.
Psychogenic Mechanism- This mechanism implies that the phantom pain was imagined or invented, existing as a
manifestation of an underlying emotional issue (like depression, etc.). Although, stress, anxiety, exhaustion, and
depression are believed to exacerbate PLP cross-sectional studies on the relationship between psychological symptoms
and PLP have not been able to prove a correlation.

Reference: Bishnu Subedi and George T. Grossberg, Phantom Limb Pain: Mechanisms and Treatment Approaches,
Pain Research and Treatment, vol. 2011, Article ID 864605, 8 pages, 2011. doi:10.1155/2011/864605

b) Choose one mechanism from the list above and research a primary literature article that supports that mechanism.
Give a brief summary of the paper (including hypothesis, important main finding(s), and main conclusion). Please site
source.
Central Neural Mechanism

Hypothesis: Anesthesia-induced pain reduction should lead to a corresponding reduction in cortical reorganization. In
cases in which anesthesia would not abolish phantom limb pain, cortical reorganization should remain unchanged.

Main Findings: Neuroelectric source imaging was used to determine changes in cortical reorganization in
somatosensory cortex after anesthesia of an amputation stump produced by brachial plexus blockade in six phantom limb
pain patients and four pain-free amputees. Three of six phantom limb subjects experienced a virtual elimination of current
phantom pain attributable to anesthesia that was mirrored by a very rapid elimination of cortical reorganization in
somatosensory cortex. Cortical reorganization remained unchanged in three phantom limb pain amputees whose pain
was not reduced by brachial plexus blockade and in the phantom pain-free amputation controls.

Main Conclusion: The above findings suggest that cortical reorganization and phantom limb pain might have a
strong causal relationship. The paper goes on to explain the functional relationship between cortical reorganization and
phantom limb pain (Central Neural Mechanism) and suggests that phantom limb pain could be modulated by behavioral
or pharmacological interventions that modify cortical reorganization.

Reference: Birbaumer, Niels, et al. "Effects of regional anesthesia on phantom limb pain are mirrored in changes in
cortical reorganization." The journal of neuroscience17.14 (1997): 5503-5508.