j****1
发帖数: 81 | 1 I'm a second year Phd student and I'm doing my prelim now. I don't know if
it's okay to post my proposal abstracts online for people to comment on.
This is one of the proposals I have to write for my prelim. I hope people on
this board could give me some feedback, especially ones that have relevant
knowledge. Thanks very much.
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Cancer stem cells are considered to be the source of tumor recurrence. Colon
cancer is the second most common cause of death from cancer in the United
States. Hippo signaling has recently been proposed to contribute to various
kinds of human cancer. Recently, Hippo signaling has also been demonstrated
to cross talk with Wnt signaling at various levels. Vermeulen L et al. has
shown that different levels of Wnt signal activation correlates with
colorectal cancer stemness. Therefore, considering the role of Hippo
signaling and Wnt signaling in contributing to intestinal growth, it is
interesting to see how Hippo signaling is functioning in the context of
colon cancer stem cells marked by hyperactive Wnt signaling.
Aim 1: Establish whether there is cross talk between Hippo signaling and Wnt
signaling in colon cancer stem cells. We will first use bioinformatics tool
to search in the available databases whether there is a crosstalk between
Hippo signaling and Wnt signaling. We will also do microarray study to
specifically study the possibility of crosstalking between Wnt signaling and
Hippo signaling in FACS sorted TOP-GFP high colon cancer cells.
Aim 2: how Hippo signaling is modulating Wnt signaling responding to
extracellular ligands. We will find out what components in the Hippo
signaling pathway is crosstalking with Wnt signaling, by first testing the
established players such as YAP or TAZ. We will first do Western blot to
find out whether YAP/TAZ is up-regulated or down-regulated in TOP-GFP high
colon cancer cells. We will then either over-express or knock down YAP/TAZ,
depending on the expression level, to see what effects will that have on the
stemness of colon cancer stem cells, using a variety of assays such as
colony formation assay or xenograft assay.
Aim 3: whether Hippo signaling can be used as another colon cancer stem cell
marker. We will find out whether Hippo signaling, particularly YAP or TAZ
can be used as another marker for colon cancer stem cells, just like breast
cancer stem cells. We will engineer a construct with YAP/TAZ responsive
promoter driving the expression of GFP and using lentivirus as a tool to
stably transduce unsorted colon cancer cells, after which we will FACS sort
the GFP high fraction of colon cancer cells and GFP low fraction of colon
cancer cells. We will then test the relative malignancy of the two groups of
colon cancer cells, again using colony formation assay and xenograft assay.
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I primarily have two concerns about aim 1. The first is that I fear it's
been studied a lot already, about the cross-talk between Wnt signaling and
Hippo signaling, but my proposal is to study the crosstalk in the colorectal
cancer stem cell context. The second is that I fear it's not specific
enough; i.e., I did not propose to study whether it's Wnt feeding into Hippo
or Hippo feeding into Wnt. Thanks again. | S**********e
发帖数: 1789 | 2 如果你们系要求独立完成， 不能找人讨论， 那你已经到了被开除的边缘了。
on
relevant
【在 j****1 的大作中提到】
: I'm a second year Phd student and I'm doing my prelim now. I don't know if
: it's okay to post my proposal abstracts online for people to comment on.
: This is one of the proposals I have to write for my prelim. I hope people on
: this board could give me some feedback, especially ones that have relevant
: knowledge. Thanks very much.
: ----------------------------------------------------------------------------
: -----------------------
: Cancer stem cells are considered to be the source of tumor recurrence. Colon
: cancer is the second most common cause of death from cancer in the United
: States. Hippo signaling has recently been proposed to contribute to various
| j****1
发帖数: 81 | 3 Well, they said in the departmental guideline that "You are strongly advised
to have your written proposal read critically by another student or postdoc
, but beware the "friend" who only has good things to say: What you seek is
constructive criticism, and the ability to provide constructive criticism is
an acquired skill many of your peers may lack."
At another place in the guideline "Most importantly - Find a mentor or guide
to help you think through your ideas. Discuss the science over and over
again; pick at the flaws in your hypotheses and experiments and correct them
through successive iterations of presentation and critique. This process is
best done with a faculty member who is willing to be critical (in the best
sense of the word). This faculty member need not be a member of the
Examination Committee. DO NOT prepare this proposal without input from
experienced scientists. You need not go over the proposal in depth with
multiple advisors (too many cooks...) but proposals prepared without
significant input from others tend to fare poorly. You are welcome (in fact,
advised) to call experts outside of the University who are working the in
field. They are usually flattered and helpful. Remember, this is how real
life science is done."
So I think it's OK to ask for inputs online.
【在 S**********e 的大作中提到】
: 如果你们系要求独立完成， 不能找人讨论， 那你已经到了被开除的边缘了。
:
: on
: relevant
| A******y
发帖数: 2041 | 4 You should talk to your PI about your aims. Are you suppose to write a R01
like grant? If you are going to do that, here is my suggestion.
Aim 1: You are proposing a fishing expedition. It is sure way to get low
score on your grant. Propose specific pathways or interacting partners for
your aim 1. The pathway has to be novel somehow.
Aim 2: Do you need some preliminary data on this? What you purposed is
like
generating preliminary data.
Aim 3: Interesting...but please check your model on testing stemness.
I'm not an expert in cancer stem cell, just looking at your puroposal as a
scientist point of view. | j****1
发帖数: 81 | 5 Thanks very much for your comment. I'm doing this for my Phd qualifying exam
, and my PI said he's not supposed to help me.
R01
for
【在 A******y 的大作中提到】
: You should talk to your PI about your aims. Are you suppose to write a R01
: like grant? If you are going to do that, here is my suggestion.
: Aim 1: You are proposing a fishing expedition. It is sure way to get low
: score on your grant. Propose specific pathways or interacting partners for
: your aim 1. The pathway has to be novel somehow.
: Aim 2: Do you need some preliminary data on this? What you purposed is
: like
: generating preliminary data.
: Aim 3: Interesting...but please check your model on testing stemness.
: I'm not an expert in cancer stem cell, just looking at your puroposal as a
| H*g
发帖数: 2333 | 6 I took a glance at what you have here. I could only contribute my thoughts
regarding of the proposal, but not about oncology.
1. The logic flow of the first 4 sentences in the Abstract is terrible. How
about saying something like (maybe wrong in science and need to be better
rephrased) "Colon cancer is the second most common cause of death from
cancer in the United States. One of factors that contribute to this high
severity is due to its high rate of recurrence. As the source of cancer
recurrence, the cancer stem cells have been suggested to be regulated by
multiple signaling pathways, especially the ... pathway."
2. Move the entire Aim 1 and its results to "Preliminary data" section and
use the results, together with any other data YOU collected, to form your
hypothesis. You cannot develop your hypothesis the way as described in the
last sentence of the first paragraph in your abstract.
3. Aim 2 somewhat depends on the result of Aim 1. In Aim 1, you would like
to know "whether there is a crosstalk between " the two signaling pathways;
while in Aim 2, you want to "find out what components in the Hippo signaling
pathway is crosstalking with the wnt". What if the result of your aim 1 is
No, there is no crosstalk? Then your entire proposal falls apart~ Form your
hypothesis on solid data.
4. Your Aim 3 is not testing the same hypothesis as the previous two aims.
This generally suggests, that from the results of the first two aims, you
feel you could already form a conclusion, which is apparently not. Delete it
or move it into a different proposal.
5. You "fear it's been studied a lot already"? Do not put your prelim exam
at this risk.
Good luck.
exam
【在 j****1 的大作中提到】
: Thanks very much for your comment. I'm doing this for my Phd qualifying exam
: , and my PI said he's not supposed to help me.
:
: R01
: for
| j****1
发帖数: 81 | 7 Thank you very much for the comments. I revised abstract. It's attached
below.
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Colon cancer is the second highest cause of cancer deaths in the United
States (Markowitz & Bertagnolli, 2009). It is well known that APC (
adenomatous polyposis coli) loss predisposes patients to colon cancer, which
has been primarily attributed to the over activation of Wnt signaling.
Although controversial, cancer stem cells (CSCs) are considered to be the
source of tumor recurrence. Originally described as the signaling pathway
regulating organ size, Hippo signaling has recently been proposed to
contribute to various kinds of human cancer (Pan, 2010). Hippo signaling has
even been reported to confer CSC features in breast cancer (Cordenonsi et
al., 2011). Recently, Hippo signaling has also been reported to cross talk
with Wnt signaling in the context of colon cancer, both Wnt signaling turns
on YAP gene expression (Konsavage et al., 2012) and beta-catenin forms
complex with YAP to drive the transcription of an array of genes (Rosenbluh
et al., 2012). However, it is still unknown whether Hippo signaling
crosstalks with Wnt signaling in colorectal cancer stem cells. Vermeulen L
et al. reported that different levels of Wnt signal activation correlates
with colorectal cancer cell stemness, mainly by measuring the tumorigenicity
using xenograft assays (Vermeulen L et al., 2010). Therefore, we will test
if and how Hippo signaling is functioning in the context of colon cancer
stem cells marked by hyperactive Wnt signaling in this proposal. In our
proposed model, YAP could functionally replace beta-catenin in CSC
proliferation and tumorigenicity.
Aim 1: Establish whether there is cross talk between Hippo signaling and Wnt
signaling in colon CSCs. Rosenbluh et al. has made it clear that in beta-
catenin active colon cancer cell lines, YAP is essential and knocking down
YAP has comparable effects as knocking down beta-catenin (Rosenbluh et al.,
2012). Based on this, we will do RT-PCR experiments to see the relative
expression level of YAP in FACS sorted TOP-GFP high/low colon CSCs. We will
also overexpress/knock down YAP in TOP-GFP high/low colon CSCs to test if
that will increase/reduce the tumorigenicity of colon CSCs, assessed by
proliferation assays, colony formation assays and xenograft assays.
Aim 2: Define how YAP responds to HGF. Vermeulen L et al. reported that in
response to HGF stimulation, beta-catenin undergoes reduced phosphorylation
at Thr 41 and Ser 45 (associated with beta-catenin degradation) and
increased phosphorylation at Ser 552 (associated with increased
transcriptional activity). In addition to that, there is increased TOP-GFP
reporter activity and increased colonogenicity and tumorigenicity (Vermeulen
L et al., 2010). We will test how YAP is responding to HGF stimulation by
treating FACS sorted TOP-GFP high/low colon CSCs with HGF and perform cell
fractionation experiments to study the phosphorylation and nuclear
translocation status of YAP upon HGF stimulation. In addition, because
Vermeulen L et al. reported coinjection of TOP-GFP low cells with
myofibroblasts can reinstall the tumorigenicity of TOP-GFP low cells, we
will try knocking down YAP in TOP-GFP low cells and test if that can prevent
the reinstallment.
Aim 3: Determine whether YAP expression level can be used as another CSC
indicator. We will find out whether YAP can be used as another indicator for
colon CSCs, just like breast cancer stem cells (Cordenonsi et al., 2011).
To do this, we will engineer a construct with YAP/TEAD responsive promoter
driving the expression of GFP and using lentivirus as a tool to stably
transduce unsorted colon cancer cells, after which we will FACS sort the GFP
high fraction of colon cancer cells and GFP low fraction of colon cancer
cells. We will then test the relative malignancy of the two groups of colon
cancer cells, again using colony formation assay and xenograft assay.
How
【在 H*g 的大作中提到】
: I took a glance at what you have here. I could only contribute my thoughts
: regarding of the proposal, but not about oncology.
: 1. The logic flow of the first 4 sentences in the Abstract is terrible. How
: about saying something like (maybe wrong in science and need to be better
: rephrased) "Colon cancer is the second most common cause of death from
: cancer in the United States. One of factors that contribute to this high
: severity is due to its high rate of recurrence. As the source of cancer
: recurrence, the cancer stem cells have been suggested to be regulated by
: multiple signaling pathways, especially the ... pathway."
: 2. Move the entire Aim 1 and its results to "Preliminary data" section and
| j****1
发帖数: 81 | 8 Here is another abstract, because I have to write two abstracts for my
qualifying exam.
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Not only are neural stem cells important in learning and memory, they are
also potential therapeutic approaches for neurodegenerative and other neural
pathological diseases. Adult neurogenesis occurs in the subventricular zone
(SVZ) of the lateral ventricle and subgranular zone (SGZ) in the
hippocampus (Suh et al., 2009). Relatively little is known about the
metabolic control of stem cells. Recently, Lkb1, a gene regulating
metabolism has also been shown to regulate hematopoietic stem cells
proliferation (Nakada et al., 2010). More recently, it has also been shown
that genes regulating de novo lipogenesis regulate the proliferation of
neural stem and progenitor cells (NSPCs) (Knobloch et al., 2013). However,
whether it’s the de novo generated lipids or other functions of the genes
that regulate the proliferation of NSPCs is not clear, nor is the detailed
mechanism. Therefore, in this grant, we aim to address these questions.
Aim 1. Whether it's the complex fatty acids that fuel the proliferation of
NSPCs. There are many potential ways through which fatty acids can regulate
the proliferation of NSPCs. However, the possibility that it's some other
functions besides lipogenesis of Spot14 that promotes the proliferation of
NSPCs also exists. To shed some light into the underlying mechanism, we will
feed Fasnflox/flox and Spot14flox/flox mice with a high-fat diet (known to
suppress de novo lipogenesis gene expression) after a series of tamoxifen
injection.
Sub aim 1.1 Because the membrane is composed of a relatively constant ratio
of aliphatic fatty acids over cholesterol (Nohturfft et al., 2009), if it’s
solely membrane biogenesis that can promote the proliferation of NSPCs,
there should be concomitant up-regulation of cholesterol synthesis
accompanying fatty acid synthesis. We will test this by examining the
expression level of cholesterol synthesis genes such as HMGCR (HMG CoA
reductase). We will also overexpress/knock down these genes to see if that
can phenocopy/reduce the proliferation of Spot14 over-expressing cells,
respectively.
Sub aim 1.2 Nuclear receptor PPAR gamma has been shown to promote
neurogenesis. The ligands for PPAR gamma such as linoleic acid can be
generated endogenously from fatty acids. The fatty acids thus produced could
be functioning through the PPAR gamma pathway. To test this hypothesis, we
will be utilizing PPAR gamma agonists/antagonists to see if that could
phenocopy/prevent the proliferation of Spot14 over-expressing cells. In
addition to that, we will use PPAR gamma knockout cells, or siRNA, to look
at loss of function.
Aim2, How NSPCs transition from low-proliferating state to high-
proliferating state, i.e., how low-proliferating cells turn down Spot14
expression and switch to high-proliferating state and how this transition is
responding to extracellular signals is not clear. SREBP-1c has been shown
to regulate Spot14 expression at the transcriptional level (Mater et al.,
1999). A series of growth factors influence the expression of SREBP-1c,
among which is VEGF (Nohturfft et al., 2009). Considering the vascular niche
of neurogenesis (Suh et al., 2009), this is an especially attempting
candidate. Therefore, we will test whether VEGF can influence the
proliferation of NSPCs through regulating SREBP-1c and Spot14 expression. We
will first establish the link between SREBP-1c and Spot14 in NSPCs by over-
expressing SREBP-1c. We will then establish the link between VEGF and SREBP-
1c by treating NSPCs with VEGF. |
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