Sample Proforma for PhD Examination
Sample Thesis Title:
“The effect of herbal-derived antioxidants on the contractility properties
and function of the heart under oxidative stress studied using an isolated cardiomyocyte
model”.
(Sample Comments are shown in blue italics)
Introduction
- Was the literature relevant to the topic reviewed
in a critical manner?
Comment: Yes.
More mention could have been made to other data using adult heart cardiomyocytes
in addition to neonatal-derived cardiomyocytes.
- Was
previous work presented within an overall conceptual framework and in
a systematic way?
Comment: Yes. However the recent work of Wang, Das and Smith (2005)
on the novel antioxidants derived from some temperate plants, should
have
been included.
- Were the hypotheses to be tested and the aims of the research
clearly
stated?
Comment: Mostly. Aims could have been separated better and a numbering
system used.
- Are the nature and extent of the putative original contributions
clear?
Comment: Mostly.
General Comment on the Introduction:
The introduction is generally well written and covers the main areas of
the research topic. It lacks a few recent references related to the possible
mechanism of action of such herbal antioxidants in relation to key indicators
of cell functionality. This and other parts of the thesis would benefit
from a thorough spelling and syntax (sentence structure) check.
ASSESSMENT - Introduction
| A |
B |
C |
D |
| EXCELLENT |
GOOD |
AVERAGE |
POOR |
Acceptable -
With very minor editing |
Acceptable -
With minor revisions |
Acceptable -
With major revision |
Not acceptable -
Major revision and edit – see comments |
Methods
- Were the methodologies employed detailed adequately?
Comment: The examiner acknowledges the difficulty in maintaining, calcium-tolerant,
adult cardiomyocytes over extended periods of time for contractility
measurements. However, greater care and optimisation of the short-term
culture conditions
would have greatly extended the number of cells available for experimentation
at any one time.
- Were the methodologies appropriate and their use justified?
Comment: The molecular probes to directly report on the generation
of free radical in the intracellular environment have been commercially
available
for some time. Such a strategy would have been far better than
much
older, less rigorous or specific, TBA (thiobarbituric acid) titrations.
- Was
the instrumentation used appropriately?
Comment: Fluorescence measurements using an indicator of intracellular
calcium ion fluxes needed to be standardized to provide actual
concentrations which could be related to free versus bound forms
of calcium ions.
Voltages used to induce contractility were excessive and resulted in very
high levels
of cardiomyocyte mortality, reducing the collection of good data.
- Were
quantitative methods adequately validated? (e.g. accuracy, precision,
etc.)
Comment: Statistical treatment of some of the data is very weak.
Intra- and inter-assay variations should have been better validated.
Bonferroni
or other multiple comparison tests were not used appropriately
in some sections.
General Comment on the Methods Section:
This section was reasonable well presented, however, the methods used to
undertake multiple studies on single cells with respect to increasing
doses of the candidate agent, together with the washout protocol, were
not described.
ASSESSMENT - Methods
| A |
B |
C |
D |
| EXCELLENT |
GOOD |
AVERAGE |
POOR |
Acceptable -
With very minor editing |
Acceptable -
With minor revisions |
Acceptable -
With major revision |
Not acceptable -
Major revision and edit – see comments |
Results Chapters
- Were results presented in adequate detail?
Comment: Data representation could have been integrated better by combining
many of the similar data sets into composite graphs and figures.
Standard figure symbols as used in international journals should
have been used.
The actual numbers of inter- and intra-sample replicates in certain
data sets were confusing.
- Were limitations inherent in the studies recognised
and stated?
Comment: To a certain degree the candidate noted that the extent
of replication (ie n= values) was on average quite low. This was
in part
due to the fact
acknowledged above that the short-term cell culture conditions had
not been adequately optimised.
- Were variables which might influence the study
recognised and either
controlled or measured?
Comment: The study would have benefited if the absolute range of the responses
of the particular bioassay system employed had been rigorously determined
prior to the experimental aspect of testing of herbal extracts.
- Were
the conclusions reached justifiable in the light of the data and the way
they were analysed?
Comment: Mostly. A number of the extracts tested gave highly significant
results with respect to the magnitude of their individual responses. Thus
the conclusions
were justified and broadly reflected the thesis aims.
- Was full use of the
collected data made?
Comment: Some data were only discussed in a very peripheral manner. Greater
commentary on the similarity of responses with certain extracts sharing
similar structures could have been made.
- Where appropriate, was due credit
given to previous workers for ideas developed?
Comment: Yes. Adequate referencing and brief discussion on the work
of others were included.
General Comment on the Results Section:
This section was reasonably well presented, however, the results could
have been better presented if a section detailing the optimisation and
validation of the experimental set-up with compounds known to have predictable
effects on cardiomyocyte contractility (ie positive and negative controls)
were presented before the new experimental data.
ASSESSMENT - Results
| A |
B |
C |
D |
| EXCELLENT |
GOOD |
AVERAGE |
POOR |
Acceptable -
With very minor editing |
Acceptable -
With minor revisions |
Acceptable -
With major revision |
Not acceptable -
Major revision and edit – see comments |
General Discussion
- Was new material reported?
Comment: Yes. Some of the extracts tested have not previously been
reported as being cardioactive.
- Would the new material be perceived as a valuable
addition to a field of knowledge?
Comment: Further confirmatory experimental validation work would
be necessary particularly on the whole, perfused working heart
model before
its potential
value could be ascertained.
- Do the conclusions overturn or challenge
previous beliefs?
Comment: No. They are consistent in that a full survey of cardioactive
compounds from numerous sources of bioactives still remains incomplete.
- Were
the findings compared to the known literature and placed in context?
Comment. Yes. The discussion was logical and compared and contrasted
the results of the present work with that previously published in
the specific
area and in the wider context of antioxidant effectors and protectors
of cellular function.
- Does the work open up new ideas for future research?
Comment: The bioactives contained in these tested herbal-derived extracts
may also have application in other physiological and pathophysiological
conditions besides their putative effects on cardiac and cardiovascular
function, eg in inflammation and neoplasia.
ASSESSMENT - General Discussion
| A |
B |
C |
D |
| EXCELLENT |
GOOD |
AVERAGE |
POOR |
Acceptable -
With very minor editing |
Acceptable -
With minor revisions |
Acceptable -
With major revision |
Not acceptable -
Major revision and edit – see comments |
General Comment on the Thesis (180-200 words)
| A |
B |
C |
D |
| EXCELLENT |
GOOD |
AVERAGE |
POOR |
Acceptable -
With very minor editing |
Acceptable -
With minor revisions |
Acceptable -
With major revision |
Not acceptable -
Major revision and edit – see comments |
Summary:
The broad aims of this thesis were to develop a cellular model for studying
reperfusion injury in order to investigate the putative reported protective
effects of various antioxidants found in different herbal extracts. Heart
cells (cardiomyocytes) were isolated from both adult and neonatal cardiac
tissue from the rat and the effects of the test substances were studied
by following the contractile properties of such cells while subjecting
then to a variety of oxidative stresses thought to mimic reperfusion
injury in the heart. The more lipid soluble herbal extracts appeared
to induce the greatest protection from injury induced by the stressors
used.
Positive points:
The finding that two of the herbal extracts tested were able to give a
synergistic protective effect at very low concentrations, and that this
effect occurred rapidly and was of long duration, is an interesting finding
with possible future human clinical applications. The breadth of approaches
used by the student in this study was particularly impressive.
Negative points:
Statistical treatment of the data was inappropriate and more significant
outcomes from the data were compromised. Adequate controls particularly
with regard to the “vehicle” solutions for administration
of agents were lacking. There were many spelling, layout and sentence
construction errors (particularly placement of the verb within qualifying
statements) which made reading and interpretation difficult.
Five Questions the candidate could be asked at an interview or a viva
session.
Question 1.
What differences would you expect in the contractility properties to agonists
and antagonists for cardiomyocytes isolated from the rat heart in your experimental
studies to that of heart cells that may be obtained from biopsied human myocardial
tissue during, for example, bypass surgery? How might this impact were such
treatments to be contemplated in human studies related to “heart health”?
Question 2.
In the data for herbal extract #15, you show a dose curve which exhibits a
maximum response and then returns to baseline values with increasing doses.
Can you please explain this data and the possible effects an excess of this
extract (antioxidants) could be having in your experimental system?
Question 3.
Explain how the differences between chronic in vivo administrations of herbal
extracts to that of acute in vitro additions in your studies may have come
about in terms of their differential effects on cardiomyocyte contractility?
Question 4.
The common finding that the more lipid soluble herbal extracts appear to exhibit
greater efficacy might imply a role for the cell membrane in facilitating
their effects. Could you explain likely mechanisms whereby this could occur?
Question 5.
Do you believe your results would have any future implications to the possibility
of using antioxidants present in herbal-derived extracts in a clinical setting
given that proof-of-principal, as opposed to anecdotal evidence is normally
required to endorse further clinical trials and testing of such drugs and
remedies? (Answer with examples).
