DSpace Collection:http://theses.ncl.ac.uk/jspui/handle/10443/5172024-03-28T23:40:59Z2024-03-28T23:40:59ZCell wall-deficiency in Staphylococcus aureus and its role in antibiotic resistanceFuller, Elizabeth Rhttp://theses.ncl.ac.uk/jspui/handle/10443/16712013-06-03T08:11:52Z2008-01-01T00:00:00ZTitle: Cell wall-deficiency in Staphylococcus aureus and its role in antibiotic resistance
Authors: Fuller, Elizabeth R
Abstract: Cell Wall-Deficiency in Staphylococcus aureus and its Role in Antibiotic Resistance.
Elizabeth R. Fuller.
Cell wall-deficient bacteria (CWDB) induced from Staphylococcus aureus
ATCC 9144 (Oxford strain) were generated on medium with elevated osmolality in
the presence of sublethal levels of penicillin G. On removal of antibiotic pressure the
cell wall-competent (CWC) revertants along with these CWDB exhibited high-level
penicillin and methicillin resistance, which was stable in the revertants. The revertants
looked visually different, had an altered Gram stain and growth rate. Their matrixassisted
laser desorption/ionisation time-of-flight (MALDI-TOF) `fingerprint' was
also altered and they were more resistant to lysis by lysostaphin in comparison to the
wild-type. Reversed-phaseh igh-performance liquid chromatography( RP-HPLC)
showed that the revertants' cell walls had shorter glycan chains and more
pentaglycine cross-bridges.
A rapid,r eproduciblem ethodu sing liquid mediaw ase stablishedu singt he
same medium and sublethal levels of penicillin G. The revertants produced using this
method had the same characteristics as those cells produced from the original method.
The high-level resistance seen in the revertants was homogenous and confirmed to be
due to the transient CWD state, along with not being strain-specific. Transmission
electron microscopy showed that the CWD cells and the revertant cells, when grown
in penicillin, had a very disordered cell wall with areas where the cell wall appeared
absent and were indistinguishable.
The revertant cells were mecA-negative,ß -lactamase-negativea nd did not
contain any mutations in the coding regions of pbp genes. The CWD cells and
revertant cells, when grown in penicillin, were resistant to lysis by lysostaphin but
were very sensitive to lysis with Triton X- 100. These data indicate that the resistant
cells are not dependent upon an intact cell wall for osmotic stability and they are able
to switch readily to this mode of growth in the presence of penicillin G.
Description: MD2008-01-01T00:00:00ZStudies on whole-body nitrogen turnover, protein synthesis and breakdown in man using 15N glycineClarke, Davidhttp://theses.ncl.ac.uk/jspui/handle/10443/16662013-05-28T11:23:35Z1994-01-01T00:00:00ZTitle: Studies on whole-body nitrogen turnover, protein synthesis and breakdown in man using 15N glycine
Authors: Clarke, David
Abstract: The experimental work described in this thesis was conducted in the surgical research
laboratories of Dr Francis D. Moore in the Peter Bent Brigham Hospital (now the
Brigham & Women's Hospital) Boston, Massachusetts, USA, between 1978-1979. It
formed part of an ongoing programme of research into protein metabolism in man;
specifically to measure total body nitrogen turnover and hence protein synthesis and
breakdown, initially in normal volunteers receiving various intravenous feeding regimens,
and subsequently in patients. The previous year, 1977, had seen the publication of
'Substrate Interaction in Intravenous Feeding' by Bruce Wolfe et al., from the same
laboratories. This was an extensive piece of work incorporating many studies and
compared nitrogen balance data together with biochemical, hormonal and plasma amino
acid data in normal men fed intravenously with a variety of regimens.
Shortly afterwards a series of protein turnover studies was embarked upon, using the uN
glycine method, and in collaboration with Dr Vernon Young of the Massachusetts Institute
of Technology. The first experiments were essentially a repeat of the studies described by
Wolfe et al. (vide supra) but in addition nitrogen turnover, protein synthesis and
breakdown were estimated using a continuous 60 hour infusion of uN glycine and
measuring enrichment of urinary urea with uN when a plateau was reached. Normal
volunteers were studied firstly on normal oral diet and then on a
iv
succession of intravenous regimens:- amino acids alone (AA), amino acids plus 'high dose'
glucose (AA + HOG), amino acids plus fat emulsion (AA + FE), amino acids plus 'low
dose' glucose (AA + LOG), amino acids, fat emulsion and low dose glucose
(AA + LDG+ FE), and finally 'low dose' glucose alone (LOG).
The studies on normal diet, AA and AA+HOG were conducted by Andrew Sim (a
Glasgow/Harvard exchange fellow) and Bruce Wolfe. The author took no practical role in
these experiments, but was responsible for analysis of the data and the protein metabolism
calculations, and was a co-author when the work was published in 1979 (Sim et al.,
Glucose Promotes Whole-Body Protein Synthesis from Infused Aminoacids in Fasting
Man, Lancet i, 68-71).
Subsequently, the author did the experiments using AA + LOG + FE, AA + FE, and
AA + LOG and LOG. The results on these four regimens were incorporated in a paper
presented in 1979 at the Tripartite Meeting of the Surgical Research Society at Oxford
under the title 'Isotope Studies of substrate interaction in parenteral nutrition', and also at
the 2nd European Congress on Parenteral and Enteral Nutrition at Newcastle upon Tyne in
1980, and later published as 'The Effect of Fat Infusion on Protein Metabolism' (Acta.
Chir. Scand., Suppl. 507, 475-484, 1981). When the studies on the various intravenous
feeding regimens were completed, attention was turned to the possible distorting effects of
variables such as exercise and diet
v
on the behaviour of the isotope equilibrium curve and plateau. Such effects, if present,
might have significance when studies were carried out on patients at a later stage in the
research programme. Because each study lasted 48-60 hours, changes might occur either
unintentionally or as a result of the needs of clinical management, and if they affected the
plateau would alter the resultant calculations of turnover, synthesis and breakdown. Such a
potential source of error clearly needed investigation.
A pilot study was done in two subjects, later repeated on each, to observe any effects on
the curve and plateau of both doubling protein intake and bicycle exercise. Subsequently,
more extensive studies were done varying the protein and energy intakes, both orally and
intravenously, allowing a detailed analysis of curve perturbation, and introducing the
concept of basal catabolic rate.
Finally, protein turnover, synthesis and breakdown were estimated seven times in four
seriously ill patients.
All of the studies mentioned above form the basis of the thesis. Unfortunately, the
gestation period of this thesis has been long. There are two main reasons for this. Firstly,
the work done was part of a five-year programme of research, with the intention of
publishing a paper in a scientific journal at the completion of each stage. This was done
vi
with the first three regimens (normal diet, AA and AA + HDG) but not with the last four
(AA + FE, AA + LOO+ FE, AA + LOG, LDG), although the results were presented at two
scientific meetings. Shortly after returning to the United Kingdom the author was
appointed a consultant surgeon and this career move assumed priority. Secondly, although
it was intended to publish the perturbation studies, it proved impossible to reduce the size
of the text to a manageable level suitable for publication in the form of a scientific paper.
However, despite the long interval since the experiments were done, no similar work has
been published. In particular, virtually no attention has been paid to intentional
perturbation. Also, whereas there was a spate of interest in protein turnover studies in the
late 1970s and early 1980s, virtually no publications have appeared since 1985. It seems
that the potential applications of the method are considered exhausted, and interest has been
lost, rather as it was in the 1950s following a short flurry of activity exploring the first
cumbersome technique. Hence, it seemed all the more pertinent, even at this late stage, to
publish the work in the form of a thesis which could describe in chronological order the
continuum of studies as briefly mentioned above. In order to preserve such a progression,
the following Introduction contains, with few exceptions, only references up to the time
that the experimental studies were commenced, 1978, but the subsequent Discussion(s) in
the various sections will attempt to include the relevant literature up to the present time.
Description: M.D.1994-01-01T00:00:00ZFunctional analysis of TSPY and its role in prostate carcinogenesisOmar, Mahmoud Mustafahttp://theses.ncl.ac.uk/jspui/handle/10443/9052010-09-28T14:18:30Z2005-01-01T00:00:00ZTitle: Functional analysis of TSPY and its role in prostate carcinogenesis
Authors: Omar, Mahmoud Mustafa
Abstract: Testis specific protein Y chromosome encoded (TSPY) is a multicopy gene located on
the human Y chromosome. It has been reported that the human genome harbours
between 20 to 40 copies of the gene. Mammalian homologues of human TSPY were
also found to be repetitive. The gene is expressed in human foetal and adult testis. The
precise function of the expressed TSPY gene product is not fully defined, but it has
been postulated to regulate proliferation of testicular spermatogonia. Up-regulation of
TSPY expression has been detected in gonadoblastoma, testicular cancer and prostate
cancer. The contribution of abnormal expression of TSPY to prostate carcinogenesis
has not been investigated. Prostate cancer (CaP) and benign prostate hyperplasia
(BPH) are the most common diseases of the human prostate. Prostate cancer is a
disease of the elderly and is the second most common cancer and second most
common cause of death from cancer among men in UK.
In this thesis, the role of TSPY in prostate carcinogenesis was studied in four related
aspects. First, TSPY protein and transcript expression pattern in CaP compared to
Benign Prostate Hyperplasia (BPH) was studied using a combination of
immunohistochemistry and mRNA in situ hybridisation. Second, the ability of TSPY
to regulate cellular proliferation was investigated by transfection experiments of the
prostate cancer LNCaP cell line. Third, TSPY genomic copy number in prostate
cancer was characterised and compared to BPH. Finally, the downstream genes
regulated by TSPY were investigated using high density microarray gene profiling
method.
An anti-human TSPY polyclonal antibody was developed for immunodetection of
TSPY expression level in resected prostate tissues. In total, 72 cases of patients with
prostate cancer and 20 cases of patients affected with BPH were studied by
immunohistochemistry. TSPY was predominantly detected in the prostatic epithelium.
In the benign gland, TSPY expression was limited to the basal cells compartment.
TSPY expression was significantly up-regulated in prostate cancer when compared to
BPH (P<0.0001). Furthermore, increased TSPY expression level was associated with
aggressive disease (tumour with high Gleason score; P<0.02) and the presence of
bone metastasis at the time of diagnosis (P<0.028). To address the functional role of
3
TSPY in prostate cancer, FLAG-TSPY was cloned and stably transfected into LNCaP
cells. The presence of transfected TSPY increased LNCaP proliferation by two fold
compared to empty vector control, consistent with a mitogenic function in CaP
(P<0.0001).
An absolute quantitative real time PCR based on Taqman assay was established and
validated. TSPY genomic copy number was determined from comparing 161 samples:
CaP-serum (n=47), resected tumour (n=31); BPH-serum (n=27), resected prostate
(n=13) and control-serum (n=45). Of the clinical samples analysed, interpersonal
variability of TSPY copy numbers was observed with the majority of cases containing
between 20 to 50 TSPY copies per genome. Although, there were variability in TSPY
copy numbers among individuals, there was no statistically significant correlation
between TSPY copy number (serum and prostatic tissue) and the development of
prostate cancer.
Studying LNCaP stably transfected with TSPY and empty vector control, the key
genes mediating the functional effect of TSPY were identified using Affymetrix
oligonucleotide microarray method. In total, 332 genes have been altered 1.5 to 90
fold due to the effect of TSPY over-expression. Ten genes were selected and the gene
expression levels were confirmed using semi-quantitative RT-PCR method. Gene
clustering analysis has indicated changes in genes that regulate cellular differentiation
(NRDG1, NF2, C-MAF and BMP11), apoptotic gene (BAX), cell cycle gene (cyclin
G), detoxification gene (GST-2A) and genes linked to adhesion (PCDH7a, PLOD2
and IRS 1).
In summary, TSPY is over-expressed in prostate cancer. This abnormal expression is
linked to prostate cancer progression and metastasis. The up-regulation of TSPY
expression is unlikely to be due to increased copy number. Expression of exogenous
TSPY in prostate cancer cells enhanced cellular proliferation. The gene meditates its
effect by down-regulating the expression pattern of selected differentiation, apoptosis
and adhesion genes. Hence, over-expression of TSPY contributes to prostate
carcinogenesis.
Description: PhD Thesis2005-01-01T00:00:00ZThe role of interleukin-17 and RANKL in the regulation of bone destruction in arthritisAbusrer, Salmahttp://theses.ncl.ac.uk/jspui/handle/10443/8662010-07-23T12:58:24Z2004-01-01T00:00:00ZTitle: The role of interleukin-17 and RANKL in the regulation of bone destruction in arthritis
Authors: Abusrer, Salma
Abstract: Arthritis is a common disease characterised by chronic inflammation as well as bone and
cartilage destruction. Proinflammatory cytokines such as interieukin-17( IL-17), aT cell derived
cytokine, and Oncostatin M (OSM) a microphage cytokine, are elevated in
rheumatoid arthritis (RA). The aim of this study was to characterise the effects of IL-17
and OSM on the expression of receptor activator of NF-kappaBl and (RANKL) and its
decoy receptoro steoprotegeri(nO PG)by mesenchymalil neage cells in vitro, in particular
synovial fibroblasts and the human osteo sarcoma cell line (MG-63 and SaOS-2) that are
potentially involved in this process as they regulate osteoclastogenesis. In addition, the
ability of IL-17 to support osteoclastogenesinis vitro was assessed.
Human synovial fibroblasts( SFB)from RA and OA patients were contrasted with cell lines
MG-63 and SaOS-2 and were treated with IL-17 and/or OSM for up to 48 hr. The
expression and production of RANKL and OPG over a time course was assessed in these
cells. To investigate osteoclastogenesis peripheral blood mononuclea cells (PBMCs)were
cultured with IL-17 either alone or with Macrophage colony stimulating factor (M-CSF).
Osteoclastogenesis was analyzed after 21 days by tartrate-resistant acid phosphatase
(TRAP),positive multinucleated cell counts, and resorption of ivory slices.
This study demonstrated a direct role for IL- 17 in bone and cartilage catabolism through the
induction of RANKL and OPG mRNA levels at 6 hr and 48 hr in SFB RA and OA, MG-63
and SaOS-2. Furthermore the combination of IL-17 and OSM showed that the expression
of RANKL and OPGw as down-regulated. Co-incubation of IL-17 with or without M-CSF
significantly enhanced TRAP+ve multinucleated cell formation. Furthermore cultures of
PBMC with IL-17 on ivory slices showed significantly increased resorption and when cocultured
with synovial fibroblasts from RA and OA patients further significant resorption.
The addition of OPG to IL-17 cultures significantly inhibited the formation of resorption
lacunae. The effect of IL-17 on bone resorption in vitro is mostly RANKL-dependent.
This finding may lead to the conclusion that IL-17 significantly induces
osteoclasto genesis
in vitro. Therefore,I L-17 represents a key cytokine involved in the exacerbation of
inflammatory joint disease and is an important target for anti-cytokine
therapies. Also
these results provide proof of the concept that OPG production can result in effective
therapies.
Description: PhD Thesis2004-01-01T00:00:00Z