313 Projects in 2003
Academic Consultant PROJECT TISSUE
Dr
Luis Filgueira OSTEOCLASTS (#1)
Expression of
tartrate resistant acidic phosphate by osteoclasts
STUDENTS: Labs
on THURSDAY (2-5)
Ms Simone BENNETT
Ms Leony FRANSISKA
Ms Wajma PADSHAH
Mr Lloyd WHITE
Mr Konrad VARGAS
Associate
Professor Paul McMenamin EYE (#2)
The cause of corneal
lesions in abattoir acquired pig eyes
STUDENTS: Labs
on MONDAY (1-4)
Ms Holly CHINNERY
Ms Jelena KEZIC
Ms Natalie LA CAVA
Mr Simon MANLEY
Professor
Arusalem Dharmarajan REPRODUCTIVE TISSUES (#3)
The role of caspase 14
in reproductive tissues
STUDENTS:
Labs on MONDAY (1-4)
Ms Clare BERRY
Mr Daniel KAM
Ms Sarah MARTIN
Ms Sally MCLAREN
Ms Zahida SHARIFI
Associate
Professor Brendan Waddell PLACENTA (#4)
Leptin receptor
expression in human placental choriocarcinoma cells
STUDENTS: Labs
on THURSDAY (2-5)
Mr Byron MINAS
Ms Trina SHAH
Ms Olivia TASLIM
Ms Felicity THORPE
Professor
Miranda Grounds SKELETAL MUSCLE (#5)
Immunostaining for extracellular matrix molecules in geriatric skeletal
muscle
STUDENTS:
Labs on THURSDAY (2-5)
Mr Brendon GAIRNS
Ms Vivian LAM
Ms Hannah RADLEY
Ms Sophie WILLIAMS
Ms Zahida SHARIFI
Professor Miranda Grounds HEART
(#6)
Cardiomyocyte identification and proliferation in vivo
STUDENTS:
Labs on MONDAY (1-4)
Ms Mei CHOW
Ms Susan CLIFFORD
Ms Monica DOBROMIRSKI
Mr Simon MAHONEY
Ms Selina LIM
Cell and
Tissue Organisation 313 – 2003
PROJECT #1: OSTEOCLASTS
Expression of tartrate resistant acidic phosphate by osteoclasts (TRAP):
A comparison between classical and fluorescence histochemistry methods.
Academic consultant Luis Filgueira
Osteoclasts (OC) are distinct macrophages responsible for bone resorption (SL Teitelbaum 2000). It is possible to generate functional OC in vitro starting with human monocytes and culturing them in the presence of a cytokine cock-tail containing RANK-ligand and M-CSF (HM Massey 1999, J Scopes 2001). OC express TRAP in their lysosomal vesicles. TRAP expression is characteristic for OC. There is a standard histochemical method to detect TRAP expression, but a method for TRAP detection through fluorescence microscopical techniques has not yet been developed.
Question: Is there a fluorescence microscopy method for TRAP detection?
Aims of the project:
3 hypotheses will be tested:
Experiments
Experimental setting: Monocytes will be isolated from human blood through a density gradient separation and adherence. They will be cultured in the presence of human serum, RANK-L and M-CSF to differentiate to osteoclast.
All experiments will be repeated for at least 2 times
References (copies of these will be provided to you):
Cell and Tissue Organisation 313 – 2003
PROJECT
#2 Macrophages
The cause of corneal lesions in abattoir acquired pig eyes
Academic supervisor A/Professor Paul McMenamin in collaboration with Dr Season Yeung
Contact details for Season Yeung are room 1.71, phone 9380 1507, syeung@anhb.uwa.edu.au
Aim: To find the cause of corneal lesions in abattoir acquired pig eyes
Background: A Masters student with Assoc. Prof. Paul McMenamin, Dr Season Yeung, is studying the distribution of immune cells in the porcine cornea and limbus. In his studies Season has noted that upto 2/3 of the pig eyes from the abbattoir have a pinkish lesion on the cornea which render them useless for his research. The cause of the lesions are unknown. They resemble a band ketatopathy.
The aim of this project will be to investigate the pathology of these lesions.
Eyes will have to be collected from the abattoir, fixed, processed and embedded in paraffin. Sections of the eyes can be stained with a number of histochemical stains to try to discover the nature of the lesions (blood, parasites, oedema, lipid accumulation, epithelial damage, vitamin deficiency are just some potential causes that come to mind).
Students will have to learn basic histological methods and some eye anatomy and eye pathology.
The expected outcome is a diagnosis of the corneal pathology.
Students will liaise with Dr Season Yeung during Dr McMenamin’s absence (away 5-31 March and May 3-19).
References. (3 books)
Cell and Tissue Organisation 313 – 2003
PROJECT #3 Reproductive tissues
Localisation of caspase-14 in reproductive tissues
Academic supervisor Professor Arusalam Dharmarajan (Dharma)
Caspases are cysteine proteases that cleave their substrates after an aspartate residue. Until now 13 members of this family have been described in mammalia and most of them are known to play a role in apoptosis or inflammation.[Earnshaw, 1999 ] Involvement of caspases in processes such as lens epithelium differentiation and erythropoiesis have been reported as well. Previous studies have identified caspase-14 as a caspase with a very short prodomain that is processed during normal epidermal differentiation without requirement of any apoptotic or inflammatory stimuli necessary to activate other caspases.[Van_de_Craen, 1998] Moreover, caspase-14 was shown to remain unprocessed under apoptotic conditions, indicating that it does not participate in the classical apoptotic pathway. To date, however, the role of caspase-14 in the reproductive tissues has not been examined. Our previous studies have demonstrated a role for other caspases in the corpus apoptosis.
Question: Does caspase-14 play a role in the regression of hormonally dependent reproductive tissues (Ovary, Mammary gland and Uterus)?
Aims of the Project:
Experiments:
Basically, the caspase-14 protein will be localized in the tissues of interest by immunocytochemistry. The tissues have already been collected at different stages of pregnancy. Depending on the results obtained and availability of time, quantitation of caspase-14 protein expression will be carried by Western Blot analysis.
References:
Earnshaw WC, martins LM and Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates and functions during apoptosis. Ann Rev Biochemistry 68:383-424
Van de Craen M, Van Loo G, Pype S, Van Criekinge W, Van den brande I, Molemans F, Fiers W, Declercq W and Vandenabeele P (1998) Identification of a new caspase homologue:caspase-14. Cell Death Diff, 5:838-846
Please look at the following web page to get an idea about research activities in my laboratory.
www.anhb.uwa.edu.au/staff/dharma/default.htm
Cell and Tissue Organisation 313 – 2003
Leptin Receptor Expression in the Placenta – a Tale of Two Antibodies.
Academic supervisor A/Professor Brendan Waddell in collaboration with Dr Peter Mark
Introduction
The leptin receptor (Ob-R) is a member of the cytokine receptor superfamily which plays an important role in mammalian body weight homeostasis and energy balance. Several isoforms of the Ob-R which differ in length and signalling capabilities have been reported (Ob-R a-f) [reviewed in (1)]. The major isoforms of the leptin receptor in the human [2; 3] and rodent [4;5] placenta are Ob-Ra (short form believed to be involved in leptin transport across biological membranes) and Ob-Rb (long form responsible for mediating leptin’s intracellular signalling capabilities). The Ob-R long (Ob-Rb) form of the receptor has been found in the choroid plexus and in the hypothalamus, which has been proposed as a control centre for satiety and energy expenditure.
Mutations in either the mouse Ob-R (db/db) or leptin (ob/ob) genes have been shown to result in early-onset obesity. Metabolic abnormalities attributable to these genotypes include hypercorticoidemia (high levels of corticosteroids), hyperinsulinemia (high levels of insulin), insulin resistance (non-responsiveness to insulin) hyperglycemia (high blood glucose levels), altered CNS activity, reduced metabolic rate of brown adipose tissue and a large increase in white adipose tissue.
The BeWo choriocarcinoma cell line(human placental trophoblast-like cells) provide a useful model for dissecting placental metabolic pathways without having the need to access human placental tissue [3]. These cells can be induced to undergo syncytialisation (fusion to form multinucleated cells that mimic the outermost layer of the placenta i.e. the maternal/placental interface).
To analyse Ob-R expression, we have available two antibodies: K-20 (Santa Cruz) which displays a high affinity for Ob-Rb [5]; and PA1-053 (Affinity BioReagents) which binds more strongly to Ob-Ra [6]. These two antibodies will be used in parallel to investigate changes in localisation and expression levels of Ob-R isoforms between days 16 and 22 of rat pregnancy (term=day 23) and to investigate expression profile changes in BeWo cells following the process of syncytialisation.
Hypotheses
Research Plan
The pattern and intensity of Ob-R staining in the rat placenta will be qualitatively assessed at days 16 and 22 of rat pregnancy using antibodies K-20 and PA1-053. Serial sections through rat placentas from these gestational days will be immunostained with the two antibodies and the expression patterns compared.
BeWo cells grown on coverslips will be treated with either vehicle (ethanol) or 20 m M forskolin for 3 days to induce syncytialisation. These cells will then be immunostained using the antibodies and the effect of syncytialisation on leptin receptor patterns of expression determined
References
Cell and Tissue Organisation 313 – 2003
Immunostaining for extracellular matric molecules in geriatric skeletal muscle
Academic supervisor Professor Miranda Grounds http://www.anhb.uwa.edu.au/staff/Grounds/default.htm
In collaboration with Ms Monique Berendse, Dr Catriona Lloyd, Ms Thea Shavlakadze and Dr Jason White
There are many morphological (1) and other (2) changes in ageing skeletal muscle. This project is focussed on the extracellular matrix (ECM) lying outside the muscle fibres. Changes in amount and composition of extracellular matrix in ageing muscle have many consequences and contribute to decreased vascularity with altered blood supply, capillary density, rigidity and pathology in old muscles (reviewed in 2).
The ECM in skeletal muscle includes both interstitial connective tissue and the external (basal) lamina which is in intimate contact with satellite cells and myofibres. A general increase in interstitial fibrous connective tissue is associated with ageing: the amount of endomysial collagen doubles between 3 and 26 weeks of age in mice (3), there is a marked increase in fibronection in very old muscles (4) and increasing fibrosis occurs in regenerating muscles of older animals (5, 6). Apart from the interstitial connective tissue, an increase in the basal/external lamina encircling satellite cells occurs with age (1, 8). In muscle diseases such as Duchenne Muscular Dystrophy and the animal dystrophies there is a marked increase in extracellular collagen and altered forms of collagen with time (3).
The aim of this project is to use a range of antibodies to identify many ECM related molecules in frozen tissue sections (LS and TS) of various muscles from young and very old mice, to determine any marked changes in ECM composition and amount with age.
References
Other related references from the lab – see http://www.anhb.uwa.edu.au/staff/Grounds/default.htm
Cell and Tissue Organisation 313 – 2003
Cardiomyocyte identification and proliferation in vivo
Academic supervisor Professor Mirand Grounds
In collaboration with Dr Cecilia Prele and Ms Marilyn Davies, cprele@cyllene.uwa.edu.au
Background
Growth of the mammalian heart is generally characterised by cell division of muscle cells during the embryonic stages of life, followed by post-natal entry into a post-mitotic state. Thus, growth of the heart during normal development and in cases of cardiac disease requires enlargement, rather than proliferation of post-mitotic cardiac myocytes. It has long been considered that proliferation of cardiac myocytes ceases permanantly soon after birth and there is no replacement with new cardiomyocytes after damage. This inability to repair damaged heart muscle contrasts markedly with the situation in salamander and zebra fish (1)
However, it now appears that there is a low level of myocyte proliferation (accompanied by cytokinesis) in the mammalian heart (2-4). The identification of proliferating populations of cardiomyocytes in normal post-natal hearts raises the possibility of expanding such cells in vivo or of stimulating such cells for cell transplantation to repair heart muscles damaged after ischaemia and cardiovascular disease (3,4). We are interested in identifying replicating cardiomyocytes on tissue sections.
It can be very difficult to distinguish replicating cardiomyocytes from other replicating mononucleated cells in the heart (2). To overcome these problems, a transgenic mouse (MHC-nLAC) has been generated that expresses the nLAC reporter gene under the regulation of the a -cardiac myosin heavy chain (MHC) promoter. The nLAC is expressd only in the nuclei of cardiac myocytes and has been used (in combination with tritiated thymidine of replicating nuclei) to confirm the identity of replicating cardiomyocytes (2).
Aim
To distinguish cardiomyocytes from non-cardiomyocytes in tissue sections.
Project
We will use antibody staining techniques to distinguish cardiomyoctes from other cells in heart tissue of normal (non-trangenic) mice (e.g. anti-desmin, pan-laminin and vimentin antibodies further details will be provided). In addition we will use the LAC marker for cardiomyocytes in transgenic mice.
References