Type-II Ribosome Inactivating Proteins from Abrus precatorius: Cytotoxicity and Mechanism of Cell Death A/B toxins produced by bacteria and plants are among the deadliest molecules known. The plant type-II ribosome inactivating proteins (RIPs) are prototype of A/B toxins. They are two subunit proteins with a toxic A subunit that harbors an RNA N-glycosidase activity and a lectin like B subunit which allows toxin entry into cells. The toxicity of A chain is due to its RNA-N-glycosidase activity which cleaves the bond between the ribose sugar and the adenine at position 4324 as demonstrated in rat liver ribosomes. The B- chain, a lectin, binds to the cell surface receptors terminating in galactose sugars and allows toxin entry into cells. The seeds of the subtropical climber Abrus precatorius contain two RIPs: the potent toxic lectin abrin and the relatively less toxic Abrus agglutinin. The toxic property of RIPs has widespread applications in the field of agriculture and medicine. The cells of our body commit suicide in response to genetic or environmental cues by the process, apoptosis or programmed cell death which results in the safe clearance of the dead cells without affecting the extra-cellular milieu. Apoptosis is essential for development, tissue homeostasis, and defense against pathogens. It involves the interplay of multiple pathways that are initiated and executed by a family of proteases termed caspases. Several plant type-I and type-II RIPs as well as bacterial toxins have been shown to induce apoptosis in cultured cell lines. Though many agents that inhibit macromolecular synthesis in cells induce DNA fragmentation and morphological changes associated with apoptosis, the link between protein synthesis inhibition by these toxins and apoptosis remains elusive. Though extensive studies have been carried out on several RIPs for e.g. ricin and shiga toxin, only few reports are available in literature on the mechanisms of toxicity exhibited by abrin, a type-II RIP, of South-East Asian origin. Earlier studies from the laboratory have focused on the sensitivity and mechanism of abrin induced cell death in Jurkat, a cell line of haematopoietic lineage and its variants. In the same direction, the objectives of my study were: (1) To delineate the structure-function relationship of Abrus agglutinin-I in comparison with abrin, (2) To establish monoclonal antibodies to the A subunit of abrin, analyzing their neutralizing effect on abrin toxicity in vitro and in vivo and (3) To delineate the pathway and determine the kinetics of apoptosis induced by abrin on cell lines of epithelial lineage. The thesis will be presented in three four chapters. The first chapter, ‘Introduction’, begins with a brief history of RIPs, followed by the description of their distribution and classification. The transport of toxins which is a unique property of this class of proteins is discussed in detail and supported with appropriate figures. Also, information pertaining to the structure of abrin and apoptosis induced by RIPs is written in brief. In the second chapter of the thesis the structural and functional studies of Abrus agglutinin-I (APA-I) as compared to abrin are discussed. Abrin and APA-I share a high degree of homology, however, previous reports by Liu et al., indicate that APA-I is many fold less toxic in cell free systems as compared to abrin. In our studies, APA-I was found to be less toxic on cultured cell lines. The IC50 value of protein synthesis inhibition by abrin was found to be 0.4 ng/ml for both Jurkat and MCF-7 cell lines. A 20-1000 fold difference was observed in the sensitivity of these cell lines to APA-I. The extent of apoptosis induced by APA-I in A3I9.2 a caspases-8 mutant Jurkat variant cell line was comparable to abrin indicating that the apoptosis induction by APA-I might not be through the extrinsic pathway. instead, our studies showed that APA-I induced apoptosis followed the mitochondrial pathway of cell death, in a caspase dependent manner similar to that of abrin. Unlike other agglutinins like wheat germ agglutinin, the agglutinating ability of the agglutinin-I had no role in the apoptosis induced. Protein synthesis inhibition appeared to be mandatory for the apoptosis induced by APA-I. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison to that of the reported structure of abrin. The substitution of Asn200 in abrin with Pro199 in agglutinin-I seems to be a major cause for the decreased toxicity. This perhaps is not a consequence of any kink formation by Pro residue in the helical segment, as reported by others earlier but due to fewer interactions that proline can possibly have with the bound substrate. Passive immuno-neutralization by administration of neutralizing antibodies is widely used as therapy against poisoning by various toxins. In case of type-II RIPs like ricin, antibodies to the toxic subunit were proven to have better protective efficacy than those to the lectin subunit. Neutralizing antibodies to abrin are not reported in literature. Therefore, a panel of monoclonal antibodies (mAbs) to the recombinant A chain of abrin was developed in our laboratory and characterized, which is presented in the third chapter of the thesis. Of these, D6F10 a high affinity antibody, exhibited neutralizing effect on abrin induced cytotoxicity on different cell lines tested. Antibodies may neutralize biological toxins in multiple ways; our studies suggested that mAb D6F10 interferes in the earliest event i.e. attachment of the toxin to the cell surface. Significantly, with the administration of mice with mAb D6F10 the prophylactic effect of the mAb could be demonstrated. In chapter 4, the sensitivity, kinetics of proteins synthesis inhibition and the mechanism of abrin induced cell death in cell lines of epithelial lineage is presented. Both sensitivity and kinetics of MCF-7/pv, Ovcar3, and T47D cells appeared comparable while, a variant culture of MCF-7 over-expressing caspases-3 was 50 times more sensitive to abrin. There was no significant difference in the binding of abrin between MCF-7/pv and MCF-7/C3+ cells. Previous studies in our laboratory indicated that abrin induced apoptosis is a caspases-3 dependent process. Also, in several systems it has been shown that caspases-3 is an indispensable molecule for apoptotic cell death. To test the absolute requirement of caspase-3, we examined abrin-induced apoptosis in a human breast cancer cell line MCF-7/pv reportedly deficient in caspases-3. Unlike other molecules like cisplatin, apoptosis induced by abrin in the MCF- 7/pv cells was found to be caspase -3 independent. However faster kinetics of apoptosis is observed, indicating that there is amplification of the apoptotic signals in the presence of caspases-3 resulting in an early onset of DNA fragmentation. The kinetics of protein synthesis inhibition and apoptosis follows similar kinetics in Jurkat cells while there is a time lapse between the two events in epithelial cells. Even with very high concentrations of abrin no detectable apoptosis was observed within 24 h in epithelial cells. The onset of fragmentation occurs after 24 h in the cell lines tested as opposed to Jurkat where it is observed as early as 6 h. Inhibition of caspases rescued the toxins from DNA fragmentation suggesting that the toxin does not cause direct nuclear damage in the cell line which does not involve the activation of caspases.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 229-253.

Spinal cord injury induces anatomical plasticity throughout the nervous system, including distant locations in the brain. Several types of injury-induced plasticity have been identified, such as neurite sprouting, axon regeneration and synaptic remodeling. However, the molecular mechanisms involved in anatomical plasticity after injury are unclear, as is the extent to which injury-induced plasticity in the brain is conserved across vertebrate lineages. Here, I used lampreys to identify the molecular mechanisms in mediating anatomical plasticity, because lampreys undergo anatomical plasticity and functional recovery after a complete spinal cord transection. Due to their robust roles in neurite outgrowth during neuronal development, I examined synapsin and synaptotagmin for their potential involvement in anatomical plasticity after injury. I found increased synapsin I mRNA throughout the lamprey brain as well as increased protein levels of synapsin I, phospho-synapsin (Ser 9) and synaptotagmin in the lamprey hindbrain after injury, suggestive of anatomical plasticity. Anatomical plasticity was confirmed at the ultrastructural level, where I found increased neurite density in the lamprey hindbrain after injury. Other molecular mechanisms that promote anatomical plasticity have been previously identified, such as cyclic AMP (cAMP). However, the cellular mechanisms and the molecular targets of cAMP in mediating anatomical plasticity are unclear. My investigation of cAMP revealed that cAMP enhanced the number of regenerated axons beyond the lesion site in lampreys after injury. For the first time in a spinal cord injury model, I found cAMP prevented the death of axotomized neurons that normally have a high tendency to die after injury. In addition, cAMP promoted more regenerating axons to re-grow in straighter paths rather than turning rostrally towards the brain stem. At the molecular level, I found cAMP increased synaptotagmin protein level at the regenerating axon tips, suggestive of enhanced axon elongation. Taken together, my results show that neurite sprouting in the brain and the cAMP-enhanced axon regeneration are conserved responses in vertebrates after spinal cord injury. In addition, my results suggest that at least some developmental pathways are activated during injury-induced and cAMP-enhanced anatomical plasticity. Further understanding of these pathways will provide insights for improving recovery after spinal cord injury. / text

Η μυο-ειδική πρωτεΐνη δεσμίνη, αποτελεί μέλος των πρωτεϊνών του κυτταροσκελετού των ενδιαμέσων ινιδίων και εκφράζεται στους λείους και τους γραμμωτούς μυς. Στους συσταλτούς μύες, το πλέγμα του κυτταροσκελετού της Δεσμίνης περιβάλλει τους Ζ-δίσκους διασυνδέοντάς τους, ενώ παράλληλα συνδέει μεταξύ τους τις συσταλτές περιοχές της μυικής ίνας με την σαρκοπλασματική μεμβράνη, με διάφορα οργανίδια και με τον πυρήνα. Για να προσδιορίσουμε τους ακριβείς μηχανισμούς δράσης της δεσμίνης χρησιμοποιήσαμε το σύστημα υβριδισμού των ζυμών – yeast two hybrid screen system – προκειμένου να ανιχνεύσουμε πρωτεΐνες που αλληλεπιδρούν με τη δεσμίνη. Χρησιμοποιήσαμε ως «δόλωμα» αλληλουχίες των άκρων του μορίου της δεσμίνης του αμινο-τελικού και το καρβόξυ-τελικού. Μελετώντας τις πρωτεΐνες που προέκυψαν, διαπιστώσαμε ότι το αμινο-τελικό άκρο της δεσμίνης αλληλεπιδρά με διάφορες μιτοχονδριακές πρωτεΐνες. Με το ίδιο σύστημα αποκαλύψαμε αλληλεπιδράσεις της δεσμίνης με λυοσωματικές πρωτεΐνες όπως η καθεψίνη D και η προσαποσίνη οι οποίες αλληλεπιδρούν με το αμινοτελικό άκρο της δεσμίνης. Η καθεψίνη D είναι μια λυοσωματική πρωτεάση, που οδηγείται και ωριμάζει πλήρως στα λυοσώματα ενώ η προσαποσίνη είναι ένα πρόδρομο λυοσωματικό μόριο με πρωτεόλυση του οποίου, εντός του λυοσώματος, προκύπτουν οι σαποσίνες Α έως D. Η καθεψίνη D αποτελεί δείκτη καταστάσεων αυτοφαγία και τελευταία φαίνεται ότι επεμβαίνει σε φαινόμενα απόπτωσης επάγωντάς την κατά περίπτωση. Η αλληλεπίδραση της δεσμίνης με την καθεψίνη D επιβεβαιώθηκε και με βιοχημικές τεχνικές (in vitro) όπως η συνεργιστική ανοσοκαθίζηση /ανοσοκατακρήμνιση (co-immuno-precipitation) και η τεχνική GST pull-down. Μετά και από αυτές τις in vitro αποδείξεις, φαίνεται πως μάλλον συμβαίνει ευθεία αλληλεπίδραση μεταξύ της δεσμίνης και της καθεψίνης D. Γι’ αυτό, και με βάση όσα είναι γνωστά για την καθεψίνη D, προτείνουμε μια νέα λειτουργία του κυτταροσκελετού της δεσμίνης πιθανόν στην μετακίνησης και τη δημιουργία των λυοσωμάτων αλλά και έναν νέο ρυθμιστικό ίσως ρόλο της, σε διαδικασίες αυτοφαγίας και απόπτωσης, μέσω της πρόσδεσής της με σημαντικά μόρια ρυθμιστές τέτοιων διαδικασιών.................... / Desmin is the muscle - specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. In order to identify the exact mechanisms of desmin’s action, we performed a yeast two-hybrid screen for desmin-interacting proteins. For this purpose, we used as baits the two non helical terminal regions of the desmin molecule, the amino (head)- and the carboxy (tail)- terminal domain. We have found that the head domain of desmin potentially interacts with two new groups of proteins, mitochondria and lysosome related. Specifically, in the second category, we have revealed an association of the head domain of desmin with Cathepsin D (one of the lysosomal proteinases) and prosaposin (a single precursor which gives rise to Saposins A-D by proteolytic cleavage in lysosomes and is also referred to as sphigolipid activator proteins). In addition to its targeting to lysosomes, Cathepsin D is also involved in apoptosis and autophagy processes. This protein interaction result has been retested. The interaction between cathepsin D and desmin has also been further confirmed both with reverse yeast transformation as well as biochemical assays such as co-immunoprecipitation and GST pull down assay. The above described strong evidence of direct interaction between desmin and cathepsin D, has allowed us to propose a novel function of desmin IFs in lysosomal trafficking and/or as a new regulator of autophagy and apoptotic cell death.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

The fate of DNA from dead cells is an important issue when interpreting results from root canal infections analysed by the PCR technique. DNA from dead bacterial cells is known to be detectable long time after cell death and its stability is dependent on many different factors. This work investigated factors found in the root canal that could affect the recovery of microbial DNA. In an ex vivo experiment, DNA from non-viable gram-positive Enterococcus faecalis was inoculated in instrumented root canals and recovery of DNA was assessed by PCR over a two-year period. DNA was still recoverable two years after cell death in 21/25 teeth. The fate of DNA from the gram-negative bacteria Fusobacterium nucleatum and the gram-positive Peptostreptococcus anaerobius was assessed in vitro. DNA from dead F. nucleatum and P. anaerobius could be detected by PCR six months post cell death even though it was clear that the DNA was released from the cells due to lost of cell wall integrity during the experimental period. The decomposition rate of extracellular DNA was compared to cell-bound and it was evident that DNA still located inside the bacterium was much less prone to decay than extracellular DNA. Free (extracellular) DNA is very prone to decay in a naked form. Binding to minerals is known to protect DNA from degradation. The fate of extracellular DNA was assessed after binding to ceramic hydroxyapatite and dentine. The data showed that free DNA, bound to these materials, was protected from spontaneous decay and from enzymatic decomposition by nucleases. The main conclusions from this thesis were: i) DNA from dead bacteria can be detected by PCR years after cell death ex vivo and in vitro. ii) Cell-bound DNA is less prone to decomposition than extracellular DNA. iii) DNA is released from the bacterium some time after cell death. iv) Extracellular DNA bound to hydroxyapatite or dentine is protected from spontaneous decomposition and enzymatic degradation.

Apoptosis (programmed cell death) and exercise immunology have been the focus of research for the past five years. Trained athletes are particularly susceptible to a wide variety of viral and bacterial infections and this has been related to oxidative damage which is a mediator of apoptosis. Apoptosis, a normal physiological mechanism has also been implicated in the pathogenesis of a wide-variety of diseases. To date, the link between apoptosis and exercise has not been shown by established methods or ultrastructurally. The objective of the study was t.o determine the effects of a single bout of high intensity exercise on lymphocyte DNA and antioxidant status in trained athletes. The study was carried out in two phases. In the first phase, 11 trained athletes were subjected to a treadmill run to exhaustion using a ramp protocol to determine their maximum oxygen uptake (V02 max). Fifteen millimetres of blood was collected before exercise, immediately after exercise, 24 hours and 48 hours after exercise. Whole blood (4 ul) was used in the determination of DNA damage in lymphocytes using the single cell gel electrophoresis (SCGE) assay. The remaining blood was centrifuged and used for the following: Vitamin C concentration was determined by the 2,4 dinitrophenylhydrazine method, vitamin E concentration was determined by the High Pressure Liquid Chromatography (HPLC) method and lipid peroxides were determined by the measurement ofhydroperoxides. In the second phase, 3 trained athletes who had participated in phase 1, were subjected to a V02 max. test. Blood samples (10 ml) were collected before and immediately after exercise, 24 hours and 48 hours later. Lymphocytes were isolated using Histopaque 1077. An in situ cell death detection kit, Fluorescein was used for the detection and quantification of apoptosis in lymphocytes at a single cell level, based on labelling of DNA strand breaks. Analysis was carried out using flow cytometry. Lymphocytes were also prepared for Transmission Electron Microscopy (TEM) using conventional techniques. The results showed that immediately after exercise there was a non-significant decrease in vitamin C concentrations (p=o, 16), and a non-significant increase in vitamin E (p=0,82) and lipid peroxide concentrations (p=0,21). There was no significant difference in all 3 levels over the 48 hour period, when compared to the pre-exercise values. The SCGE assay revealed that the immediate post exercise samples showed DNA damage in lymphocytes of all subjects as evidenced by fluorescent strands of DNA outside the cell while DNA damage was observed in only one subsequent sample. In the pre-exercise samples, DNA was visualised as a central core, whereas in all samples taken after exercise, DNA was located at the periphery or confined to one pole of the cell. The pattern of DNA distribution seen in the SCGE assay over the 48 hour period were characteristic features of apoptosis. Flow cytometric analysis showed an increase in apoptosis in lymphocytes immediately after exercise with a further increase after 24 hours. After 48 hours the numbers decreased to control levels. TEM showed that majority of cells were normal before exercise while other lymphocytes were smaller with indented nuclei. Immediately after exercise the lymphocytes displayed features of indented nuclei and microsegregation, cell shrinkage, swelling of the endoplasmic reticulum, mitochondria and Golgi. These changes persisted after 24 hours but were not observed after 48 hours when most of the cells showed normal morphology. The ultrastructural changes observed were also characteristic features of apoptosis. These results suggest that high intensity exercise may cause an increase in apoptosis as evidenced by DNA damage in the SCGE assay and fully supported by the results achieved during flow cytometry and by the ultrastructural changes observed. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Parkinson's disease (PD) is one of the most common motor neurodegenerative diseases, affecting 1-2% of the world's population over the age of 65. It is characterised by a loss of dopamine neurons within the substantia nigra, which is an integral part of the basal ganglia (BG) where dopamine is the most important modulating neurotransmitter. As the BG is primarily involved with the execution of movement, the lack of dopamine input results in dysfunctional motor control. The current PD treatment, L-DOPA, improves these motor symptoms, however only provides patients 5 to 10 years of improved quality of life before debilitating side effects, often worse than the original symptoms, begin. The neuropeptide substance P (SP) is found in high concentration in the substantia nigra, and BG in general, where it is involved in dopamine release. In the late stages of PD, SP content within the substantia nigra and BG is decreased, thus implicating SP in the pathophysiology of PD. However, SP production has not been examined in the early stages of PD when dopaminergic degeneration is first initiated. This thesis therefore sought to characterise the role of SP in dopaminergic degeneration in an experimental model of early PD, the 6-hydroxydopamine model in rats. In contrast to the prevailing dogma that a decline in SP is associated with neurodegeneration in PD, this thesis demonstrates that SP is actually increased within the striatum in early PD, particular in perivascular tissue and within surviving dopaminergic neurons during the degenerative process. Increasing exposure of the dopaminergic neurons to SP, either by inhibition of substance P breakdown with Captopril or by direct injection with SP, exacerbated the disease progression as indicated by more profound neurogenic inflammation, functional deficits and increased dopaminergic cell death. However, when SP was inhibited by treatment with a SP NK₁ receptor antagonist, dopaminergic neurons were conserved, the inflammatory response was reduced and motor function was returned to near normal levels. We conclude that SP is increased in early PD, and that increased SP plays an important role in the degenerative process, specifically, in the genesis of BBB breakdown and initiation of neurogenic inflammation. Treatment with an NK1 antagonist may thus represent a novel therapeutic approach to early stage Parkinson’s disease. / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Parkinson's disease (PD) is one of the most common motor neurodegenerative diseases, affecting 1-2% of the world's population over the age of 65. It is characterised by a loss of dopamine neurons within the substantia nigra, which is an integral part of the basal ganglia (BG) where dopamine is the most important modulating neurotransmitter. As the BG is primarily involved with the execution of movement, the lack of dopamine input results in dysfunctional motor control. The current PD treatment, L-DOPA, improves these motor symptoms, however only provides patients 5 to 10 years of improved quality of life before debilitating side effects, often worse than the original symptoms, begin. The neuropeptide substance P (SP) is found in high concentration in the substantia nigra, and BG in general, where it is involved in dopamine release. In the late stages of PD, SP content within the substantia nigra and BG is decreased, thus implicating SP in the pathophysiology of PD. However, SP production has not been examined in the early stages of PD when dopaminergic degeneration is first initiated. This thesis therefore sought to characterise the role of SP in dopaminergic degeneration in an experimental model of early PD, the 6-hydroxydopamine model in rats. In contrast to the prevailing dogma that a decline in SP is associated with neurodegeneration in PD, this thesis demonstrates that SP is actually increased within the striatum in early PD, particular in perivascular tissue and within surviving dopaminergic neurons during the degenerative process. Increasing exposure of the dopaminergic neurons to SP, either by inhibition of substance P breakdown with Captopril or by direct injection with SP, exacerbated the disease progression as indicated by more profound neurogenic inflammation, functional deficits and increased dopaminergic cell death. However, when SP was inhibited by treatment with a SP NK₁ receptor antagonist, dopaminergic neurons were conserved, the inflammatory response was reduced and motor function was returned to near normal levels. We conclude that SP is increased in early PD, and that increased SP plays an important role in the degenerative process, specifically, in the genesis of BBB breakdown and initiation of neurogenic inflammation. Treatment with an NK1 antagonist may thus represent a novel therapeutic approach to early stage Parkinson’s disease. / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009

Tendonopathy, resulting in the loss of mechanical strength of a tendon, is a serious health problem affecting many people. The common symptom of tendonopathy is pain – patients' daily activities, their participation in sport and exercise, and their ability to work are greatly compromised. Tendonopathy is considered to be a degenerative disorder caused by repetitive injury of the tendon. The most common tendon lesions are Achilles tendon rupture, lateral epicondylitis (tennis elbow) and rotator cuff tear. However, in spite of its clinical significance, our knowledge about tendonopathy is still very poor. This research was undertaken to investigate the pathology of tendonopathy. It is proposed that apoptosis, autophagic cell death and myofibroblasts play a role in the progression of tendonopathy in the rotator cuff; the aim of this study was therefore to determine if this was indeed the case. Tendon tissues were collected from 30 patients suffering from rotator cuff tears. A terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL assay) was performed to detect apoptosis. Autophagic cell death of the tenocytes in the ruptured rotator cuff tendon was detected by immunohistochemical staining for ubiquitin. Myofibroblasts were identified immunohistochemically with anti-alpha-smooth muscle actin (anti--SMA) antibody. The distribution of apoptosis, autophagic cell death and myofibroblasts, as well as the total cell density, were assessed respectively and were correlated using a four-category (i.e. graded from 0-3) degeneration of collagen matrix. – 6 – The results showed that apoptosis, autophagic cell death and myofibroblasts were observed in all of the samples. The highest percentage of autophagic cell death was evidenced in the Grade 2 matrix, while the percentage of apoptosis increased significantly with the increase of matrix degeneration from Grade 0-3; a similar pattern was found for myofibroblasts. The total cell numbers varied among the matrix grades, with the maximum and minimum percentages occurring in Grades 1 and 3, respectively. It can be concluded that apoptosis, autophagic cell death and myofibroblasts might be closely related to the damage of the extracellular matrix (ECM) structure.