Stroke is the 3rd leading cause of death and the leading cause of major disability worldwide. Currently, there are no neuroprotective drugs approved for the acute treatment of ischaemic stroke. The vast majority of stroke therapeutics failed in clinical trials due to toxic side effects and/or a clinically irrelevant therapeutic window. This thesis is focused on exploiting the delayed neurodegeneration that occurs in the compromised penumbra, as these cells may be capable of being saved by therapeutic intervention in a clinically obtainable window. In order to investigate the ischaemic cascade and be able to draw conclusions that are applicable to humans, the international gold standard animal model for cerebral ischaemia, the filament insertion middle cerebral artery occlusion (MCAO) model, was established at the University of Otago. This model was validated under new laboratory conditions and employed adult male Sprague Dawley rats. After testing multiple occlusion lengths, it was concluded that a 2hr ischaemic period was sufficient to produce a consistent infarct of optimal size. It has been well documented that neuroinflammation contributes to much of the delayed progression of neural injury post-stroke. Therefore, the catechin (-)-epigallocatechin gallate (EGCG), which is an anti-inflammatory, anti-oxidant and free-radical scavenging agent was investigated in the MCAO model of stroke. 50mg/kg i.p. of EGCG or saline was administered immediately post-MCAO and animals were sacrificed at 72hr post-filament insertion. The results confirmed that treatment with EGCG was neuroprotective and non-toxic. However, EGCG also induced an over 50% increase in the risk of haemorrhagic conversions. The anti-platelet effects of EGCG and lack of toxicity suggests that the catechin may prove to be an efficacious prophylactic for stroke. The contrary findings for EGCG treatment led to the re-evaluation of the neuroinflammatory pathway for alternate mechanisms to target therapeutic interventions. The temporal profile of the primary inducible enzymes nitric oxide synthase (NOS), cyclooxygenase (COX) and arginase (and their isoforms) were quantified 0, 3 and 7 days post-stroke. In both hemispheres, total NOS activity exhibited a significant and sustained up-regulation to 7 days post-occlusion. In the ipsilateral hemisphere at least half of the total increase was accounted for by inducible NOS (iNOS) expression. Arginase, which competes with NOS for L-arginine, demonstrated a delayed but significant increase in activity by day 7 in the infarcted hemisphere, thereby correlating well with the downward slope of NOS activity (illustrating the switch in the conversion pathway). COX activity was observably increased in the ipsilateral hemisphere, but the up-regulation did not reach significance by day 7. Alternately, the contralateral hemisphere displayed a significant decrease in activity by day 3. These results give conclusive evidence that the contralateral hemisphere is NOT an appropriate internal control and imply that NOS and COX inhibitors may prove to be efficacious for a much longer therapeutic window than current treatments. However, the delayed induction of COX activity may also indicate that this enzyme has a finite therapeutic window, as it may also stimulate remodelling of surviving neural networks. The prolonged up-regulation of inflammatory mediators implies that there may be an induction of an autoimmune component to the response. Therefore, the thymus (T) lymphocyte activation was quantified up to 14 days post-stroke. Cluster of differentiation (CD) 3⁺ T lymphocytes (equally contributed to by CD4⁺ and CD8⁺ T cells) exhibited a significant and sustained up-regulation in the infarcted region from day 3 up to at least day 14 post-ischaemia. Quantitative analysis of all cells present post-stroke determined that immune cells make up an average of 73% of all cells present in the 'peak' ischaemic areas. The CD4⁺ T helper cell response was delineated by double immunohistochemical labelling. Interferon-γ positively labelled with CD4⁺ T cells at days 3, 7 and 14 post-insult detailing a Th1-driven pro-inflammatory response. This evidence indicates that the autoimmune response is critical post-ischaemia and that it may be highly susceptible to modification by anti-inflammatory therapeutic intervention. The primary downstream effect of the pro-inflammatory/immune cascade is apoptosis. The main organelle responsible for the 'go, no go' response to apoptotic factors is the mitochondria. In order to distinguish whether mitochondrial dysfunction was initiated shortly after ischaemia induction or if it was delayed, like the inflammatory/immune response, to a clinically relevant window, the temporal profile of mitochondrial complex inactivation was studied. It was found that mitochondrial membrane viability was impaired by day 3, followed by a significant decrease in respiratory complex activation and an increase in tissue injury by oxidative stress by 7 days post-ischaemia. These results indicate that targeting the early decrease in membrane viability or mitochondrial permeability transition pore opening combined with anti-apoptotic therapeutics, may attenuate the proceeding mitochondrial impairment in oxidative phosphorylation, reactive oxygen species generation and subsequent cell death cascades. The current investigations into the temporal profile and quantitative contributions of the inflammatory, immune and apoptotic mechanisms post-stroke highlight potential strategies for modulation by acute stroke therapeutics. Furthermore, the general knowledge amassed from these studies dictates the necessity of a new approach to therapeutic intervention. The acknowledgement of so many contributing systems suggests that in addition to a thrombolytic, a combination therapy involving multiple neuroprotectants should be employed to account for the multifaceted nature of the sequelae of ischaemic stroke.

歯科基礎医学会, 金子 知生 = Tomoo Kaneko, 歯に矯正力を加えた際の圧迫側歯周組織の三次元的様相について = Three-dimensional situation of periodontal tissue at pressure side incident to orthodontic tooth movement, 歯科基礎医学会雑誌, 36(2), APR 1994, pp.170-186 / Hokkaido University (北海道大学) / 博士 / 歯学

Graduation date: 1998

La formation de néo-vaisseaux sanguins est un processus impliqué dans de nombreuses pathologies, telles que le développement de carcinomes cutanés et la néo-vascularisation choroïdienne caractéristique de la forme exsudative de la dégénérescence maculaire liée à lâge (DMLA). Dans ces deux cas, lactivation du réseau vasculaire est un facteur de mauvais pronostic. Lanalogie entre ces deux pathologies est renforcée par le développement récent dapproches thérapeutiques anti-angiogènes. Ces approches ciblent principalement le VEGF et les molécules associées. Malgré lefficacité de ces molécules sur la pathologie ciblée, leur administration systémique engendre des effets secondaires non négligeables. Notre travail a pour but détudier limplication dautres acteurs moléculaires dans ces pathologies, en vue de développer des stratégies thérapeutiques alternatives ou complémentaires. Parmi les principales molécules impliquées lors de langiogenèse, les protéases et leurs inhibiteurs sont des cibles potentielles pour le développement de nouveaux traitements. Il était initialement admis que les protéases (MMPs, protéases à sérine) sont des facteurs pro-angiogènes et leurs inhibiteurs (TIMPs, PAI-1) des facteurs anti-angiogènes. Dans ce contexte, lhypothèse thérapeutique évidente était dinhiber les protéases et de protéger ou dactiver leurs inhibiteurs. Cependant, lorsque nous avons entamé ce travail, ce concept a été remis en question. En effet, un niveau élevé de PAI-1 a été détecté dans de nombreux cancers et représente un facteur de mauvais pronostic. Par ailleurs, les inhibiteurs des MMPs nont présenté aucun effet lors dessais cliniques, certains stimulant même la croissance tumorale. Il est important de noter que ces premiers inhibiteurs étaient des inhibiteurs à large spectre bloquant laction non seulement des MMPs, mais aussi des membres de familles proches. Une détection fine des rôles joués par les MMPs et linhibiteur PAI-1 sest avérée indispensable. Nous avons, dès lors, focalisé notre travail sur deux thèmes : létude de PAI-1 et létude du rôle individuel de quelques MMPs. Il a précédemment été démontré que linhibiteur PAI-1 exerce un rôle pro-angiogène lors du développement de carcinomes cutanés et de néo-vascularisation choroïdienne. PAI-1 exerce donc un effet paradoxal et complexe sur langiogenèse. Bien que le rôle de PAI-1 au cours de langiogenèse bourgeonnante était bien documenté, son implication au cours de la vasculogenèse nétait pas connue. Nos résultats démontrent que le développement des carcinomes cutanés nécessite la migration des cellules stromales adjacentes aux cellules tumorales. Par contre, la néo-vascularisation choroïdienne, également dépendante de PAI-1, requiert le recrutement des cellules issues de la moelle osseuse. Publications 1 et 2. La seconde partie de notre travail est consacrée aux métalloprotéinases matricielles. Nos résultats montrent les rôles opposés ou synergiques des MMPs. Les MMP-2 et -9 sont des protéases pro-angiogènes agissant de concert au cours de linvasion des carcinomes. A lopposé, la MMP-19 exerce une fonction anti-angiogène et nos travaux suggèrent que cette MMP contribuerait à la stabilité des vaisseaux matures et au maintien physiologique des tissus, son expression étant diminuée lors de linvasion tumorale. Publications 3, 4 et 5.

The bending of an orthotropic cusped plate in energetic and weighted Sobolev spaces has been considered. The existence and uniqueness of generalized and weak solutions of admissible boundary value problems (BVPs) have been investigated.

The aim of this thesis was to identify genetic factors involved in frontotemporal lobar degeneration (FTLD), a neurodegenerative disorder clinically characterised by a progressive change in personality, behaviour and language. FTLD is a genetically complex disorder and a positive family history is found in up to 40% of the cases. In 10-20% of the familial cases the disease can be explained by mutations in the gene encoding the microtubule associated protein tau (MAPT). In the first study we describe the clinical and neuropathological features of a Finnish family with FTLD caused by a mutation in MAPT. We also provide evidence that the pathogenic mechanism of this mutation is through altered splicing of MAPT transcripts. Recently, mutations in the gene encoding progranulin (PGRN) were identified as a major cause of FTLD. In the second study we describe a Swedish family with FTLD caused by a frameshift mutation in PGRN. We provide a clinical and neuropathological description of the family, as well as evidence that the pathogenicity of this mutation is through nonsense-mediated decay of the mutant mRNA transcripts and PGRN haploinsufficiency. In the third study we describe a novel PGRN splice site mutation and a previously described PGRN frameshift mutation, found in a mutation screen of 51 FTLD patients. We describe the clinical and neuropathological characteristics of the mutation carriers and demonstrate that haploinsufficiency is the pathogenic mechanism of the two mutations. In the fourth study we investigate the prevalence of PGRN and MAPT gene dosage alterations in 39 patients with FTLD. No gene dosage alterations were identified, indicating that variations in copy number of the PGRN and MAPT genes are not a common cause of disease, at least not in this FTLD patient collection.

The bank vole (Microtus arvalis) develops glucose intolerance both when kept in captivity and in the wild state. Glucose intolerant bank voles kept in captivity exhibited polydipsia, polyuria, hyperglycemia, hyperinsulinemia, islet autoantibodies and a markedly changed islet structure resembling so–called hydropic degeneration. Islets showing hydropic degeneration have reduced β–cell mass. However, the relative islet size to total pancreas area was not changed. Pancreatic islet isolated from glucose intolerant bank voles had an altered islet function showing signs of being exposed to an increased functional demand on their β–cells. Also, islets from male bank voles seem more affected than the islets from females. Islets isolated from glucose tolerant male bank voles cultured for 5 days at 28 mM glucose did not reveal any change in insulin gene expression or insulin biosynthesis rate. However, islets from female bank voles displayed a glucose concentration dependent response. This suggests that there is gender difference in that, islets of female more easily than islets of males adapt to elevated glucose concentration. Furthermore, islets isolated from glucose tolerant males had reduced insulin gene expression after exposure to proinflammatory cytokines for 48 hrs. This effect seemed to be NO-independent since only a minor elevation of nitrite accumulation in the medium was seen, and the use of iNOS inhibitor could not counteract the cytokine effect. The observed response seen in bank vole islets upon exposure to various glucose concentrations or proinflammatory cytokines is similar to those seen in studies of human islets. The bank vole may therefore represent a novel animal model for the study of diabetes. An unresolved issue is the role of the Ljungan virus which is found in the bank vole colony. Bank voles developing glucose intolerance display features of both human type 1 and type 2 diabetes, where environmental factors seems to play an important role as determinant. Our findings suggest that bank voles bred in the laboratory may develop more of a type 2 diabetes. However, bank voles caught in nature instead may rather develop a type 1 form of the disease.

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.

The pharmacological development of non-steroidal contraceptives has yet to be achieved. Arresting oocyte maturation without blocking ovulation has been evaluated using different inhibitors of the phosphodiesterase 3A (PDE3A). Unfortunately, PDE3A is also expressed in the heart and blood vessels, and inhibition of PDE3A in oocytes can produce cardiovascular side effects. We reviewed the literature on available PDE3 inhibitors and selected cilostazol (CLZ), which is an FDA approved therapeutic. CLZ has the ability to decrease cellular adenosine uptake and consequently antagonizes side effects of PDE3A inhibition in vital organs. CLZ inhibited oocyte meiotic maturation in vitro. CLZ has more degenerative impact on arrested oocytes than matured oocytes, indicating that prolonged meiotic arrest of oocytes is harmful. Administration of CLZ any time from 9h before the ovulatory stimulus to 4h after the stimulus resulted in ovulation of immature oocytes. Controlling CLZ dose, time of CLZ administration, and time of oocyte collection resulted in ovulation of oocytes at different meiotic stages. Oral administrations of CLZ in naturally cycling mice were also observed to block pregnancy whereas remating of those previously treated females resulted in normal offspring and litter sizes. Therefore, CLZ does not only have a wide margin of contraception but also is reversible. Ovulated immature oocytes were observed to have higher rates of advanced chromatin configuration and cortical granule distribution, normal spindle and chromosomal organization, maturation, and in vitro fertilization (IVF) than ovarian immature oocytes. Ovulated metaphase I oocytes that were matured in vitro or in vivo had higher IVF rates than ovulated mature oocytes. Ovulated germinal vesicle (GV) oocytes that were in vitro matured also showed higher IVF rates but when in vivo matured, they had lower IVF rates than ovulated mature oocytes because of the high degeneration and low fertilization rates associated with in vivo maturation of GV oocytes. In summary, CLZ merits further evaluation as a non-steroidal contraceptive and is capable of producing oocytes of various meiotic stages with advanced developmental features.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.