Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to a-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and realtime quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/ macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.

Newly designed pyrrolo[2,1-c][1,4]benzodiazepines tricyclic skeleton has shown potential clusters of cannabinoid receptors CB1/CB2 selective ligands. CB2 plays a critical role in microglial-derived neuroinflammation, where it modulates cell proliferation, migration, and differentiation into M1 or M2 phenotypes. Beginning with computer-based docking studies accounting the recently discovered X-ray crystal structure of CB2, we designed a series of PBD analogs as potential ligands of CB2 and tested their binding affinities. Interestingly, computational studies and theoretical binding affinities of several selected (S,E)-11-[2-(arylmethylene)hydrazono]-PBD analogs, have revealed the presence of potential selectivity in binding attraction toward CB1 and CB2. Reported here is the discovery of the first representatives of this series of selective binding to CB2. Preliminary data showed that this class of molecules display potential binding efficacy toward the cannabinoid receptors tested. Intriguingly, initial cannabinoid binding assay showed a selective binding affinity of 4g and 4h showed Ki of 0.49 and 4.7 μM toward CB2 receptors while no binding was observed to CB1. The designed leads have shown remarkable stability pattern at the physiological pH magnifying their therapeutic values. We hypothesize that the PBD tricyclic structure offers the molecule an appropriate three-dimensional conformation to fit snugly within the active site of CB2 receptors, giving them superiority over the reported CB2 agonists/inverse agonists. Our findings suggested that the attachment of heterocyclic ring through the condensation of diazepine hydrazone and S- or N-heterocyclic aldehydes enhances the selectivity of CB2 over CB1. [Figure not available: see fulltext.].

No description available.

The World Health Organization (WHO) estimates that one billion people worldwide suffer from central nervous system (CNS) disorders [1]. One major issue in treating these disorders is inadequate drug penetration which can be attributed to an effective blood-brain-barrier that limits passage across blood vessels. Low diffusivity of large classes of drug compounds restricts transport across blood vessel walls and subsequent passage through surrounding brain tissues. Tissue transport is emerging as an increasingly important area of research in drug delivery since the vast majority of therapeutic agents must traverse this space before reaching their targets.

Increasing health care costs is an international problem from which South Africa is not excluded. Prescription medication contributes most to these high health care costs, and methods to reduce their costs to society are implemented worldwide. In South Africa, such a method is a managed care reference medicine price list, as introduced by a PBM (pharmacy benefit management) company. This step had some cost implications in the private health sector in South Africa, and these implications were investigated in this study. Central nervous system (CNS) medicine items are among the top ten medicine items claimed and represent a substantial amount of the costs of all medicine items claimed during the study period. Antidepressants, a subdivision of the CNS agents, comprise the largest share of CNS agents claimed and CNS costs, and were therefore investigated more closely. The objective of this study was to analyse the usage patterns and costs of central nervous system medicine items, and more specifically, the antidepressants, against the background of the implementation of a managed care reference medicine price list in the private sector of South Africa. This study was conducted as a retrospective, non-experimental quantitative research project. The study population consisted of all medicine items claimed as observed on the database over the two-year study period of May 2001 to April 2002 (pre-MPL) and May 2002 to April 2003 (post-MPL). Data were provided by MedschemeTM/lnterpharm, and the Statistical Analysis System® SAS 8.2® was used to extract the data from the database. The central nervous system agents had a prevalence of 8.10% (N=49098736) and a total cost of R757576976.72 over the two-year study period. The cost per CNS item increased by 5.98% or R11.50 per CNS item in the year after MPL implementation, and the cost per prescription containing CNS medicine items increased by 4.09% or R9.07 per prescription. CNS agents are classified into ten sub-pharmacological groups, according to the MIMSC3 (Snyman, 2003:13a). One of these sub-pharmacological groups, antidepressants, comprised 33.97% of all CNS medicine items claimed (N=3978364) and 45.53% of all costs associated with CNS medicine items (N=R757576976.72) over the study period. The number one antidepressant claimed was amitriptyline, a tricyclic antidepressant. Of the antidepressants with generic substitutes, all with the exception of clomipramine, were prescribed at generic substitution rates of more than 50%. After the MPL implementation, generic antidepressant products were more frequently prescribed (16.48% increase, N=617190), although patient co-payments did not decrease immediately. Some innovator products had price reductions after the implementation of the MPL. This study indicates that cost minimisation analyses and retrospective drug utilisation reviews are valuable tools in the evaluation of managed care medicine price lists. / Thesis (M. Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2005.

Increasing health care costs is an international problem from which South Africa is not excluded. Prescription medication contributes most to these high health care costs, and methods to reduce their costs to society are implemented worldwide. In South Africa, such a method is a managed care reference medicine price list, as introduced by a PBM (pharmacy benefit management) company. This step had some cost implications in the private health sector in South Africa, and these implications were investigated in this study. Central nervous system (CNS) medicine items are among the top ten medicine items claimed and represent a substantial amount of the costs of all medicine items claimed during the study period. Antidepressants, a subdivision of the CNS agents, comprise the largest share of CNS agents claimed and CNS costs, and were therefore investigated more closely. The objective of this study was to analyse the usage patterns and costs of central nervous system medicine items, and more specifically, the antidepressants, against the background of the implementation of a managed care reference medicine price list in the private sector of South Africa. This study was conducted as a retrospective, non-experimental quantitative research project. The study population consisted of all medicine items claimed as observed on the database over the two-year study period of May 2001 to April 2002 (pre-MPL) and May 2002 to April 2003 (post-MPL). Data were provided by MedschemeTM/lnterpharm, and the Statistical Analysis System® SAS 8.2® was used to extract the data from the database. The central nervous system agents had a prevalence of 8.10% (N=49098736) and a total cost of R757576976.72 over the two-year study period. The cost per CNS item increased by 5.98% or R11.50 per CNS item in the year after MPL implementation, and the cost per prescription containing CNS medicine items increased by 4.09% or R9.07 per prescription. CNS agents are classified into ten sub-pharmacological groups, according to the MIMSC3 (Snyman, 2003:13a). One of these sub-pharmacological groups, antidepressants, comprised 33.97% of all CNS medicine items claimed (N=3978364) and 45.53% of all costs associated with CNS medicine items (N=R757576976.72) over the study period. The number one antidepressant claimed was amitriptyline, a tricyclic antidepressant. Of the antidepressants with generic substitutes, all with the exception of clomipramine, were prescribed at generic substitution rates of more than 50%. After the MPL implementation, generic antidepressant products were more frequently prescribed (16.48% increase, N=617190), although patient co-payments did not decrease immediately. Some innovator products had price reductions after the implementation of the MPL. This study indicates that cost minimisation analyses and retrospective drug utilisation reviews are valuable tools in the evaluation of managed care medicine price lists. / Thesis (M. Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2005.

Adenosine is an essential purine nucleoside of particular importance within heart and brain. The widespread and diverse actions of adenosine, driven by activation of cell surface receptors, include regulation of sleep/arousal and neuroprotective properties. The mechanisms involved in regulating adenosine concentrations remain poorly understood but are critical to signaling pathways as they determine the availability of adenosine at corresponding receptors within the extracellular space. The equilibrative nucleoside transporter (ENT) family, bi-directional, Na+-independent nucleoside transporters, are key components in both the release and uptake of adenosine. This study has been conducted to investigate ENT3, a novel member of the ENT family. Our work has demonstrated ENT3 to be expressed throughout brain, located in cortex, cerebellum, striatum and hippocampus, at similar levels. Neurons and astrocytes, but not microglia, showed intracellular ENT3 localisation. This was confirmed by differential centrifugation, of cortex and cerebellum, which suggests ENT3 to be found within the cytoplasm.

Le glutamate est accumulé dans des vésicules synaptiques par des transporteurs vésiculaires du glutamate appelés VGLUT1-3. VGLUT1 et VGLUT2 sont utilisés par les neurones glutamatergiques «classiques» corticaux et sous-corticaux. VGLUT3 est présent dans des sous-populations de neurones utilisant d’autres neurotransmetteurs que le glutamate. Dans la cochlée, VGLUT3 permet la transmission glutamatergique entre les cellules ciliées internes et les neurones du nerf auditif. Le travail mené par l’équipe du Pr Puel a permis de découvrir l’implication de VGLUT3 dans une pathologie héréditaire de l’audition chez l’Homme. Une mutation p.A211V du gène codant VGLUT3 humain est responsable d’une surdité progressive à transmission autosomique. Il s’agit de la première mutation d’un VGLUT associé à une pathologie humaine. Mon travail de thèse a consisté à caractériser l’impact de cette mutation sur le SNC d’une lignée de souris exprimant cette mutation. Nous avons observé que cette mutation avait des effets complexes sur VGLUT3. La mutation p.A211V entraine une baisse marquée de l’expression de VGLUT3 dans les terminaisons nerveuses qui semble liée à une dégradation accélérée de VGLUT3. 20% d’expression résiduelle de VGLUT3 suffisent à assurer la majeure partie des fonctions du transporteur. L’activité de VGLUT3 ne semble donc pas être linéairement corrélée à son expression. De plus, la réduction de VGLUT3 au niveau des synapses semble s’accompagner d’une réduction du nombre de vésicules VGLUT3-positives et d’une réduction du nombre de copies de VGLUT3 par vésicule. Dans l’ensemble, mon travail de thèse a permis d’acquérir une meilleure connaissance de la régulation de VGLUT3. / Glutamate is the major excitatory neurotransmitter in the Central Nervous System (CNS) and is accumulated into synaptic vesicles by proton-dependent transporters named VGLUT1-3. VGLUT1 and VGLUT2-positive neurons are respectively found in cortical and subcortical glutamatergic neurons. In contrast, VGLUT3 is localized in a small population of neurons using other neurotransmitter than glutamate i.e.: cholinergic interneurons in the striatum, subpopulation of GABAergic interneurons in the hippocampus and cortex and serotoninergic neurons. Furthermore, VGLUT3 is also expressed by sensory inner hear cells (IHCs).In the cochlea, VGLUT3 accumulates glutamate into synaptic vesicles of the IHCs. A mutation of the gene that encodes VGLUT3 is responsible for a progressive, high-frequency deafness. It is the first mutation of a VGLUT that was demonstrated to be responsible for a human pathology.We investigated the effects of the p.A211V mutation on VGLUT3 in the CNS of a mouse line expressing this mutation. We observed that this mutation had complex effects on VGLUT3. The p.A211V mutation causes a 80% decrease of VGLUT3 in nerve endings. 20% residual expression of VGLUT3 is sufficient to fulfill most part of its functions. Contrary to prevailing views, VGLUT3 global activity is not linearly correlated to VGLUT3 quantity. Futhermore, VGLUT3 reduction seems to be associated with a diminution of VGLUT3-positive vesicles accompanied by an homogenous reduction of VGLUT3 copy number per vesicle.Overall, my thesis allowed to acquire a better understanding of the regulation of VGLUT3. This work will deepen our understanding of the involvement of VGLUTs in various pathologies.

Made available in DSpace on 2018-06-27T18:56:03Z (GMT). No. of bitstreams: 3 Diego Becker Borin.pdf: 1553948 bytes, checksum: b94d052c85f74f3bcc5e4cadbcb71e67 (MD5) Diego Becker Borin.pdf.txt: 134530 bytes, checksum: f3ad94a2052d1806ff21e9c0ce7733fb (MD5) Diego Becker Borin.pdf.jpg: 3538 bytes, checksum: b4b8b2603cafaeb4736f56efe30f7f1d (MD5) Previous issue date: 2013-03-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Creatine is a biomolecule endogenously synthesized from amino acids which can also be obtained through the diet. But it does not permeate easily through the blood-brain barrier (BBB), so the brain must supply their needs through the synthesis of creatine in the central nervous system (CNS) by itself. Creatine has a major role in maintaining stable levels of adenosine triphosphate (ATP) thus keeping the whole body in a proper condition. Neurodegenerative diseases leads to decreases in ATP levels, which compromises cellular metabolism, generating the increase of reactive species that could lead to neuronal cell death. Therefore, it becomes important to increase creatine levels in neurodegenerative diseases, so it could provide new alternative treatments for these diseases. This study aimed to develop and characterize liposomes with creatine, as well as investigate its protective effect in ischemia and reperfusion (I/R) brain model. Two methodologies were used for the preparation of liposomes, the lipid film hydration and ethanol injection. Due the best results were obtained by the ethanol injection method, this was chosen for the preparation of liposomes in in vivo tests. The rats were tested using I/R by clamping bilateral carotid arteries and different groups were treated with creatine (30 mg/kg) in free form (Liv+I/R) and liposomed (Lip+I/R) and compared with the control group and I/R. The animals motor activity, exploratory and memory abilities were evaluated through field tests and passive avoidance, 24 and 48 hours after I/R, respectively. After 72 hours, the animals were euthanized and the brains removed for biochemical determination of levels of reactive species and ascorbic acid (AA), and the biochemical breakdown activity of superoxide dismutase (SOD) and catalase (CAT). The suspensions of liposomes used for the in vivo treatment had homogenous average particle diameter (154 ± 6.9 nm), low polydispersity index (0.211 ± 0.019), pH near neutral (around 6.7), negative potential Zeta (-21 ± 1.8 mV) and association rate around 31%. The results of field tests have shown that I/R caused a change in exploratory activity of animals and increased oxidative stress in the brain of rats subjected to I/R. Liposomes creatine protected the change in exploratory activity and increased oxidative stress in rat cortex. Already in free form, creatine was not able to protect against these changes. With these results, we can conclude that the production of liposomes containing creatine technological feasibility presented, demonstrating the potential to increase the bioavailability of creatine into the CNS via the BBB. / A creatina é uma biomolécula sintetizada endogenamente a partir de aminoácidos, que também pode ser obtida através da dieta. Porém, ela não permeia facilmente através da barreira hematoencefálica (BHE), assim o cérebro deve suprir suas necessidades através da síntese de creatina no próprio sistema nervoso central (SNC). A grande importância da creatina para o funcionamento correto do organismo está em manter estáveis os níveis de adenosina trifosfato (ATP). Doenças neurodegenerativas levam a déficits de ATP, o que compromete o metabolismo celular, gerando espécies reativas (ERs) capazes de levar as células neuronais à morte. Assim, torna-se importante aumentar os níveis de creatina em doenças neurodegenerativas, podendo gerar novas alternativas de tratamento para estas doenças. Desta forma, o presente trabalho teve como objetivo desenvolver e caracterizar lipossomas de creatina, bem como, investigar o seu efeito protetor em um modelo de isquemia e reperfusão (I/R) cerebral. Foram utilizadas duas metodologias para o preparo dos lipossomas, a hidratação do filme lipídico e a injeção de etanol. Devido aos melhores resultados obtidos pelo método de injeção de etanol, este foi escolhido para o preparo dos lipossomas a serem aplicados nos testes in vivo. Os ratos foram submetidos ao processo de I/R pelo clampeamento bilateral das artérias carótidas, sendo os diferentes grupos tratados com a creatina (30 mg/kg) na forma livre (Liv+I/R) e lipossomada (Lip+I/R), sendo comparados com os grupos controle e I/R. A atividade locomotora, exploratória e a memória dos animais foram avaliadas através dos testes de campo aberto e da esquiva passiva, 24 e 48 h após a I/R, respectivamente. Após 72 h, os animais foram eutanasiados e os cérebros removidos para as determinações bioquímicas dos níveis de ERs e ácido ascórbico (AA), e a atividade das enzimas superóxido dismutase (SOD) e catalase (CAT). As suspensões de lipossomas utilizadas para o tratamento in vivo apresentaram diâmetro médio de partícula homogêneo (154 ± 6,9 nm), com baixo índice de polidispersão (0,211 ± 0,019), pH próximo a neutralidade (em torno de 6,7), potencial Zeta negativo (-21 ± 1,8 mV) e taxa de associação em torno de 31%. Os resultados do teste de campo aberto demonstraram que a I/R causou alteração na atividade exploratória dos animais. Além disso, o estudo demonstrou aumento no estresse oxidativo no cérebro de ratos submetidos a I/R. Os lipossomas de creatina protegeram a alteração na atividade exploratória e o aumento do estresse oxidativo em córtex de ratos. Já a creatina na forma livre não foi capaz de proteger contra estas alterações. Através destes resultados, pode-se concluir que a produção de lipossomas contendo creatina apresentou viabilidade tecnológica, demonstrando potencial para aumentar a biodisponibilidade de creatina para o SNC através da BHE.

Glioblastoma (GBM) is the most common and deadliest of the malignant primary brain tumors in humans, with a reported 5-year survival rate of only 6.8% despite years of extensive research. Failure to improve local tumor control rates and overall patient outcome is attributed to GBM's inherent therapeutic resistance. Marked heterogeneity, extensive local invasion within the brain parenchyma, and profound immunosuppression within the tumor microenvironment (TME) are some of the unique features that drive GBM therapeutic resistance. Furthermore, tumor cells are sequestered behind the blood-brain barrier (BBB), limiting delivery of effective therapeutics and immune cell infiltration into the local tumor. Electroporation-based therapies, such as irreversible electroporation (IRE) and second generation, high-frequency IRE (H-FIRE) represent attractive alternative approaches to standard GBM therapy given their ability to induce transient BBB disruption (BBBD), achieve non-thermal tumor cell ablation and stimulate local and systemic anti-tumor immune responses without significant morbidity. The following work explores the use of H-FIRE to overcome GBM-induced therapeutic resistance and improve treatment success. Chapter 1 opens with an overview of GBM and known barriers to treatment success. Here, we emphasize the utility of spontaneous canine gliomas as an ideal translational model for investigations into novel treatment approaches. Chapter 2 introduces novel ablation methods (i.e. IRE/H-FIRE) capable of targeting treatment-resistant cancer stem cells. The focus of Chapter 3 is to highlight IRE applications in a variety of spontaneous tumor types. In Chapter 4, we investigate the feasibility and local immunologic response of percutaneous H-FIRE for treatment of primary liver tumors using a spontaneous canine hepatocellular carcinoma (HCC) model. In chapter 5, we characterize the mechanisms of H-FIRE-mediated BBBD in an in vivo healthy rodent model. In Chapter 6, we characterize the local and systemic immune responses to intracranial H-FIRE in rodent and canine glioma models to enhance the translational value of our work. Collectively, our work demonstrates the potential for H-FIRE to overcome therapeutic resistance in GBM, thereby supporting its use as a novel, alternative treatment approach to standard therapy. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadliest form of primary brain cancer in humans, with only 6.8% of people surviving 5-years after their diagnosis. GBM is characterized by a number of unique features that make it resistant to standard treatments, such as surgery, radiation and chemotherapy. Examples include: (1) extensive invasion of tumor cells into the brain, making complete removal via surgery very difficult; (2) tumor cells are protected by a structure called the blood-brain barrier (BBB), which restricts the entry of most drugs (i.e. chemotherapy) and many immune cells, into the brain, thereby preventing them from reaching tumor cells; (3) tumor cells produce substances that block the immune system from being able to detect the tumor itself, which allows it to continue to grow undetected. High-frequency irreversible electroporation (H-FIRE) represents a new approach for the treatment of GBM. H-FIRE uses electric pulses to temporarily or permanently injure cell membranes without the use of heat, which allows for very precise treatment. The following work explores the ways in which H-FIRE can interfere with specific GBM features that drive its resistance to treatment. Here, we demonstrate that H-FIRE is capable of temporarily disrupting the BBB and characterize the mechanisms by which this occurs. This allows for drugs and immune cells within the blood to enter the brain and access the tumor cells, particularly those extending beyond the visible tumor mass and invading the brain. We also illustrate the potential for H-FIRE treatment within the brain to stimulate local and systemic immune responses by causing the release of proteins from injured cells. Similar to a vaccine, these proteins are recognized by the immune system, which becomes primed to help fight off cancer cells within the body. The end result is an anti-tumor immune response. Collectively, this work supports the use of H-FIRE as an alternative treatment approach to standard therapy for GBM given its potential to overcome certain causes of treatment resistance.