O glioblastoma multiforme (GBM) é uma doença grave e sem tratamento eficaz, especialmente pelos agentes terapêuticos disponíveis causarem reações adversas importantes nas doses terapêuticas. O metotrexato (MTX) é um fármaco citotóxico utilizado para tratar diversas neoplasias, no entanto, sua utilização é limitada pela baixa biodisponibilidade e reações adversas. A nanotecnologia tem sido utilizada para aumentar a eficácia dos medicamentos antitumorais, com o intuito de direcioná-los para o sítio de ação e reduzir os efeitos adversos. Nesse sentido, realizamos ensaios com nanocápsulas lipídicas de MTX (LNC MTX) para avaliar os mecanismos de captação em linhagens celulares de glioblastoma e micróglia, além investigar a eficácia terapêutica da LNC MTX em ensaios in vitro e in vivo. Inicialmente, ensaios de microscopia de fluorescência, empregando bloqueadores farmacológicos específicos para transportes de membrana, mostraram que as LNC MTX marcadas com Rodamina B penetram em células tumorais GL261 por endocitose, dependente de caveolinas, e em células de micróglia da linhagem BV2 por fagocitose e macropinocitose. Os tratamentos com LNC MTX ou solução de MTX (em concentrações correspondentes) em células GL261 inibiram a proliferação; aumentaram a fragmentação de DNA, mas, somente as LNC induziram a morte celular por necrose e diminuíram o número de células na fase G1/G0 do ciclo celular. Na linhagem celular BV2, os tratamentos com LNC MTX ou solução de MTX inibiram a proliferação, reduziram a quantidade de células na fase G1/G0 do ciclo celular, aumentaram a fragmentação de DNA e induziram morte celular por apoptose e apoptose tardia. Os ensaios in vivo de microscopia intravital mostraram que a LNC MTX atravessa a barreira Hematoencefálica (BHE) de camundongos fêmea C57Bl/6 após administração intravenosa ou oral, sem danificar a sua estrutura. O tamanho do glioblastoma in vivo foi reduzido em animais tratados com LNC MTX por via oral em relação aos animais tratados com salina. Esta redução não foi detectada em animais tratados com solução de MTX. Em conjunto, os dados obtidos mostram que a LNC MTX penetram em células de glioma e da glia e causam toxicidade, atravessam a BHE in vivo e sugerem que a nanoencapsulação do MTX pode ser uma estratégia importante para o tratamento do glioblastoma. / Glioblastoma multiforme (GBM) is a serious disease and no effective treatment is availabe, especially because the drugs cause significant adverse reactions in therapeutic doses. Methotrexate (MTX) is a cytotoxic drug used to treat many neoplasms, however, their use is limited by the low bioavailability and adverse reactions. Nanotechnology has been used to increase the effectiveness of antitumor drugs in order to direct them to the site of action and to reduce adverse effects. Accordingly, we carried out an experimental approach with MTX lipid nanocapsules (MTX LNC) to evaluate the uptake mechanisms in glioblastoma and microglia cell lines, and the therapeutic efficacy of MTX LNC in vitro and in vivo systems. Initially, fluorescence microscopy assays employing specific pharmacological blockers for membrane transport showed that the MTX LNC stained with Rhodamine B penetrated into GL261 tumor cells by caveolae-mediated endocytosis, and in BV2 microglia cells by phagocytosis and macropinocytosis. Treatment with MTX solution or MTX LNC (at corresponding concentrations) on GL261 cells inhibited the proliferation; increased DNA fragmentation, but only the LNC induced cell death by necrosis and decreased the number of cells in the G1/G0 phase of the cell cycle. In BV2 cells, treatment with MTX solution or MTX LNC inhibited proliferation, reduced number of cells in the G1/G0 phase of the cell cycle, increased DNA fragmentation and cell death, induced by apoptosis and late apoptosis. Intravital microscopy study showed that the MTX LNC across the Blood-Brain Barrier (BBB) of C57BL/6 female mice after intravenous or oral administrations, without damaging its structure. The area of glioblastoma in vivo was reduced in animals oral treated with MTX LNC comparing to saline treated mice. This reduction was not observed in animals treated with MTX solution. Together, the data herein obtained show that MTX LNC penetrate the cell membrane and cause cell toxicity on glioma and neurons lineage, cross the BBB and suggest that the nanoencapsulation of MTX can be an important strategy for the treatment of glioblastoma.

Sepse e suas complicações (sepse grave e choque séptico) ainda são a principal causa de morte nas unidades de terapia intensiva em todo o mundo. Estudos clínicos e experimentais têm demonstrado que na fase inicial da sepse a concentração plasmática de arginina vasopressina (AVP) está elevada. No entanto, durante o processo fisiopatológico os níveis plasmáticos da mesma permanecem inadequadamente baixos, apesar de haver hipotensão persistente. Uma das hipóteses sugeridas para essa deficiência relativa de AVP é a apoptose de neurônios vasopressinérgicos. Nosso objetivo foi identificar elementos envolvidos na morte celular hipotalâmica, além de avaliar o comportamento de células gliais e da barreira hematoencefálica (BHE) durante a sepse. Ratos Wistar foram submetidos à sepse por ligadura e punção cecal (CLP) ou não manipulados (naive) como controle e então divididos em dois grupos. No primeiro, foram perfundidos e os cérebros coletados para imunohistoquímica. Outro grupo foi decapitado para a retirada de sangue para dosagem de interferon- gama (IFN-?) e encéfalo para análise da expressão de proteínas no hipotálamo ou nos núcleos supraópticos (SON) e paraventriculares (PVN). Um terceiro foi separado para investigação da permeabilidade da BHE. Apesar de aumento da imunomarcação de CD8 e MHC-I no SON dos animais sépticos, não encontramos indícios de morte celular mediada por células imunes. No SON e PVN de animais sépticos, a expressão de fatores envolvidos na ativação da via extrínseca de apoptose (tBID, caspase-8 clivada) se manteve inalterada, enquanto fatores anti-apoptóticos relacionados à via intrínseca (BCL-2, BCL-xL) estavam diminuídos no hipotálamo. No SON destes animais a micróglia assumiu uma morfologia associada à sua ativação, concomitante com o aumento plasmático de IFN-?. Houve rompimento transitório da BHE no hipotálamo após 6 horas do CLP. Os resultados indicam que a via intrínseca de apoptose parece ser a responsável pela morte celular que é observada nos núcleos vasopressinérgicos e essa condição está temporalmente associada à ativação microglial e rompimento da BHE / Sepsis and its complications (severe sepsis and septic shock) remain as the main cause of death in intensive care units worldwide. Clinical and experimental studies have shown that in the early phase of sepsis the plasma concentration of arginine vasopressin (AVP) is increased. However, during the pathophysiological process the plasma levels remain inadequately low, despite of persistent hypotension. One of the hypothesis suggested for this relative deficiency is the apoptosis of vasopressinergic neurons. Our objective was to identify elements involved in the hypothalamic cellular death and evaluate the modifications of glial cells and blood-brain-barrier (BBB) during sepsis. Wistar rats were submitted to sepsis by cecal ligation and puncture (CLP) or non-manipulated (naïve), as control and then divided in two groups. In the first one, they were perfused and brains were collected for immunohistochemistry. In another one they were decapitated for blood collection and further plasma interferongama (IFN-?) analysis by ELISA. Brain was also collected for apoptosis-related proteins expression analysis in the hypothalamus or in the supraoptic (SON) and paraventricular (PVN) nuclei. A third set was separated for the investigation of BBB permeability. Despite of increased immunostaining for CD8 and MHC-I in the SON of septic animals, we did not find evidence of cell death mediated by immune cells. In the SON and PVN of septic animals, the expression of proteins involved in the activation of the extrinsic apoptosis pathway (tBID, cleaved caspase-8) was not altered, whereas anti-apoptotic factors related to the intrinsic pathway (BCL-2, BCLxL) were decreased. In the SON of these animals, microglia assumed a morphology related to its activation, associated with the increase of plasma IFN-?. There was a transitory breakdown of BBB in hypothalamus after 6 hours following CLP. The results indicate that the intrinsic apoptosis pathway seems to be responsible for the cell death observed in vasopressinergic nuclei and this condition is temporally associated with microglial activation and BBB leaking

Orientador: Maria Alice da Cruz Hofling / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T15:24:51Z (GMT). No. of bitstreams: 1 Mendonca_MoniqueCulturatoPadilha_M.pdf: 7402848 bytes, checksum: 64244a22fab0eb94170d857f28da8e50 (MD5) Previous issue date: 2013 / Resumo: O fator de crescimento endotelial vascular (VEGF), o principal regulador da angiogênese e da permeabilidade vascular, foi recentemente reconhecido como neurotrófico, neurogênico e neuroprotetor, sendo, portanto, regulado positivamente em muitos processos neuropatológicos. Neste modelo experimental de quebra da barreira hematoencefálica (BHE) pelo veneno da aranha Phoneutria nigriventer (PNV), a expressão do VEGF e seus receptores tirosina-quinase, Flt-1 e Flk-1 e de seus RNAs mensageiros foi investigada no hipocampo e cerebelo de ratos Wistar (Rattus norvegicus) por imunohistoquímica (IHQ), western blotting (WB) e reação em cadeia da polimerase em tempo real (qPCR). Paralelamente, a integridade da BHE foi avaliada através da expressão das proteínas da via paracelular, Ocludina e ?-catenina, e da principal proteína da membrana basal, a Laminina, que estão presentes no endotélio na interface sangue-cérebro. O estudo foi realizado em ratos de 14 dias (neonatos) e de 8-10 semanas (adultos jovens) para avaliar diferenças em função da idade na funcionalidade da BHE e na possível mediação dos efeitos neurotóxicos do PNV pelo VEGF. A via escolhida para administração de PNV (1,7 mg/kg em 0,5ml de salina 0,9%) foi intraperitoneal, devido sua administração mais favorável nos animais neonatos. Os tempos de 2, 5 e 24 horas após a administração de PNV visaram investigar a expressão das proteínas, RNAs mensageiros e uma possível mediação pelo VEGF na fase aguda do envenenamento. A administração do PNV provocou sinais imediatos de intoxicação nos animais, os quais foram mais severos e imediatos nos neonatos do que nos adultos. No hipocampo, os dados do WB mostraram aumento da expressão de VEGF, Flt-1 e Flk-1 e respectivos RNAs mensageiros, que foram concomitantes com o desenvolvimento de edema perivascular e diminuição da expressão da Ocludina, ?-catenina e Laminina. Os dados da IHQ mostraram que a imunorreatividade de VEGF ocorreu nos corpos dos neurônios piramidais e dendritos nos subcampos CA1, CA2, CA3 e giro denteado do hipocampo, em contraste com a marcação nuclear de Flt-1 e Flk-1. O PNV aumentou visivelmente a imunomarcação das três proteínas. No cerebelo, os dados do WB e IHQ mostraram que o PNV induziu aumento na expressão dos componentes do sistema VEGF/Flt-1/Flk-1. Células presentes na camada granular e molecular que não mostraram marcação nos animais controles passaram a ser positivas nos animais xviii envenenados, principalmente as células de Purkinje. Os animais adultos apresentaram aumentos mais proeminentes no sistema VEGF/Flt-1/Flk-1 do cerebelo do que os recémnascidos; nestes, o PNV induziu diminuição na expressão da Ocludina, ?-catenina e Laminina, enquanto nos adultos a diminuição ocorreu apenas na Ocludina e ?-catenina. O veneno da aranha P. nigriventer contem peptídeos neurotóxicos que causam excitabilidade na neurotransmissão nervosa por modificarem a fisiologia dos canais de sódio, potássio e cálcio. Uma vez que a dinâmica das alterações descritas diferiu entre regiões cerebrais e entre animais neonatos e adultos jovens, sugerimos particularidades regionais e de funcionalidade, tanto da BHE, como dos neurônios imunorreativos em resposta ao PNV. Estes resultados são os primeiros a demonstrar alterações do sistema VEGF/Flt-1/Flk-1 no hipocampo e cerebelo que cursam com sinais neuroexcitotóxicos dos animais que foram tratados com o veneno / Abstract: Vascular endothelial growth factor (VEGF), a major regulator of developmental angiogenesis and vascular permeability, was recently recognized as neurotrophic, neurogenic and neuroprotector, hence being upregulated in many neuropathological processes. In this experimental model of blood brain barrier (BBB) breakdown by the Phoneutria nigriventer spider venom (PNV), the expression of VEGF and its receptor tyrosine kinases, Flt-1 and Flk-1 and their mRNAs was investigated in the hippocampus and cerebellum of Wistar rats (Rattus norvegicus) by immunohistochemistry (IHC), western blotting (WB) and real time polymerase chain reaction (qPCR). Simultaneously, the BBB integrity was assessed through expression of paracellular pathway proteins, ?- catenin and Occludin, and the main basement membrane protein, Laminin, which are present in the endothelium blood-brain interface. The study was performed in rats by 14 days (neonates) and 8-10 weeks (young adults) to assess differences related to age in the BBB functionality and the possible mediation of the PNV neurotoxic effects by VEGF. The via chosen for PNV administration (1.7 mg/kg in 0.5 ml of 0.9% saline) was intraperitoneally, due to more favorable application in neonate animals. The times of 2, 5 and 24 hours after PNV administration aimed to investigate the expression of proteins, mRNAs, and possible mediation by VEGF in acute envenomation. The PNV administration provoked immediate signs of intoxication in animals, which were more severe and immediate in neonates than in adults. In hippocampus, the WB data showed increased expression of VEGF, Flt-1 and Flk-1 and their mRNAs, which were concomitant with the development of perivascular edema, and decreased expression of Occludin, ?-catenin and Laminin. IHC data show that VEGF immunoreactivity occurred in the bodies and dendrites of pyramidal neurons in the subfield CA1, CA2, CA3 and dentate gyrus of the hippocampus, in contrast with nuclear staining of Flt-1 and Flk-1. The PNV visibly increased immunostaining of the three proteins. In cerebellum, the WB data and IHC showed that the PNV induced an increase in the expression of VEGF/Flt-1/Flk-1 system components. Cells in the granular and molecular layer showed no marking in the control animals became positive in animals envenomed, especially Purkinje cells. Adult animals showed increases more prominent in the VEGF/Flt-1/Flk-1 system of the cerebellum than xx neonates; in these animals, the PNV induced decrease in expression of Occludin, ?-catenin and Laminin, while in adults the decrease occurred only in Occludin and ?-catenin. The venom of the spider P. nigriventer contains peptides that cause excitability in the nervous neurotransmission by modifying the physiology of sodium, potassium and calcium channels. Since the dynamics of the changes described differed between brain regions and among neonates and young adult animals, we suggest regional specificities and functionality of BHE and immunoreactive neurons in response to PNV. These results are the first to demonstrate changes in the hippocampus and cerebellum VEGF/Flt-1/Flk-1 system that courses with neuroexcitotoxic signs of animals that were treated with venom / Mestrado / Farmacologia / Mestra em Farmacologia

Dans les systèmes biologiques, les barrières tissulaires permettent le transport sélectif de molécules du sang au tissu approprié. Un exemple de barrière tissulaire est la barrière hémato-encéphalique (BHE). La BHE protège le cerveau du sang et maintient l'homéostasie du microenvironnement du cerveau, ce qui est essentiel à l'activité et à la fonction neuronale. La caractérisation de cette BHE est importante, car un dysfonctionnement de cette barrière est souvent révélateur de toxicité ou de maladie. Bien que le nombre d'articles publiés dans le domaine du développement et de la caractérisation de la BHE ait été multiplié ces dernières années, la validité des modèles utilisés est encore un sujet de débat. L'avènement de l'électronique organique a créé une occasion unique pour coupler les mondes de l'électronique et de la biologie, à l'aide de dispositifs tels que le transistor électrochimique organique (OECT). OECT constitue un outil très sensible et économique pour diagnostiquer l’intégrité d’une barrière tissulaire. Dans cette étude, nous avons tout d’abord développé trois différents modèles de BHE. Nous avons optimisé l’adhésion des cellules endothéliales cérébrales sur la matière active du transistor. Nous avons ainsi pu établir l'intégration des OECTs avec des cellules immortalisées humaines micro vasculaires cérébrales endothéliales (h CMEC/D3) en tant que modèle in vitro de BHE. Nous avons démontré que la fonction de tissu de la BHE peut être détectée en utilisant cette nouvelle technique. En outre, par cette technique, une perturbation de la barrière (par exemple, provoquée par un composé toxique) pourra être détectée électriquement au moyen d'une mesure de courant. / In biological systems many tissue types have evolved a barrier function to selectively allow the transport of matter from the lumen to the tissue beneath; one example is the Blood Brain Barrier (BBB). The BBB protects the brain from the blood and maintains homeostasis of the brain microenvironment, which is crucial for neuronal activity and function. Characterization of the BBB is very important as its disruption or malfunction is often indicative of toxicity/disease. Though the number of published papers in the field of in vitro BBB has multiplied in recent years, the validity of the models used is still a subject of debate.The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the Organic ElectroChemical Transistor (OECT), which provide a very sensitive way to detect minute ionic currents in an electrolyte as the transistor amplifies the gate current.In this study, we test three different type of BBB in order to develop a stable BBB model. We optimize the adhesion of brain endothelial cell on OECT conducting polymer. We show the integration of OECTs with immortalized human cerebral microvascular endothelial cells as a model of human blood brain barrier, and demonstrate that the barrier tissue function can be detected. Moreover, by this technique, a disruption in the barrier (e.g. caused by a toxic compound) is assessed electrically through a measurement of the drain current. Results show the successful development and validation of an in vitro BBB model. Dynamic monitoring of the barrier properties of the BBB barrier tissue was possible using the OECT.

Cerebral ischaemia by mechanism of thrombosis is one of the leading causes of disability and/or death worldwide, the outcome thereof increasing in severity with advancing age. Cerebral ischaemia triggers a cascade of events including inflammation, blood-brain barrier disruption and apoptosis. It is well known that oestrogen is neuroprotective through various mechanisms including the interruption of inflammation, regulation of thrombosis and delay of apoptosis. This creates a strong factorial interconnection in predicting the consequences of cerebral ischaemia. Since platelets have a central role in thrombosis and inflammation, their ultrastructure being altered in conditions of inflammatory and thrombotic derivation, the question arises whether chemical analysis of coagulation factors and ultrastructural analyses of platelet morphology may provide further insight into the role of oestrogen during ischaemic insult associated with stroke. Accordingly, an exclusively hyperglycaemic modification of the two-vessel occlusion model for inducing experimental cerebral ischaemia was established, since pre-ischaemic hyperglycaemia is known to intensify the outcome of cerebral ischaemic injury. Consequent neural tissue injury levels were correlated for three experimental groups (males, cyclic and acyclic females) of Sprague Dawley rats at vital times, to the presence of oestrogen as well as changes in coagulation factors and ultrastructure. This design allowed for an association to be formed between cerebral ischaemia, inflammation and thrombotic potential. Collectively the results strongly suggest that oestrogen is indeed neuroprotective through various actions including roles in the regulation of thrombosis and inflammation, targeting neural cells through the inhibition of apoptosis and exerting anti-inflammatory and antioxidant effects. It is evident that under the influence of oestrogen in cyclic females, there is reduced neural tissue injury as well as a lesser degree of inflammation evident in coagulation factor analysis and platelet activation morphology when compared to males and acyclic females. Oestrogen therefore exerts positive effects on the outcome of cerebral ischaemia through mechanisms which regulate inflammation, thrombosis and apoptosis. Furthermore it is unmistakeable that neural injury is closely shadowed, if not preceded, by inflammatory changes in the coagulation system, particularly manifested in platelet ultrastructure. It is therefore suggested that platelets may be used successfully to follow the progression of events of cerebral ischaemia and possibly assist in the assessment of treatment strategies and their effects on haemostasis. This research advances the understanding that inflammation is evident soon after ischaemic insult and if such inflammation is not curbed, necrosis of platelets and more severe injury to neural tissue may follow. Therefore, the development of agents which not only target thrombosis, but also which control inflammation must be explored to advance treatment strategies. It is proposed that even before it is determined whether a stroke has been caused by thromboembolism or haemorrhage; it will be beneficial to immediately target inflammation in order to prevent most severe consequences in human patients. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Anatomy / unrestricted

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of t h e Rf extract was able to partially alleviate excess ROS-induced effects. / National Research Foundation (NRF)

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Cerebral small vessel pathology is now known to be associated with the development of cognitive impairment and mild motor impairments such as gait disturbance in a variety of neurodegenerative diseases. This dissertation explores the hypothesis that blood brain barrier dysfunction is an early event in cerebral ischemia and contributes to the development of cerebral small vessel disease (CSVD). A common rodent model of CSVD is permanent bilateral common carotid artery occlusion in the rat. This model was used to study several aspects of the progression of CSVD including the timecourse of blood brain barrier permeability changes following the onset of ischemia, gait disturbance, the expression of tight junction proteins and cytokine expression. It was determined that BBB permeability was elevated for 2 weeks following BCCAO and ischemic rats displayed lower gait velocity. There was no change in expression of TJ proteins. However, ischemic rats had higher levels of some proinflammatory cytokines and chemokines in brain tissue with no obvious changes in plasma levels. The mechanisms underlying the increase in BBB permeability were studied in vitro using artificial barriers made of confluent rat brain microvascular endothelial cells. Cerebral ischemia has been reported to cause an increase in plasma toxicity, likely by elevating the numbers of circulating microparticles (MPs). MPs isolated from the plasma of ischemic rats were applied to artificial barriers where it was found that they act mainly as vectors of TNF-α signaling. MPs induce activation of caspase-3 and the Rho/Rho kinase pathways. It is concluded that most of the increase in barrier permeability is due to apoptosis and disassembly of actin cytoskeleton and disruption of adherens junctions IV and not an increase in transcellular transport. The effects of treatment with the type III phosphodiesterase inhibitor cilostazol on dye extravasation in the brain, glial activation, white matter damage and motor performance were evaluated. It was determined that cilostazol could improve the increased BBB permeability and gait disturbance and microglial activation in optic tract following BCCAO. Also, the effects of treatment with cilostazol on plasma toxicity in vivo (24h and 14d following BCCAO) and artificial barriers (in vitro) were assessed. It was found that cilostazol could reduce plasma toxicity at 24h and improve increased endothelial barrier permeability that is induced by MP treatment respectively. In summary BBB dysfunction occurs in the rat model of chronic cerebral hypoperfusion with no differences in expression of TJ proteins. There is a mild motor disturbance in the form of lower gait velocity following BCCAO. Cytokines released in brain tissue may be associated with pathological consequences following BCCAO while there is no significant difference in plasma levels and circulating MPs may play a role in BBB dysfunction.

For decades the opioid receptors have been an attractive therapeutic target for the treatment of pain. Since the first discovery of enkephalin, approximately a dozen endogenous opioid peptides have been known to produce opioid activity and analgesia, but their therapeutics have been limited mainly due to low blood brain barrier penetration and poor resistance to proteolytic degradation. One versatile approach to overcome these drawbacks is the cyclization of linear peptides to cyclic peptides with constrained topographical structure. Compared to their linear parents, cyclic analogs exhibit better metabolic stability, lower offtarget toxicity, and improved bioavailability. Extensive structure-activity relationship studies have uncovered promising compounds for the treatment of pain as well as further elucidate structural elements required for selective opioid receptor activity. The benefits that come with employing cyclization can be further enhanced through the generation of polycyclic derivatives. Opioid ligands generally have a short peptide chain and thus the realm of polycyclic peptides has yet to be explored. In this review, a brief history of designing ligands for the opioid receptors, including classic linear and cyclic ligands, is discussed along with recent approaches and successes of cyclic peptide ligands for the receptors. Various scaffolds and approaches to improve bioavailability are elaborated and concluded with a discourse towards polycyclic peptides.

Oligonucleotide-based drugs hold great promise for the treatment of many types of diseases, ranging from genetic disorders to viral infections and cancer. The problem is that efficient delivery across the cell membrane is required for oligonucleotides to have their desired effect. Cell-penetrating peptides (CPPs) provide a solution to this problem. CPPs are capable of transporting cargoes such as drugs or nucleic acids for gene therapy into the cell, either by covalent conjugation to the cargo or by non-covalent complex formation. This thesis is focused on the development of a class of peptides called PepFects, peptides with fatty acid modifications capable of forming nanoparticle-sized complexes with oligonucleotides. These complexes are efficiently internalized by many different cell types and are generally non-toxic and non-immunogenic. We have developed a number of novel PepFect peptides and a quantitative structure-activity model to predict the biological effect of our peptides. In addition, the involvement of scavenger receptors class A in the endocytic uptake of PepFect complexes as well as other CPPs and polymeric transfection agents was studied. Lastly, we have developed a series of PepFect peptides for delivery across the blood-brain barrier and a model system mimicking the blood-brain barrier in order to evaluate the passage of these peptides. The general aim of this thesis is to improve the understanding of intracellular delivery of oligonucleotides with PepFect peptides from both a chemical and a biological viewpoint, and further improve the efficacy of this delivery system with the long-term goal of making it useful in clinical settings.