Breast cancer metastasizes to the brain in 15-30% of all breast cancer cases, and metastasis is the predominant cause of breast cancer-related deaths. Patients with HER2-enriched and triple-negative breast cancers (TNBCs) are more likely to develop brain metastases. While targeted therapies exist for HER2-enriched breast cancers, there are no effective treatments for TNBCs. Thus, a greater understanding of how these cancers spread to the brain is critical. In order to spread to the brain, disseminated breast cancer cells must overcome 2 major steps—crossing the blood-brain barrier (BBB) and survival and successful colonization of the distinctive and mostly cellular brain environment. Here, we report a novel role for breast cancer cell surface receptor, Syndecan-1 (Sdc1), a heparan sulfate proteoglycan, in promoting breast cancer cell transmigration across the BBB. We found that when we silenced Sdc1 expression in a highly metastatic TNBC cell line, MDA-MB-231, these cells exhibited reduced migration across an in vitro BBB model system. Further, in an in vivo experimental model of metastasis, mice injected with MDA-MB-231 Sdc1 KD (knock-down) cells developed less brain metastases than mice injected with control non-silencing (NS1) cells. Conversely, we found that overexpression of Sdc1 in a metastatic triple-negative mouse mammary carcinoma cell line, 4T1, led to an increase in brain metastases compared to empty vector control-treated mice. We predicted that a secreted factor(s) facilitated BBB disruption that allowed for Sdc1-mediated BBB transmigration, and found that silencing Sdc1 led to decreases in the production and/or release of various cytokines and chemokines implicated in BBB permeability and transmigration. In addition to supporting BBB transmigration, through an in vitro tissue section adhesion assay, we found that Sdc1 also facilitates adhesion of breast cancer cells to the brain, and not to the liver or lungs, revealing specificity for the brain. Further, we report that Sdc1 is expressed in 81% of breast cancer patient brain metastases in our tissue microarray study and that patients with TNBC and high Sdc1 expression have shorter disease-free survival based on a study performed using data from The Cancer Genome Atlas. Taken together, we predict that breast cancer cell Sdc1-regulated cytokines and chemokines promote BBB permeability and/or support transmigration to facilitate breast cancer metastasis to the brain. We also provide evidence for breast cancer-secreted extracellular vesicles, namely exosomes, in supporting the formation of a pro-metastatic brain environment. We compared exosomes derived from the metastatic 4T1 mouse mammary carcinoma cell line to a non-metastatic counterpart, the 67NR cell line, to assess their microRNA and protein composition and their effect(s) on recipient astrocytes, known mediators of brain metastasis. We found that there are inherent differences in both the microRNA and protein cargo from the metastatic 4T1 cells compared to the non-metastatic 67NR cells, whereby the metastatic 4T1 cells contained various tumor-promoting microRNAs and proteins, and also contained 4.5-fold more protein than the non-metastatic 67NR cells. Mouse astrocytes treated with the metastatic 4T1 exosomes exhibited a shift towards a pro-metastatic phenotype, characterized by upregulation of pro-inflammatory genes, and genes associated with astrocyte reactivity and cancer, whereby 67NR exosome-treated astrocytes exhibited a response profile that overlapped with untreated controls. Overall, these findings reveal an important role for exosomes in driving changes in the brain microenvironment to create a site conducive for cancer growth. Together, both studies help to elucidate how breast cancer cells can invade and colonize the unique brain environment.

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by degeneration and loss of spinal cord motor neurons leading to denervation and muscular atrophy. It is caused by defects in the Survival Motor Neuron 1 gene (SMN1) and it is classified by age of onset and motor milestones into three main types which strongly correlate with the copy number of its homologous gene, SMN2. SMN2 expresses markedly less full‐length protein than SMN1, provoking disease manifestations. The essential neuropathological feature in SMA is motor neuron death. Previous studies in SMA foetal samples showed early pathological changes in spinal cord suggesting that the disease is a developmental disorder. Studies in mouse SMA models support that neuromuscular junctions (NMJs) may play a significant role in the disease, although this implication has not yet been addressed in humans. AIMS AND METHODOLOGIES: To better understand the mechanisms of SMA disease, a comprehensive histologic, immunohistochemical and ultrastructural analysis of the muscle and neuromuscular junctions in prenatal and postnatal SMA samples was carried out. To further correlate neuropathological findings with early developmental manifestations of the disease, foetal motility between 11‐14 gestational weeks was recorded and analyzed by 2D ultrasound in pregnancies predicted to develop SMA. RESULTS: At 12 weeks, most SMA myotubes were smaller than controls, indicating a delay in muscle maturation. At this stage, the presence of early acetylcholine receptor (AChR) clusters in developing SMA muscles suggested that pre‐patterned and nerve‐independent AChR clustering would not be affected by the disease. However, as development proceeded in the following weeks, early degeneration of nerve terminals was present associated with a dispersion of AChRs and abnormal preterminal accumulation of vesicles. These findings pointed towards a defect in maintenance of the initial innervation in developmental SMA muscle. Furthermore, postnatal muscle of type I SMA patients showed persistence of the foetal subunit of acetylcholine receptors, suggesting a continuous delay in maturation of neuromuscular junctions. Therefore, if nerve terminals are unable to efficiently maintain functional NMJs, a dying‐back process leading to motor neuron degeneration and loss may appear, with the consequent increase in programmed cell death. Despite all these early neuropathological findings, we did not observe qualitative differences in foetal movements between unaffected and SMA prenatal cases. The synaptic defects in SMA at this stage of development, therefore, might be compensated by several mechanisms. During perinatal and postnatal periods compensation would no longer be present, resulting in the drastic SMA pathology and clinical manifestations. CONCLUSIONS: These developmental studies open new possibilities to improve our knowledge of presymptomatic SMA stages. Early therapeutic strategies should be investigated to reverse the process of denervation, maintain activity of the NMJ, and improve maturity of the motor endplates. / INTRODUCCIÓN: La atrofia muscular espinal (AME) es una enfermedad neuromuscular infantil caracterizada por la muerte de las neuronas motoras del asta anterior de la médula espinal. Como consecuencia de ello hay una degeneración y atrofia muscular, por lo que los pacientes mueren a menudo de insuficiencias respiratorias graves. La AME se clasifica en tres tipos principales según el grado de gravedad, la edad de aparición y las pautas motoras. Se trata de una enfermedad con patrón de herencia autosómico recesivo causada por ausencia o mutaciones en el gen Survival Motor Neuron 1 (SMN1). Existe un gen homólogo, SMN2, que está presente en todos los pacientes aunque a diferencia del SMN1, produce mucha menor proteína SMN completa y, por lo tanto, no evita la aparición de la enfermedad. Sin embargo se ha demostrado una importante correlación con el tipo de AME y el número de copias de SMN2. El rasgo patológico esencial de la AME es la muerte de las neuronas motoras. Estudios hechos en muestras fetales indican que ya existen hallazgos patológicos en el estadio prenatal lo que sugiere que la AME sería un trastorno del desarrollo. Además en modelos de ratón AME se ha determinado que la unión neuromuscular tendría un papel importante en la patogenia de la enfermedad aunque en humanos todavía no existen investigaciones al respecto. OBJETIVOS Y METODOLOGÍA: Con el fin de profundizar en los conocimientos de la patogenia de la AME, en esta tesis se ha realizado un análisis histológico, inmunohistoquímico y utraestructural del músculo y la unión neuromuscular en muestras prenatales y postnatales de controles y AME. Paralelamente, se han correlacionado los resultados neuropatológicos obtenidos con el estudio de la motilidad fetal por ecografía 2D entre las 11 y 14 semanas de gestación en embarazos diagnosticados genéticamente como AME. RESULTADOS: A partir de las 12 semanas, los miotubos AME son más pequeños que los controles lo que es compatible con un retraso en la maduración muscular. En esta etapa, la presencia de receptores de acetilcolina agrupados en músculo AME sugiere que éste es capaz de formar la placa neuromuscular. Sin embargo en semanas posteriores se observa una degeneración temprana de los terminales nerviosos asociados a una dispersión de los receptores de acetilcolina y acumulación anormal de vesículas presinápticas. Esto indica que en este período uno de los principales defectos sería la falta de mantenimiento de la unión neuromuscular. El músculo postnatal AME muestra persistencia de la expresión del receptor fetal de acetilcolina que refuerza la idea de una maduración retardada de la unión neuromuscular que persiste durante todo el desarrollo. La falta de mantenimiento de las uniones neuromusculares justificaría el inicio de un proceso de muerte retrograda (“dying back process”) dando lugar a una excesiva pérdida de neuronas motoras en la médula espinal. El estudio de los movimientos fetales, sin embargo, no demostró diferencias cualitativas entre los fetos normales y los AME. La falta de correlación entre la neuropatología descubierta en los fetos con AME tipo I y la presencia de movimientos fetales normales en ese mismo grupo indica que deben existir mecanismos compensatorios en el feto AME que enmascaran las posibles consecuencias funcionales de los defectos sinápticos hallados. Estos mecanismos compensatorios desaparecerían más tarde dando lugar a las graves manifestaciones de la enfermedad en las etapas perinatal y neonatal. CONCLUSIONES: Los resultados obtenidos contribuyen al mejor conocimiento de esta enfermedad en etapas presintomáticas, y abre nuevas perspectivas para investigar estrategias terapéuticas a fin de revertir los procesos de denervación, mantener la actividad de las uniones neuromusculares y mejorar la maduración de las placas motoras.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

The majority of the population in South Africa use traditional health care to treat various mental conditions. This thesis has two main objectives; to bring together a comprehensive and detailed record of psychotropic plants used in southern Africa by indigenous peoples for medicinal or cultural purposes. Secondly, this research attempts to investigate the validity and rationale of the use of these plants by screening them in various biological assays for psychotropic activity. Plants were selected, based on their traditional use and availability, and were screened in four assays, which detect biological activity of a useful nature. A number of in vitro enzymatic and neuronal signal transduction assays were employed in this thesis, the inhibition of the serotonin reuptake transporter protein (SERT); inhibition of catabolic enzymes (e.g. acetylcholinesterase, monoamine oxidase); GABAA- benzodiazepine receptor binding. The influence of legislation, past and present, on the state of traditional medicine is highlighted. Aspects of the philosophies and practises of the various practitioners of South African traditional medicine will be discussed. An annotated list compiled from available ethnobotanical literature of plants traditionally used for central nervous system-related purposes is provided. It contains more than 330 species, from 94 families, which are currently used or have been used for cultural, medicinal and recreational purposes related to the central nervous system (CNS). Where available, information pertaining to plant part used, preparation method, dosage, route of administration, known and potentially active constituents are included. Seventy five extracts from 34 indigenous plant species used in South African traditional medicine or taxonomically related to these were investigated for their affinity to the serotonin reuptake transport protein, making use of an in vitro [3H]-citalopram serotonin reuptake transport protein binding assay. Aqueous and 70% ethanolic extracts of various plant parts were screened and 45 extracts derived from 15 plant species showed affinity. The affinity of 12 extracts from four plants was characterized as high (more than 50% inhibition at 5, 1, and 0.5 mg/ml). Plant species with high affinity to the serotonin reuptake transport protein included Agapanthus campanulatus, Boophone disticha, Datura ferox and Xysmalobium undulatum. Agapanthus campanulatus yielded high activity in aqueous extracts from leaves and flowers. B. disticha showed high activity both in aqueous and ethanolic extracts of leaves and bulbs. D. ferox showed high activity in aqueous extracts from the seeds and X. undulatum showed high activity in the ethanolic extract of the whole plant. Two compounds, buphanadrine and buphanamine, were isolated by bioassay-guided fractionation on vacuum-liquid-chromatography (VLC) and preparative thin-layer-chromatography (TLC) from B. disticha. The structures of the compounds were determined by 1H and 13C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [3H]-citalopram as a ligand. The IC50 values of buphanidrine and buphanamine were 274 ìM (Ki = 132 ìM) and 1799 ìM (Ki = 868 ìM), respectively. The two alkaloids were also tested for affinity to the 5HT1A receptor, but only showed slight affinity. Aqueous and ethanol extracts of 43 plants that are traditionally used to treat against epilepsy and convulsions were initially tested in the GABAA-benzodiazepine receptor binding assay, where the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in epilepsy and convulsions. Out of the 118 extracts tested, one aqueous and 18 ethanol extracts showed activity. The most active extracts were the ethanolic leaf extracts of Searsia tridentata, Searsia rehmanniana and Hoslundia opposita and the ethanolic corm extract of Hypoxis colchicifolia, which all showed good dose-dependent activity. A further forty-six ethanol extracts from another 35 species, both indigenous and exotic that are traditionally used predominantly as sedatives or to treat various CNS-related ailments were tested in the GABAA-benzodiazepine receptor-binding assay. Out of the 46 extracts tested, seven showed good activity and 10 showed moderate activity. The most active extracts were the ethanolic leaf extracts of Arctopus echinatus, Artemisa afra, four Helichrysum species and Mentha aquatica which all showed good dose-dependent activity. Two biflavonoids with activity in the 3H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Searsia pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of 3H-Ro 15-1788 with a Ki of 28 and 37 nM, respectively. Extracts of Searsia dentata and Searsia pentheri were not as active as the extract from Searsia pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABAA receptor benzodiazepine site. This reflected the affinities of the compounds in the [3H]-flumazenil binding assay. Mentha aquatica, a mint that is found in Europe and Africa, is used in Zulu traditional medicine for spiritual purposes. The ethanolic leaf extract showed a strong affinity to the GABA-benzodiazepine receptor. Viridiflorol from the essential oil and (S)-naringenin from an ethanolic extract was isolated by bioassay-guided fractionation using binding to the GABA-benzodiazepine site. Viridiflorol had an IC50 of 0.19 M and (S)-naringenin of 0.0026 M. Twenty plants used in Zulu traditional medicine for several CNS-related ailments were screened for MAO inhibition and specific MAO-B inhibition activity. MAO-B inhibitors are currently employed in the treatment of neurodegenerative related illnesses such as Parkinson's and Alzheimer's diseases. A photometric peroxidase linked assay was used to determine the inhibition of the oxidative deamination of tyramine by MAO isolated from rat liver. Ruta graveolens exhibited the best MAO inhibitory activity (ethyl acetate leaf extract = IC50 5 ± 1 ìg/ml, petroleum ether extract = 3 ± 1 ìg/ml) and specific MAO-B inhibition (ethyl acetate leaf extract = IC50 7 ± 6 ìg/ml petroleum ether extract = 3 ± 1 ìg/ml). Schotia brachypetala, Mentha aquatica and Gasteria croucheri also exhibited good MAO-B inhibition activity. Six extracts of varying polarity of Mentha aquatica were tested in a photometric peroxidase linked MAO bioassay. The 70% ethanol extract had highest inhibitory activity. (S)-Naringenin was isolated from the extract by bioassay guided fractionation on VLC and preparative TLC. The structure of the compound was determined by 1H, 13C and 13C-DEPT NMR and optical rotation. The IC50 values for MAO inhibition by naringenin were 342 ± 33 ìM for the rat liver mitochondrial fraction, 955 ± 129 ìM for MAO-A and 288 ± 18 ìM for MAO-B respectively. South African traditional medicine clearly utilizes many botanical species with CNS-related activity. Only a small number of the more than 330 southern African plant species reported to treat or alter the CNS have been scientifically evaluated. To date very few of the active compounds have been isolated and identified. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Multiple Sclerosis (MS) is a neurological autoimmune disease characterized by demyelination of central nervous system tissue and one way this is presented is in the demyelination of the retinal nerve, causing vision disturbance and loss (Munger et al., 2006). The thinning of the retinal nerve fiber layer (RNFL) can be measured and visualized using a noninvasive technique called Optical Coherence Tomography (OCT), which is also used to measure relative MS severity (Petzold et al., 2010). One environmental factor that has been found to have a relationship with MS is vitamin D; research findings suggest that sufficient levels of vitamin D may reduce the risk of developing MS, decrease MS severity, and may slow its progression (Ascherio et al., 2010; Munger et al., 2006; Muris et al., 2013). The mechanism by which vitamin D affects certain symptoms requires deeper investigation. This research examines the relationship between serum concentrations of 25-hydroxyvitamin D and retinal nerve fiber layer thicknesses in patients with MS. It was hypothesized that patients with sufficient vitamin D levels would have less demyelination of the retinal nerve caused by MS, and therefore would have a thicker RNFL in both eyes based on the proposed immunomodulatory role of vitamin D found in other studies. Blood samples were assayed to measure the concentration of 25-hydroxyvitamin D and OCT was used to measure RNFL thicknesses in patients with MS at the Harbor-UCLA Medical Center Neurology Clinic. Patients with sufficient levels of 25-hydroxyvitamin D had a greater mean global RNFL thickness in both eyes than in patients with insufficient levels of 25-hydroxyvitamin D; however the differences were not significant. Further research is necessary in order to determine whether or not there is a correlation between vitamin D and RNFL thickness and what role vitamin D plays in MS presentation.

Thesis (Ph. D.)--University of Lund, 1994. / Published dissertation. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains x, 102 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1994. / Published dissertation. Includes bibliographical references.

Introduction: The increase of cardiovascular disease risk has been demonstrated in subjects with HIV infection. The dysfunction in the regulation of the autonomic system has been identified as a mechanism underlying to cardiac death in this group of patients. This study aimed to analyze the association between cardiovascular risk and cardiac autonomic control in patients with HIV. Methods: The sample consisted of 25 patients with HIV, of both genders, in use of antiretroviral therapy and with undetectable viral load, treated on the of Infectious Diseases Ambulatory, from the University Hospital of Santa Maria (HUSM), between August and December of 2014. Patients with cardiovascular disease, metabolic, respiratory, neurological or kidney were excluded. The cardiovascular risk was assessed by Framingham score, used to estimate the probability of cardiovascular events in ten years. The cardiac autonomic control was assessed by measuring the heart rate variability, analyzing the following variables: 1) in the time domain were measured SDNN, triangular index (overall variability) and rMSSD (parasympathetic activity); 2) in the frequency domain understood the low frequency components (LF; sympathetic activity) and high frequency (HF; parasympathetic activity), both normalized, and the LF/HF ratio (sympatho-vagal balance). Results: The sample (12 women and 13 men) had a mean age of 48.7±10.9 years, body mass index of 25.7±5.1 kg/m2, heart rate of 72.1±13.4 bpm, respiratory rate of 16.3±3.8 bpm, systolic blood pressure of 125.2±18.7 mmHg and diastolic 83.3±12.2 mmHg. The average time since diagnosis of the disease was 10.2±5.0 years, the medication time of 7.2±4.2 years and the CD4 count of 628.6±223.8 mm3 of blood. The score obtained in the Framingham score was 9.5±5.1 and the risk of cardiovascular events was 9.5±7.9%, with 7 patients classified as low risk, 14 as intermediate risk and 4 as high cardiovascular risk. The Framingham score presented a correlation with the medication time (r=0.53), with the LFnu component (r=0.45) and with the ratio LF/HF (r=0.44), but it was inversely correlated with SDNN (r=-0.43), rMSSD (r=-0.47) and with the triangular index (r=-0.49). The risk of cardiovascular events was positively correlated with the medication time (r=0.54), with the LFnu component (r=0.45) and with the ratio LF/HF (r=0.45), but had negative correlation with SDNN (r=-0.40), rMSSD (r=-0.43) and the triangular index (r=-0.48). Conclusion: Patients with HIV classified into different bands of Framingham score, presented association between cardiovascular risk and increased sympathetic activity, decreased parasympathetic activity and sympatho-vagal balance. This demonstrates that even in patients with undetectable viral load, cardiovascular autonomic dysfunctions may be associated with cardiovascular risk in ten years. These findings point to the importance of routine assessments of cardiovascular autonomic nervous system in this population. / Introdução: o aumento do risco de doença cardiovascular tem sido demonstrado em sujeitos com infecção por HIV. A disfunção na regulação do sistema autônomo tem sido apontada como mecanismo subjacente a morte cardíaca nesse grupo de pacientes. Este estudo objetivou analisar a associação entre risco cardiovascular e controle autonômico cardíaco em portadores de HIV. Métodos: a amostra foi composta por 25 pacientes com HIV, de ambos os sexos, em uso de antirretrovirais e com carga viral não detectável, oriundos do Ambulatório de Doenças Infecciosas do Hospital Universitário de Santa Maria (HUSM), entre agosto e dezembro de 2014. Pacientes com doença cardiovascular, metabólica, respiratória, neurológica ou renal foram excluídos. O risco cardiovascular foi avaliado pelo Escore de Framingham, utilizado para estimar a probabilidade de eventos cardiovasculares em dez anos. O controle autonômico cardíaco foi avaliado pela medida da variabilidade da frequência cardíaca, analisando-se as seguintes variáveis: 1) no domínio do tempo foram mensurados o SDNN, índice triangular (variabilidade global) e rMSSD (atividade parassimpática); 2) o domínio da frequência compreendeu os componentes de baixa frequência (LF; atividade simpática) e de alta frequência (HF; atividade parassimpática), ambos normalizados, e a relação LF/HF (balanço simpato-vagal). Resultados: a amostra (12 mulheres e 13 homens) apresentavam idade média de 48,7±10,9 anos, índice de massa corporal de 25,7±5,1 kg/m2, frequência cardíaca de 72,1±13,4 bpm, frequência respiratória de 16,3±3,8 rpm, pressão arterial sistólica de 125,2±18,7 mmHg e diastólica de 83,3±12,2 mmHg. O tempo médio de diagnóstico da doença foi de 10,2±5,0 anos, o tempo de medicação de 7,2±4,2 anos e a contagem de CD4 de 628,6±223,8 mm3 de sangue. A pontuação obtida no Escore de Framingham foi de 9,5±5,1 e o risco de eventos cardiovasculares foi de 9,5±7,9%, sendo 7 pacientes classificados como baixo risco, 14 como risco intermediário e 4 como alto risco cardiovascular. A pontuação do Escore de Framingham apresentou correlação com o tempo de medicação (r= 0,53), com o componente LFnu (r=0,45) e com a relação LF/HF (r=0,44), mas correlacionou-se inversamente com o SDNN (r=-0,43), rMSSD (r=-0,47) e com o índice triangular (r=-0,49). O risco de eventos cardiovasculares esteve correlacionado positivamente com o tempo de medicação (r=0,54), com o componente LFnu (r=0,45) e com a relação LF/HF (r=0,45), porém, apresentou correlação negativa com o SDNN (r=-0,40), rMSSD (r=-0,43) e com o índice triangular (r=-0,48). Conclusão: Pacientes portadores de HIV, classificados em diferentes faixas do Escore de Framingham, apresentam associação entre o risco cardiovascular e o aumento da atividade simpática, redução da atividade parassimpática e do balanço simpato-vagal. Isso demonstra que, mesmo em pacientes com carga viral não detectável, as disfunções autonômicas cardiovasculares podem estar associadas ao risco cardiovascular em dez anos. Estes achados apontam para a importância de avaliações rotineiras do sistema nervoso autonômico cardiovascular nesta população.